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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">00947</article-id><article-id pub-id-type="doi">10.7554/eLife.00947</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Biochemistry</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group></article-categories><title-group><article-title>The ER–Golgi intermediate compartment is a key membrane source for the LC3 lipidation step of autophagosome biogenesis</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-5338"><name><surname>Ge</surname><given-names>Liang</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-4"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-5520"><name><surname>Melville</surname><given-names>David</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" id="author-5521"><name><surname>Zhang</surname><given-names>Min</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf2"/></contrib><contrib contrib-type="author" corresp="yes" id="author-1032"><name><surname>Schekman</surname><given-names>Randy</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Department of Molecular and Cell Biology</institution>, <institution>Howard Hughes Medical Institute, University of California, Berkeley</institution>, <addr-line><named-content content-type="city">Berkeley</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Levine</surname><given-names>Beth</given-names></name><role>Reviewing editor</role><aff><institution>University of Texas Southwestern Medical School</institution>, <country>United States</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>schekman@berkeley.edu</email></corresp></author-notes><pub-date date-type="pub" publication-format="electronic"><day>06</day><month>08</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e00947</elocation-id><history><date date-type="received"><day>15</day><month>05</month><year>2013</year></date><date date-type="accepted"><day>03</day><month>07</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Ge et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Ge et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife00947.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.00947.001</object-id><p>Autophagy is a catabolic process for bulk degradation of cytosolic materials mediated by double-membraned autophagosomes. The membrane determinant to initiate the formation of autophagosomes remains elusive. Here, we establish a cell-free assay based on LC3 lipidation to define the organelle membrane supporting early autophagosome formation. In vitro LC3 lipidation requires energy and is subject to regulation by the pathways modulating autophagy in vivo. We developed a systematic membrane isolation scheme to identify the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) as a primary membrane source both necessary and sufficient to trigger LC3 lipidation in vitro. Functional studies demonstrate that the ERGIC is required for autophagosome biogenesis in vivo. Moreover, we find that the ERGIC acts by recruiting the early autophagosome marker ATG14, a critical step for the generation of preautophagosomal membranes.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.001">http://dx.doi.org/10.7554/eLife.00947.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.00947.002</object-id><title>eLife digest</title><p>Cells continually adapt their behavior to accommodate changes in their environment. For example, when nutrients are abundant, cells can grow or proliferate; in times of scarcity, however, they must conserve resources for essential tasks. In particular, during periods of starvation, cells can cannibalize themselves in a process called autophagy, which literally means ‘self-eating’. Structures called autophagosomes engulf bits of cytoplasm and carry the contents to the digestive compartment of the cell, the lysosome, to be broken down into their constituent parts. This can include the degradation of proteins into amino acids, which can then be recycled into other proteins needed by the cell.</p><p>In cells, proteins are shipped to their destinations—which can be the plasma membrane or a specific organelle within the cell—via a delivery system known as the secretory pathway. This pathway begins in the endoplasmic reticulum (ER), where many of these proteins are made. From the ER, the proteins move to a compartment called the Golgi apparatus, which then sends them to their destinations, or to the lysosome to be broken down. Between the ER and Golgi they pass through a structure called the ER–Golgi intermediate compartment (ERGIC).</p><p>Although the signaling pathways that initiate autophagy are known, less is understood about the actual formation of the autophagosomes. Now, Ge et al. have developed an in vitro system to study their formation, and gone on to identify a membrane that is both necessary and sufficient to create these structures.</p><p>Previous studies have implicated a variety of membranes—including the plasma membrane and the membranes belonging to the ER, the Golgi apparatus, the lysosome and various other organelles—in the formation of autophagosomes. To identify which of these membranes might be involved, Ge et al. focused on a protein called LC3 that is a key marker for the formation of the autophagosome. This protein is recruited to the growing autophagosome by a lipid, so discovering which membranes can add a lipid to LC3 should shed light on the assembly process.</p><p>By separating the full range of organelles in a cell lysate into fractions (a process called fractionation), Ge et al. found that the ERGIC was the most active membrane to attach lipid to LC3. Additionally, the lipid was only added when signaling pathways that stimulate autophagy—such as the PI3K pathway—were activated. Together, these results provide insight into the mechanism of autophagosome formation, and the structures in the cell that participate in this process.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.002">http://dx.doi.org/10.7554/eLife.00947.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>autophagy</kwd><kwd>ER–Golgi intermediate compartment</kwd><kwd>LC3 lipidation</kwd><kwd>autophagosome</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Human</kwd><kwd>Mouse</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>Howard Hughes Medical Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Schekman</surname><given-names>Randy</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>University of California, Berkeley Miller Institute</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Schekman</surname><given-names>Randy</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Human Frontier Science Program</institution></institution-wrap></funding-source><award-id>LT000003/2012</award-id><principal-award-recipient><name><surname>Ge</surname><given-names>Liang</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution>Jane Coffin Childs Fund</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Ge</surname><given-names>Liang</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>A cell-free biochemical assay for protein lipidation identifies the ER–Golgi intermediate compartment as a key early station in the formation of an autophagosome.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>Autophagy is a conserved catabolic process underlying the self-digestion of cytoplasmic components through the formation of double-membraned vesicles termed autophagosomes. One basic role of autophagy is to turn over damaged proteins and organelles to maintain cellular homeostasis. Autophagy also allows cells to cope with stresses such as starvation, hypoxia, and pathogen infection (<xref ref-type="bibr" rid="bib44">Mizushima et al., 2008</xref>; <xref ref-type="bibr" rid="bib5">Burman and Ktistakis, 2010</xref>; <xref ref-type="bibr" rid="bib36">Levine, 2005</xref>; <xref ref-type="bibr" rid="bib76">Yang and Klionsky, 2010</xref>; <xref ref-type="bibr" rid="bib73">Weidberg et al., 2011</xref>).</p><p>In the process of starvation-induced autophagy, several upstream signals are triggered, including inhibition of the mechanistic target of rapamycin (MTOR), and activation of the Jun N-terminal kinase (JNK) and AMP kinase (AMPK) (<xref ref-type="bibr" rid="bib50">Noda and Ohsumi, 1998</xref>; <xref ref-type="bibr" rid="bib72">Wei et al., 2008</xref>; <xref ref-type="bibr" rid="bib31">Kim et al., 2011</xref>; <xref ref-type="bibr" rid="bib60">Rubinsztein et al., 2012</xref>; <xref ref-type="bibr" rid="bib30">Kim et al., 2013</xref>). These signals are conveyed to activate the serine/threonine-protein kinase complex containing the Atg1 homologs ULK1/2, ATG13, FIP200 (RB1CC1) and ATG101 (C12orf44) (<xref ref-type="bibr" rid="bib43">Mizushima, 2010</xref>). Together with upstream signals, this complex promotes the formation and membrane docking of the class III phosphatidylinositol 3 (PtdIns3)-kinase (PI3K) complex consisting of ATG14 (ATG14L/Barkor), the Atg6 homologue BECN1 (Beclin1), VPS34 (PIK3C3) and VPS15 (p150) for phosphatidylinositol 3-phosphate (PI3P) production (<xref ref-type="bibr" rid="bib51">Obara and Ohsumi, 2011</xref>). Subsequently, DFCP1 (ZFYVE1), an endoplasmic reticulum (ER)-associated PI3P binding protein, is recruited to the site of newly-generated PI3P to form omegasomes (<xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>). This is followed by two ubiquitin-like conjugation systems to produce the ATG12–ATG5 conjugate and phosphatidylethanolamine (PE)-lipidated ATG8/LC3, which initiates the formation of a preautophagosomal organelle termed the phagophore or isolation membrane (<xref ref-type="bibr" rid="bib45">Mizushima et al., 1998a</xref>, <xref ref-type="bibr" rid="bib46">1998b</xref>; <xref ref-type="bibr" rid="bib26">Ichimura et al., 2000</xref>; <xref ref-type="bibr" rid="bib15">Geng and Klionsky, 2008</xref>). The membrane then expands and engulfs cytoplasmic components. Finally, the crescent-shaped tubular membrane seals to form a double-membraned autophagosome with cytoplasmic components enclosed within the inner membrane. Fusion of the autophagosome with the lysosome leads to the breakdown of the inner membrane together with the trapped cytosolic material (<xref ref-type="bibr" rid="bib45">Mizushima et al., 1998a</xref>; <xref ref-type="bibr" rid="bib5">Burman and Ktistakis, 2010</xref>; <xref ref-type="bibr" rid="bib76">Yang and Klionsky, 2010</xref>; <xref ref-type="bibr" rid="bib73">Weidberg et al., 2011</xref>; <xref ref-type="bibr" rid="bib60">Rubinsztein et al., 2012</xref>).</p><p>A long-standing quest in the autophagy field has been to define the origin of the autophagosomal membrane. Recent data suggest a multi-membrane source model for autophagosome biogenesis. The endoplasmic reticulum (ER) supports PI3P-dependent formation of the omegasome, a cradle for phagophore generation and elongation (<xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>; <xref ref-type="bibr" rid="bib23">Hayashi-Nishino et al., 2009</xref>; <xref ref-type="bibr" rid="bib78">Yla-Anttila et al., 2009</xref>). The outer membrane of the mitochondrion may also supply lipids for the phagophore and autophagosome (<xref ref-type="bibr" rid="bib19">Hailey et al., 2010</xref>). Recently, a study by Hamasaki et al. (<xref ref-type="bibr" rid="bib20">Hamasaki et al., 2013</xref>) indicates the ER–mitochondrial junction as being required for autophagosome biogenesis, possibly reconciling these two origins. In addition, clathrin-coated vesicles from the plasma membrane have been shown to promote phagophore expansion through the SNARE protein VAMP7 and its partner SNAREs (<xref ref-type="bibr" rid="bib58">Ravikumar et al., 2010</xref>; <xref ref-type="bibr" rid="bib48">Moreau et al., 2011</xref>). Moreover, ATG9-positive vesicles cycle between distinct cytoplasmic compartments to deliver membrane to a developing autophagosome or, in yeast, to phagophore assembly sites (PAS) (<xref ref-type="bibr" rid="bib79">Young et al., 2006</xref>; <xref ref-type="bibr" rid="bib62">Sekito et al., 2009</xref>; <xref ref-type="bibr" rid="bib39">Mari et al., 2010</xref>; <xref ref-type="bibr" rid="bib49">Nair et al., 2011</xref>; <xref ref-type="bibr" rid="bib54">Orsi et al., 2012</xref>; <xref ref-type="bibr" rid="bib75">Yamamoto et al., 2012</xref>). Autophagosomes may also acquire membrane from other sources including Golgi (<xref ref-type="bibr" rid="bib16">Geng et al., 2010</xref>; <xref ref-type="bibr" rid="bib53">Ohashi and Munro, 2010</xref>; <xref ref-type="bibr" rid="bib77">Yen et al., 2010</xref>; <xref ref-type="bibr" rid="bib69">van der Vaart et al., 2010</xref>), early endosomes (<xref ref-type="bibr" rid="bib38">Longatti et al., 2012</xref>) and vesicles budding from the ER and Golgi (<xref ref-type="bibr" rid="bib21">Hamasaki et al., 2003</xref>; <xref ref-type="bibr" rid="bib83">Zoppino et al., 2010</xref>; <xref ref-type="bibr" rid="bib17">Guo et al., 2012</xref>). Although tremendous progress has been made, a direct functional link between a membrane source and autophagosome biogenesis has not been established. Furthermore, the identity of the membrane determinant that responds to a stress signal to initiate autophagosome formation is unknown.</p><p>A variety of visual techniques have been developed to define the origin of the autophagosome membrane. Here, we developed a functional approach relying on the lipidation of LC3 to assay an early stage in autophagosome biogenesis. We establish a cell-free system that reflects many of the physiological and biochemical landmarks of early events in the autophagic pathway and define the ER–Golgi intermediate compartment (ERGIC), a membrane compartment between ER and Golgi for cargo sorting and recycling (<xref ref-type="bibr" rid="bib1">Appenzeller-Herzog and Hauri, 2006</xref>), as a key membrane determinant for autophagosome biogenesis.</p></sec><sec id="s2" sec-type="results"><title>Results</title><sec id="s2-1"><title>Establishment of a regulated in vitro LC3 lipidation assay</title><p>A key step in autophagosome biogenesis is the generation of PE-lipidated LC3 by a ubiquitin-like conjugation system (<xref ref-type="bibr" rid="bib26">Ichimura et al., 2000</xref>; <xref ref-type="bibr" rid="bib28">Kabeya et al., 2000</xref>). The level of LC3 lipidation has long been a reliable measure of autophagy activity in vivo (<xref ref-type="bibr" rid="bib32">Klionsky et al., 2012</xref>). In vitro LC3 lipidation has recently been reconstituted with synthetic liposomes, recombinant LC3 and other components including the ATG12–ATG5 conjugate, ATG7 and ATG3 (<xref ref-type="bibr" rid="bib64">Sou et al., 2006</xref>; <xref ref-type="bibr" rid="bib22">Hanada et al., 2007</xref>; <xref ref-type="bibr" rid="bib52">Oh-oka et al., 2008</xref>; <xref ref-type="bibr" rid="bib63">Shao et al., 2007</xref>; <xref ref-type="bibr" rid="bib12">Gao et al., 2010</xref>). We sought to capture this modification in a more physiological context by relying on native membranes and cytosol to provide the core components of LC3 lipidation as well as any regulatory proteins that may be required for early autophagosome formation.</p><p>To establish such an assay, we mixed autophagosome precursor-deficient membranes with cytosol from normal and starved cells. Cells lacking ATG5 are deficient in starvation-induced autophagy and phagophore formation (<xref ref-type="bibr" rid="bib47">Mizushima et al., 2001</xref>). Hence they only contain unmodified LC3 (LC3-I) in both cytosol and membrane fractions (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Cytosols derived from WT cells (including WT MEF [mouse embryonic fibroblast], COS-7 [<italic>Cercopithecus aethiops</italic> fibroblast-like kidney cells], and HEK293T [human embryonic kidney 293T cells]) were highly enriched in LC3-I whereas the lipidated form of LC3 (LC3-II) sedimented with membranes (<xref ref-type="bibr" rid="bib28">Kabeya et al., 2000</xref> and <xref ref-type="fig" rid="fig1">Figure 1A</xref>). We incubated membranes from <italic>Atg5</italic> KO MEFs with cytosol from WT MEFs in the presence of GTP and an ATP regeneration system (<xref ref-type="fig" rid="fig1">Figure 1B</xref>) and observed the formation of LC3-II in a time- (<xref ref-type="fig" rid="fig1">Figure 1B</xref>) and ATP-dependent manner (<xref ref-type="fig" rid="fig1">Figure 1C</xref>).<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.003</object-id><label>Figure 1.</label><caption><title>In vitro reconstitution of endogenous LC3 lipidation.</title><p>(<bold>A</bold>) The distribution of LC3-I and LC3-II between the cytosol (C) and membrane (M) fractions from indicated cells. Cytosol and membranes from indicated cells were separated and evaluated by immunoblot (IB) with indicated antibodies. TFR, transferrin receptor (<bold>B</bold>) cell-free reconstitution of LC3 lipidation. Membranes from <italic>Atg5</italic> knockout (KO) MEFs were incubated with cytosol from wild type (WT) cells plus GTP and an ATP regeneration system (ATPR) for the indicated times. Then SDS-PAGE and immunoblot were performed to detect the generation of lipidated LC3 (LC3-II). (<bold>C</bold>) ATP dependence of in vitro LC3 lipidation. Reactions similar to (<bold>B</bold>) were performed in the absence or presence of indicated reagents followed by SDS-PAGE and immunoblot. (<bold>D</bold>) Delipidation of LC3 by ATG4B. A reaction similar to (<bold>B</bold>) was performed and the 16,000×<italic>g</italic> membranes were sedimented and solubilized with 1% TritonX-100. The indicated concentrations of ATG4B were incubated with the samples for 30 min followed by SDS-PAGE and immunoblot. Asterisk, non-specific band.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.003">http://dx.doi.org/10.7554/eLife.00947.003</ext-link></p></caption><graphic xlink:href="elife00947f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Characterization of the in vitro-lipidated LC3.</title><p>(<bold>A</bold>) LC3-II distributes in the 16,000×<italic>g</italic> membrane pellet fraction. Reactions similar to those of <xref ref-type="fig" rid="fig1">Figure 1B</xref> were performed. After the indicated times, the post-reaction mixtures were centrifuged at 16,000×<italic>g</italic> for 10 min. The pellet fractions (16k P) were collected and the supernatant fractions were further centrifuged at 100,000×<italic>g</italic> yielding the pellet (100k P) and supernatant (100k S) fractions. SDS-PAGE and immunoblot were performed with indicated antibodies. RPN1, Ribophorin 1 (<bold>B</bold>) LC3-II is tightly anchored to membranes. A reaction similar to that in (<bold>A</bold>) was performed and 16,000×<italic>g</italic> membranes were collected. The pellet was resuspended, divided into aliquots and incubated with indicated reagents followed by another 16,000×<italic>g</italic> centrifugation to separate into pellet (P) and supernatant (S) fractions, which were examined by SDS-PAGE and immunoblot. TFR, transferrin receptor.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.004">http://dx.doi.org/10.7554/eLife.00947.004</ext-link></p></caption><graphic xlink:href="elife00947fs001"/></fig></fig-group></p><p>We compared the fractionation and biochemical properties of the in vitro-generated LC3-II to its in vivo counterpart. In a crude fractionation study, we found that the in vitro-generated LC3-II partitioned in the 16,000×<italic>g</italic> membrane fraction (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>). Moreover, the in vitro product resisted extraction with urea or Na<sub>2</sub>CO<sub>3</sub> (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>) and was delipidated to LC3-I by ATG4B (<xref ref-type="fig" rid="fig1">Figure 1D</xref>), a cysteine protease that cleaves the C-terminal tail of LC3 and removes PE from LC3-II (<xref ref-type="bibr" rid="bib68">Tanida et al., 2004</xref>). These properties are shared with LC3-II generated in vivo (<xref ref-type="bibr" rid="bib28">Kabeya et al., 2000</xref>; <xref ref-type="bibr" rid="bib68">Tanida et al., 2004</xref>).</p><p>Starvation-induced lipidation of LC3 requires the ATG12–ATG5 conjugate (<xref ref-type="bibr" rid="bib47">Mizushima et al., 2001</xref>). To test the ATG5 dependence and starvation effect on in vitro LC3 lipidation, we incubated cytosols from either untreated or starved WT cells or <italic>Atg5</italic> KO MEFs with the corresponding membranes from <italic>Atg5</italic> KO MEFs (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). LC3-II formation was stimulated about threefold in incubations containing cytosol from starved WT MEFs and membranes from starved <italic>Atg5</italic> KO MEFs, compared to incubations containing cytosol and membranes from non-starved MEFs (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Cytosol from <italic>Atg5</italic> KO MEFs did not generate LC3-II when combined with membranes from <italic>Atg5</italic> KO MEFs (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). In addition, cytosols from COS-7 and HEK293T cells also reconstituted starvation-induced lipidation of LC3 (<xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). These data suggest that the cell-free LC3 lipidation is regulated by starvation-induced components in cells and is dependent on ATG5.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.005</object-id><label>Figure 2.</label><caption><title>The in vitro lipidation of LC3 is regulated by ATG5, starvation and PI3K.</title><p>(<bold>A</bold>) Starvation-promoted and ATG5-dependent lipidation of LC3. Indicated cells were either untreated or starved for 30 min. The in vitro lipidation reaction with the indicated combination of cytosols and membranes was performed. The formation of LC3-II was analyzed by SDS-PAGE and immunoblot. Asterisk, non-specific band (<bold>B</bold>) PI3K inhibitors 3-methyladenine (3-MA) and wortmannin (Wtm) inhibit LC3 lipidation. The in vitro lipidation reaction, with cytosol from starved WT MEFs and membrane from <italic>Atg5</italic> KO MEFs, was performed in the absence or presence of the indicated concentrations of 3-MA and wortmannin for 60 min. LC3 lipidation was analyzed by SDS-PAGE and immunblot. (<bold>C</bold>) PI3P dependence of in vitro LC3 lipidation. The in vitro lipidation reaction similar to (<bold>B</bold>) was performed in the absence or presence of increasing concentrations of GST-FYVE or FYVE (C/S) proteins for the indicated times. SDS-PAGE and immunoblot were performed to analyze the level of LC3-II. Quantification of lipidation activity was shown as the ratio of LC3-II to LC3-I (II/I).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.005">http://dx.doi.org/10.7554/eLife.00947.005</ext-link></p></caption><graphic xlink:href="elife00947f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.006</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Starvation-promoted lipidation of LC3 by COS-7 or HEK293T cytosol.</title><p>COS-7, HEK293T and <italic>Atg5</italic> KO MEF cells were either untreated or starved for 60 min. The in vitro lipidation reaction with indicated combination of cytosols and membranes was performed followed by SDS-PAGE and immunoblot to examine the formation of LC3-II.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.006">http://dx.doi.org/10.7554/eLife.00947.006</ext-link></p></caption><graphic xlink:href="elife00947fs002"/></fig><fig id="fig2s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.007</object-id><label>Figure 2—figure supplement 2.</label><caption><title>Purification and verification of GST-FYVEs.</title><p>(<bold>A</bold>) Purification of GST-fusion PI3P binding FYVE domains and mutants (C/S). (<bold>B</bold>) PIP Strip blot with 10 µg/ml GST-FYVE. (<bold>C</bold>) PIP Strip blot with 10 µg/ml GST-FYVE (C/S).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.007">http://dx.doi.org/10.7554/eLife.00947.007</ext-link></p></caption><graphic xlink:href="elife00947fs003"/></fig></fig-group></p><p>To test the physiological relevance of the cell-free reaction, we examined the effect of inhibitors of autophagy on the lipidation of LC3 in vitro. Starvation-induced autophagosome biogenesis requires the class III PI3K complex which contains ATG14, BECN1, VPS15, and the PI3K subunit VPS34 (<xref ref-type="bibr" rid="bib5">Burman and Ktistakis, 2010</xref>; <xref ref-type="bibr" rid="bib51">Obara and Ohsumi, 2011</xref>). Inhibition of the PI3K activity prevents autophagy. LC3 lipidation was inhibited in a dose-dependent manner by 3-methyladenine (3-MA) and wortmannin, two PI3K inhibitors of different potency but which act in the same concentration ranges to block autophagy in intact cells (<xref ref-type="fig" rid="fig2">Figure 2B</xref> and <xref ref-type="bibr" rid="bib32">Klionsky et al., 2012</xref>).</p><p>In starved cells, downstream effector proteins recognize the PI3P generated by the autophagy-specific VPS34 PI3 kinase. The FYVE domain binds to PI3P (<xref ref-type="bibr" rid="bib66">Stenmark and Aasland, 1999</xref>) and when expressed in excess blocks autophagy in the cell by sequestering PI3P (<xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>). To study the role of PI3P in the in vitro reaction, we isolated a FYVE domain derived from FENS-1 (WDFY1), an endosomal protein (<xref ref-type="bibr" rid="bib59">Ridley et al., 2001</xref>; <xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>), and included the peptide in a lipidation reaction mixture (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2A,B</xref>; <xref ref-type="fig" rid="fig2">Figure 2C</xref>). As reported in intact cells, the FYVE domain peptide inhibited LC3 lipidation in a dose-dependent manner whereas a cysteine to serine (C/S) mutation, which abolishes the ability of FYVE to bind PI3P (<xref ref-type="fig" rid="fig2s2">Figure 2—figure supplement 2A,C</xref> and <xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>), had no effect on lipidation (<xref ref-type="fig" rid="fig2">Figure 2C</xref>).</p><p>One technical limitation is that the lipidation reaction relies on the conversion of endogenous LC3-I to LC3-II. In order to control the level of substrate, we isolated tagged recombinant LC3 expressed in <italic>Escherichia coli</italic>. LC3 is synthesized as a precursor that is processed by ATG4 cleavage to expose a glycine at position 120, the site of PE attachment (<xref ref-type="bibr" rid="bib68">Tanida et al., 2004</xref>). Cell-free lipidation of recombinant T7-LC3 (aa1-120) required the glycine at position 120 and responded in a cytosol- and membrane concentration-dependent manner (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1A,B and C</xref>). Lipidated T7-LC3 sedimented along with membranes at 16,000×<italic>g</italic>, a property shared with the endogenous LC3-II generated in vitro (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1D</xref> and <xref ref-type="fig" rid="fig1s1">Figure1—figure supplement 1A</xref>).</p><p>We next evaluated the physiological requirements for lipidation using the T7-LC3 substrate. Cytosol and membranes isolated from starved cells stimulated T7-LC3 lipidation in vitro (<xref ref-type="fig" rid="fig3">Figure 3A</xref>), just as we observed with endogenous LC3 (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Likewise, PI3K inhibitors, 3-MA and wortmannin, and the PI3P blocking peptide, FYVE, blocked in vitro T7-LC3 lipidation in a dose-dependent manner (<xref ref-type="fig" rid="fig3">Figure 3B,C</xref>). Furthermore, cytosol deficient in ATG7, ATG3 or ATG5, key factors in the ubiquitin-like pathway for LC3 lipidation, failed to generate lipidated T7-LC3 in vitro (<xref ref-type="fig" rid="fig3">Figure 3D,E and F</xref>).<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.008</object-id><label>Figure 3.</label><caption><title>Recapitulation of the major regulatory pathways for autophagy by in vitro lipidation of T7-LC3.</title><p>(<bold>A</bold>) Starvation-induced lipidation of T7-LC3. HEK293T and <italic>Atg5</italic> KO MEF cells were either untreated or starved for 90 min. The in vitro lipidation reaction was performed by incubating T7-LC3 with HEK293T cytosols and <italic>Atg5</italic> KO MEF membranes with indicated treatments for the indicated times followed by SDS-PAGE and immunoblot. (<bold>B</bold>) 3-methyladenine and wortmannin inhibit T7-LC3 lipidation. The in vitro lipidation reaction was performed by incubating T7-LC3 with cytosol from starved HEK293T and <italic>Atg5</italic> KO MEF membranes in the absence or presence of the indicated drugs for 60 min followed by SDS-PAGE and immunoblot. (<bold>C</bold>) PI3P dependence of in vitro T7-LC3 lipidation. In vitro lipidation reactions similar to (<bold>B</bold>) were performed in the absence or presence of the indicated concentrations of GST-FYVEs for 60 min followed by SDS-PAGE and immunoblot. (<bold>D</bold>) Dependence on ATG5 for T7-LC3 lipidation. The in vitro lipidation reaction was performed by incubating T7-LC3 with starved cytosols as indicated and <italic>Atg5</italic> KO MEF membranes for 60 min followed by SDS-PAGE and immunoblot to analyze LC3-II in the membrane fraction. (<bold>E</bold>) Dependence on ATG3 for T7-LC3 lipidation. A similar experiment was performed using cytosols from <italic>Atg3</italic> KO and WT MEFs, and membrane from Atg3 KO MEFs. (<bold>F</bold>) Dependence on ATG7 for T7-LC3 lipidation. A similar experiment was performed using cytosols from <italic>Atg7</italic> KO and WT MEFs, and membrane from <italic>Atg7</italic> KO MEFs. (<bold>G</bold>) Dependence on ULK1 for starvation-induced T7-LC3 lipidation. The in vitro lipidation reaction was performed by incubating T7-LC3 with untreated or starved cytosols as indicated and <italic>Ulk1</italic> KO MEF membranes for 60 min followed by SDS-PAGE and immunoblot of the membrane fraction. (<bold>H</bold>) Rapamycin-induced lipidation of T7-LC3. Cells were treated with 1 µM rapamycin or a control solution for 2 hr and cytosol was incubated with membranes as in (<bold>A</bold>). (<bold>I</bold>) Torin 1-induced lipidation of T7-LC3. Cells were treated with 200 nM Torin 1 or a control solution for 90 min and incubated with membranes as above. Quantification of lipidation activity was shown as the ratio of LC3-II to LC3-I (II/I).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.008">http://dx.doi.org/10.7554/eLife.00947.008</ext-link></p></caption><graphic xlink:href="elife00947f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.009</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Purification of the T7-tagged LC3 and characterization of the lipidation.</title><p>(<bold>A</bold>) Purification of HisT7-LC3 and the G/A mutant. (<bold>B</bold>) Lipidation of HisT7-LC3. The in vitro lipidation reaction was performed by incubating HisT7-LC3 or G/A mutant with indicated cytosols and <italic>Atg5</italic> KO MEF membranes for 60 min followed by SDS-PAGE and immunoblot. (<bold>C</bold>) Cytosol and membrane dependence of HisT7-LC3 lipidation. The in vitro lipidation reaction was performed by incubating HisT7-LC3 with increasing concentrations of cytosol or membrane for 60 min followed by SDS-PAGE and immunoblot. (<bold>D</bold>) T7-LC3-II distributes in the 16,000×<italic>g</italic> membrane pellet fraction. T7-LC3 protein was generated by thrombin digestion of the HisT7-LC3 protein to remove the N-terminal His tag. The in vitro lipidation reaction was performed by incubating T7-LC3 with cytosol from starved HEK293T cells and <italic>Atg5</italic> KO MEF membranes for 60 min. A centrifugation procedure similar to the experiment in <xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref> was employed and the reaction products were evaluated by SDS-PAGE and immunoblot.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.009">http://dx.doi.org/10.7554/eLife.00947.009</ext-link></p></caption><graphic xlink:href="elife00947fs004"/></fig></fig-group></p><p>Starvation induces autophagy through an MTORC1-ULKI protein kinase regulatory scheme (<xref ref-type="bibr" rid="bib31">Kim et al., 2011</xref>). We found that cytosol from starved or untreated <italic>Ulk1</italic> KO MEFs reduced lipidation two to threefold relative to cytosol from WT cells (<xref ref-type="fig" rid="fig3">Figure 3G</xref>). Furthermore, T7-LC3 lipidation was stimulated two to threefold by two MTOR inhibitors, rapamycin (<xref ref-type="bibr" rid="bib24">Heitman et al., 1991</xref>) and Torin 1 (<xref ref-type="bibr" rid="bib37">Liu et al., 2010</xref>), known to induce autophagy (<xref ref-type="fig" rid="fig3">Figure 3H,I</xref>). Thus, for endogenous and recombinant LC3, the cell-free reaction reflects and responds to the major regulatory pathways of autophagy.</p></sec><sec id="s2-2"><title>Identification of the ERGIC as the membrane determinant that triggers in vitro LC3 lipidation</title><p>We employed the cell-free reaction as an assay to isolate the membrane responsible for LC3 lipidation. For this purpose, we devised a three-step membrane fractionation procedure and monitored enrichment of the lipidation activity with respect to a variety of membrane marker proteins and the lipid donor PE, in relation to a bulk membrane marker, phosphatidylcholine (PC) (<xref ref-type="fig" rid="fig4">Figure 4</xref>).<fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.010</object-id><label>Figure 4.</label><caption><title>Membrane fractionation scheme.</title><p>Briefly, <italic>Atg5</italic> KO MEFs were homogenized and the lysates were subjected to differential centrifugations with indicated g forces. The ability of each fraction to trigger T7-LC3 lipidation was examined. The 25,000×<italic>g</italic> (25k) pellet, which had the most activity, was selected and a sucrose gradient ultracentrifugation was performed to separate the 25k pellet to L (light) and P (pellet) fractions. The L fraction, which contained the majority of the activity to promote T7-LC3 lipidation, was further resolved on an OptiPrep gradient after which ten fractions from the top were collected and the lipidation activity was examined in each.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.010">http://dx.doi.org/10.7554/eLife.00947.010</ext-link></p></caption><graphic xlink:href="elife00947f004"/></fig></p><p>In order to separate cellular membranes, we first performed differential centrifugation to obtain four membrane pellets containing different membrane markers (<xref ref-type="fig" rid="fig4 fig5">Figures 4 and 5</xref>). The PC level of each fraction was measured and normalized so that the lipidation activity for equal amounts of PC (specific activity) could be determined (<xref ref-type="fig" rid="fig5">Figure 5A,B and C</xref>). The 25k and 100k fractions had significant lipidation activity (<xref ref-type="fig" rid="fig5">Figure 5A,B</xref>). The total activity contained in each fraction was calculated by multiplying the specific activity by the PC level (<xref ref-type="fig" rid="fig5">Figure 5D</xref>). The 25k pellet contained most (>70%) of the total activity, whereas the 100k pellet had little (<10%) total activity due to low levels of membrane. The 25k membrane was enriched in peroxisomes (PMP70/ABCD3), late endosomes (LAMP2) and cis-Golgi (GM130/GOLGA2). This fraction also contained membranes from the ERGIC (SEC22B and ERGIC53/LMAN1), plasma membrane/early endosomes (PM/Endo, TFR), ER (RPN1), ER exit sites (ERES, active sites on the ER that generate COPII-coated vesicles, SEC12), lysosomes (Cathepsin D), and ATG9 vesicles. Low levels of a mitochondrial marker (Prohibitin 1) and almost no trans-Golgi (TGN38/TGOLN2) or nuclear (Histone H4) compartments (<xref ref-type="fig" rid="fig5">Figure 5A</xref>) were detected.<fig id="fig5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.011</object-id><label>Figure 5.</label><caption><title>Separation of the total membrane by differential centrifugations.</title><p>(<bold>A–D</bold>) A differential centrifugation experiment was performed as depicted in <xref ref-type="fig" rid="fig4">Figure 4</xref>. The total PC of each fraction was measured and presented as a percentage of the total membrane (<bold>C</bold>) and adjusted to a concentration of 0.6 mg/ml. The T7-LC3 lipidation activity of each fraction was tested and immunoblot was performed to examine the generation of lipidated T7-LC3 as well as the distribution of the indicated membrane markers (<bold>A</bold>). The level of each marker in the 25k pellet fraction was calculated as a percentage of the total membrane (<bold>A</bold>). The specific activity (the ability of each membrane fraction to induce LC3 lipidation with the equal amount of PC) of each membrane fraction to trigger T7-LC3 lipidation was measured as a ratio of lipidated to unlipidated T7-LC3s (<bold>B</bold>). The total activity recovered from each fraction was calculated by multiplying the specific activity by the corresponding PC level of each fraction and shown as a percentage of the total membrane (<bold>D</bold>). Error bars represent standard deviations of at least three experiments. RPN1, Ribophorin1; TFR, Transferrin receptor; Mem, membrane; Endo, endosome.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.011">http://dx.doi.org/10.7554/eLife.00947.011</ext-link></p></caption><graphic xlink:href="elife00947f005"/></fig></p><p>To further fractionate the 25k membrane, we performed sucrose step gradient ultracentrifugation (<xref ref-type="fig" rid="fig4">Figure 4</xref>). This separated the 25k membrane to two distinct fractions, a light (L) fraction between the 1.1 M and 0.25 M layers of sucrose, and a pellet (P) fraction that sedimented to the bottom (<xref ref-type="fig" rid="fig4">Figure 4</xref>). The specific and total activity was determined as described above (<xref ref-type="fig" rid="fig6">Figure 6</xref>). Interestingly, the lipidation activity was almost exclusively retained in the L fraction, which was enriched in ERGIC, cis-Golgi, ATG9 vesicles and plasma membrane/early endosomes (<xref ref-type="fig" rid="fig6">Figure 6A,B and D</xref>). In contrast, the P fraction, which mainly consisted of ER, ERES, mitochondria, lysosomes and peroxisomes, induced very little T7-LC3 lipidation (<xref ref-type="fig" rid="fig6">Figure 6A,B and D</xref>).<fig id="fig6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.012</object-id><label>Figure 6.</label><caption><title>Separation of the 25k pellet fraction by sucrose gradient ultracentrifugation.</title><p>(<bold>A–D</bold>) A sucrose step gradient ultracentrifugation to further separate the 25k pellet fraction was performed as depicted in <xref ref-type="fig" rid="fig4">Figure 4</xref>. The total PCs of each fraction were measured and presented as a percentage of the 25k pellet membrane (<bold>C</bold>) and adjusted to a concentration of 0.6 mg/ml. The T7-LC3 lipidation activities of the L and P fraction were tested and immunoblot was performed as in <xref ref-type="fig" rid="fig5">Figure 5A</xref>. The level of each marker in the L fraction was calculated as a percentage of the total membrane (<bold>A</bold>). The specific activity of each membrane fraction was measured as in <xref ref-type="fig" rid="fig5">Figure 5B</xref>. The total activity recovered from each fraction was calculated by multiplying the specific activity by the PC level of each fraction and shown as the percentage of 25k pellet membrane (<bold>D</bold>). Error bars represent standard deviations of at least three experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.012">http://dx.doi.org/10.7554/eLife.00947.012</ext-link></p></caption><graphic xlink:href="elife00947f006"/></fig></p><p>To further refine the membrane source of T7-LC3 lipidation activity, we centrifuged the L fraction on an OptiPrep gradient and collected ten fractions from the top (<xref ref-type="fig" rid="fig4">Figure 4</xref>). The lipidation activity was distributed in fractions two through four which co-distributed with SEC22B and ERGIC53 (<xref ref-type="fig" rid="fig7">Figure 7</xref>), two ERGIC markers (<xref ref-type="bibr" rid="bib80">Zhang et al., 1999</xref>; <xref ref-type="bibr" rid="bib1">Appenzeller-Herzog and Hauri, 2006</xref>). Intriguingly, PE, the substrate for LC3 lipidation, was not enriched in the fractions that triggered T7-LC3 lipidation (<xref ref-type="fig" rid="fig7">Figure 7B</xref>). The high activity of these membrane fractions was not caused by selective enrichment of the autophagic factors directly contributing to LC3 lipidation, as all of these factors are enriched in the cytosol, or by influencing the formation of the ATG5–12–16 complex essential for LC3 lipidation (<xref ref-type="bibr" rid="bib11">Fujita et al., 2008</xref>) compared with other membrane fractions (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1</xref>). These data indicate that factors other than those directly involved in catalyzing LC3 lipidation contribute to the high lipidation activity of these membranes. We further found that the lipidation reaction triggered by the ERGIC-enriched membranes was enhanced by cytosol from starved cells and was inhibited by wortmannin and FYVE peptide (<xref ref-type="fig" rid="fig7s2">Figure 7—figure supplement 2</xref>). These data suggest that the lipidation activity of the isolated membranes is controlled by the pathway(s) that regulate autophagy in vivo.<fig-group><fig id="fig7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.013</object-id><label>Figure 7.</label><caption><title>Separation of the L fraction by OptiPrep gradient ultracentrifugation.</title><p>(<bold>A–B</bold>) An OptiPrep gradient ultracentrifugation was used to resolve membranes in the L fraction, as depicted in <xref ref-type="fig" rid="fig4">Figure 4</xref>. 10 fractions were collected. The total PCs of each fraction were measured and adjusted to a concentration of 0.6 mg/ml. The T7-LC3 lipidation activities of each fraction were tested and immunoblot was performed as in <xref ref-type="fig" rid="fig5">Figure 5A</xref>. The specific activity of each membrane fraction was measured similar to <xref ref-type="fig" rid="fig5">Figure 5</xref>. The PE level of each normalized fraction was determined. A heat map showing the relative levels of the specific activity, PE and each of the indicated markers was generated (<bold>B</bold>). In each group the fraction with the highest value was defined as 1.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.013">http://dx.doi.org/10.7554/eLife.00947.013</ext-link></p></caption><graphic xlink:href="elife00947f007"/></fig><fig id="fig7s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.014</object-id><label>Figure 7—figure supplement 1.</label><caption><title>The ERGIC membrane promotes LC3 lipidation without altering cytosolic factors.</title><p>(<bold>A</bold>) The major autophagy factors are cytosolic. Immunoblot of the fractions from <xref ref-type="fig" rid="fig7">Figure 7</xref> and the cytosol (Cyt) used for the in vitro lipidation assay was performed with indicated antibodies. (<bold>B</bold>) The formation of ATG5–12–16 complex is not altered by ERGIC. Indicated membrane fractions obtained from the membrane fractionation were incubated with cytosol for the LC3 lipidation assay. Membranes were removed by centrifugation and the supernatant fractions were collected for size exclusion chromatography on a Superpose 6 column followed immunoblot analysis with the indicated antibodies. Most of the ATG5 is conjugated with ATG12 in wild-type cells (data not shown). ERGIC, the combination of fractions 2–4 in the OptiPrep gradient fractionation; P, the pellet fraction from the sucrose gradient centrifugation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.014">http://dx.doi.org/10.7554/eLife.00947.014</ext-link></p></caption><graphic xlink:href="elife00947fs005"/></fig><fig id="fig7s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.015</object-id><label>Figure 7—figure supplement 2.</label><caption><title>The lipidation activity of the ERGIC-enriched fractions are regulated by starvation and PI3K.</title><p>(<bold>A</bold>) Lipidation of T7-LC3 from the ERGIC-enriched fractions is enhanced by starvation. An in vitro lipidation reaction similar to that in <xref ref-type="fig" rid="fig7">Figure 7</xref> was performed with HEK293T cytosols from starved (ST) and non-starved (NT) cells followed by SDS-PAGE and immunoblot. (<bold>B</bold>) Wortmannin inhibits T7-LC3 lipidation triggered by the ERGIC-enriched fraction. Fractions 2–4 from Opti-prep gradient were collected and pooled. The in vitro lipidation reaction was performed with increasing concentrations of wortmannin (Wtm) followed by SDS-PAGE and immunoblot. (<bold>C</bold>) PI3P dependence of T7-LC3 lipidation triggered by the ERGIC-enriched fraction. The ERGIC-enriched membranes were pooled as shown in (<bold>B</bold>). The in vitro lipidation reaction was performed with indicated concentrations of GST-FYVE and FYVE(C/S) followed by SDS-PAGE and immunoblot.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.015">http://dx.doi.org/10.7554/eLife.00947.015</ext-link></p></caption><graphic xlink:href="elife00947fs006"/></fig></fig-group></p><p>The data from membrane fractionation indicate that an ERGIC-enriched membrane fraction induces LC3 lipidation. To determine if ERGIC is indeed the essential membrane for LC3 lipidation, we immunodepleted ERGIC from the L fraction with an anti-SEC22B antibody (<xref ref-type="fig" rid="fig8">Figure 8A</xref>). After immunodepletion, both SEC22B and ERGIC53 membranes were reduced, whereas the plasma membrane/endosome membranes, indicated by transferrin receptor, were not affected. Significantly, the ability of the L fraction to induce T7-LC3 lipidation was reduced more than threefold (<xref ref-type="fig" rid="fig8">Figure 8A</xref>), suggesting that ERGIC contributes to the high activity of LC3 lipidation in the L fraction.<fig-group><fig id="fig8" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.016</object-id><label>Figure 8.</label><caption><title>ERGIC directly triggers in vitro LC3 lipidation.</title><p>(<bold>A</bold>) Immunodepletion of ERGIC membrane from L fraction reduces in vitro lipidation activity. The L fraction was prepared as shown in <xref ref-type="fig" rid="fig4 fig5">Figures 4 and 5</xref>. An immunodepletion experiment with indicated combinations of anti-SEC22B antibody (Ab) and blocking peptide (pep) was performed. The membranes from the flow-through were collected and the in vitro lipidation reaction was performed. Equal amounts of membrane from each group were used for the lipidation reaction. T, total membrane from the L fraction. (<bold>B</bold>) Enrichment of lipidation activity on the SEC22B-positive membranes. Immunoisolation of SEC22B positive membranes from the L fraction of <italic>Atg5</italic> KO MEFs was performed and the in vitro lipidation reaction was conducted with membranes bound to the beads as well as the total membrane (T) from the L fraction. The total membrane used was adjusted to the same amount of the membranes (based on PC content) specifically bound to the beads in the reaction. (<bold>C</bold>) Enrichment of lipidation activity on KDEL Receptor (KDELR)-positive membranes. <italic>Atg5</italic> KO MEFs were transfected with a plasmid encoding the Flag-tagged KDELR protein. 48 hr after transfection, KDELR-positive membranes were immunoisolated with anti-Flag agarose and assayed for lipidation activity as in (<bold>B</bold>). (<bold>D</bold>–<bold>F</bold>) Lipidation activity was not enriched in late endosome/lysosome, plasma membrane/endosome or ER membranes. <italic>Atg5</italic> KO MEFs were transfected with plasmids encoding LAMP1-Flag (<bold>D</bold>), Vangl2-Myc (<bold>E</bold>) or Flag-GFP-ER-TM (<bold>F</bold>). 48 hr after transfection, the 25k pellet fractions (for LAMP1-Flag and Flag-GFP-ER-TM) or the L fraction were collected and immunoisolations were performed with anti-Flag agarose or anti-Myc agarose as described above and assayed for lipidation activity. Quantification of lipidation activity was shown as the ratio of LC3-II to LC3-I (II/I).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.016">http://dx.doi.org/10.7554/eLife.00947.016</ext-link></p></caption><graphic xlink:href="elife00947f008"/></fig><fig id="fig8s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.017</object-id><label>Figure 8—figure supplement 1.</label><caption><title>Mitochondrial-associated endoplasmic reticulum membranes (MAM) are not active to trigger in vitro LC3 lipidation.</title><p>(<bold>A</bold>) Indicated membrane fractions were prepared as described by Wieckowski et al. (<xref ref-type="bibr" rid="bib74">Wieckowski et al., 2009</xref>) and in vitro lipidation was performed as shown in <xref ref-type="fig" rid="fig4 fig5 fig6 fig7">Figures 4–7</xref>. T, total membrane; S, supernatant membrane separated from crude mitochondria preparation; CM, crude mitochondria fraction; Mito, mitochondria fraction; MAM, Mitochondrial-associated endoplasmic reticulum membranes, FACL4, Long-chain acyl-CoA synthetase 4. (<bold>B</bold>) Immunoblot analysis of the MAM fractions purified from non-treated (NT) and starved (ST) cells. Asterisk, non-specific band.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.017">http://dx.doi.org/10.7554/eLife.00947.017</ext-link></p></caption><graphic xlink:href="elife00947fs007"/></fig></fig-group></p><p>To test the direct role of ERGIC on LC3 lipidation, we immunoisolated SEC22B or KDEL receptor (KDELR, another ERGIC marker [<xref ref-type="bibr" rid="bib6">Capitani and Sallese, 2009</xref>])-positive membranes (<xref ref-type="fig" rid="fig8">Figure 8B,C</xref>) for use in the in vitro lipidation assay. In both experiments, the immunoisolated membranes had substantially increased activity (about twofold or more than fivefold in the SEC22B or KDELR immunoisolated membranes, respectively) compared to the total input membrane (<xref ref-type="fig" rid="fig8">Figure 8B,C</xref>). In contrast, immunoisolation of lysosomes, plasma membrane/endosomes or ER did not enrich the lipidation activity compared to the total input membrane (<xref ref-type="fig" rid="fig8">Figure 8D,E and F</xref>). Therefore the data suggest that ERGIC is the most active membrane substrate for LC3 lipidation.</p><p>A recent report suggested that starvation induces the recruitment of ATG14 to a zone of adhesion between the ER and mitochondria, termed mitochondrial-associated endoplasmic reticulum membranes (MAM; <xref ref-type="bibr" rid="bib20">Hamasaki et al., 2013</xref>). Hamasaki et al. suggested that this zone of adhesion might trigger the formation of the autophagosome (<xref ref-type="bibr" rid="bib20">Hamasaki et al., 2013</xref>). Such a specialized patch of the ER could be an immediate precursor of the autophagosome. Markers of this adhesion were separated from the ERGIC fraction and the adhesion membrane isolated by the protocol of Wieckowski et al. (<xref ref-type="bibr" rid="bib74">Wieckowski et al., 2009</xref>) contained little lipidation activity (<xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1</xref>), suggesting this membrane alone may not be the direct template for LC3 lipidation. Consistent with Hamasaki et al., a fraction of ATG14 appeared on MAM in a starvation-stimulated manner (<xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1B</xref>).</p></sec><sec id="s2-3"><title>ERGIC is a key membrane determinant for LC3 lipidation</title><p>To further test the importance of ERGIC in LC3 lipidation, we used inhibitors to deplete ERGIC in cultured cells. H89 is a protein kinase A (PKA) inhibitor that blocks COPII-coated vesicle assembly by preventing SAR1 binding to ER membrane when used at a high concentration (<xref ref-type="bibr" rid="bib8">Chijiwa et al., 1990</xref>; <xref ref-type="bibr" rid="bib2">Aridor and Balch, 2000</xref>). Brefeldin A (BFA) is a fungal metabolite that inhibits ARF1 activation (<xref ref-type="bibr" rid="bib55">Peyroche et al., 1999</xref>). Treatment of cells with a high concentration of H89 (100 µM) led to the dispersal of ERGIC53 whereas GM130, a cis-Golgi marker, remained in the perinuclear region, suggesting that the ERGIC is disrupted but not the cis-Golgi (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). A low concentration of H89 (10 µM), which is enough to inhibit PKA, did not affect the localization of either ERGIC53 or GM130 (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). BFA treatment collapsed the Golgi into puncta colocalizing with ERGIC53 (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). Treatment of cells with 100 µM H89 after BFA treatment dispersed both ERGIC53 and GM130 (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>).</p><p>To assess the membrane fractions for retention of lipidation activity, we treated cells with the indicated drugs and the total membrane from each sample was collected and incubated with cytosol from starved cells (<xref ref-type="fig" rid="fig9">Figure 9</xref>). Membrane from cells treated with a high but not a low concentration of H89 (100 µM vs 10 µM) lost the ability to activate LC3 lipidation (<xref ref-type="fig" rid="fig9">Figure 9A</xref>). Membranes from BFA-treated cells did not show diminished lipidation activity, nor did BFA mitigate or enhance the effect of H89 treatment on lipidation activity (<xref ref-type="fig" rid="fig9">Figure 9A</xref>). Clofibrate is a peroxisome-proliferator activated receptor (PPAR) agonist that inhibits ER-to-Golgi transport and promotes retrograde transport of Golgi vesicles back to the ER through an unknown mechanism independent of PPAR activation (<xref ref-type="bibr" rid="bib9">de Figueiredo and Brown, 1999</xref>). Clofibrate treatment led to the dispersal of ERGIC53 and GM130 (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). Like H89, membranes from clofribate-treated cells failed to promote LC3 lipidation (<xref ref-type="fig" rid="fig9">Figure 9B</xref>). As controls, kbNB142-70, a PKD inhibitor (<xref ref-type="bibr" rid="bib4">Bravo-Altamirano et al., 2011</xref>), had no affect and Pitstop 2, a clathrin inhibitor (<xref ref-type="bibr" rid="bib70">von Kleist et al., 2011</xref>), only moderately decreased in vitro LC3 lipidation (<xref ref-type="fig" rid="fig9">Figure 9B</xref>).<fig-group><fig id="fig9" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.018</object-id><label>Figure 9.</label><caption><title>ERGIC is required for in vitro LC3 lipidation.</title><p>(<bold>A</bold> and <bold>B</bold>) In vivo depletion of ERGIC abolishes the in vitro lipidation of LC3. <italic>Atg5</italic> KO MEFs were treated without or with 10 µg/ml Brefeldin A (BFA) for 30 min and then incubated with the indicated concentrations of H89 for 10 min (<bold>A</bold>). Alternatively, cells were directly treated with the indicated drugs: 100 µM H89, 500 µM clofibrate, 50 µM kbNB142-70 and 50 µM Pitstop2 (<bold>B</bold>). Total membranes from the cells were collected, the lipidation reaction with T7-LC3 was performed and the products evaluated by SDS-PAGE and immunoblot. Ctr, control (<bold>C</bold>) Blocking ERGIC disruption preserved the in vitro lipidation of LC3. <italic>Atg5</italic> KO MEFs were treated with control, H89 or clofibrate (Clofi) in the absence or presence of nocodazole (Noco). Lipidation reactions with the total membranes from the treated cells were performed. (<bold>D</bold>–<bold>F</bold>) The in vitro lipidation of LC3 recovers with restoration of ERGIC. <italic>Atg5</italic> KO MEF cells were treated with BFA followed by 100 µM H89. Cells were then washed with fresh medium to remove the drugs and, at indicated intervals, samples were collected for immunofluorescence (<bold>D</bold>) or total membrane collection for the in vitro lipidation reaction (<bold>E</bold>). Quantification of the recovery of lipidation activity, ERGIC and Golgi are shown in (<bold>F</bold>). Bar, 10 µm. Quantification of lipidation activity was shown as the ratio of LC3-II to LC3-I (II/I).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.018">http://dx.doi.org/10.7554/eLife.00947.018</ext-link></p></caption><graphic xlink:href="elife00947f009"/></fig><fig id="fig9s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.019</object-id><label>Figure 9—figure supplement 1.</label><caption><title>Immunofluorescence showing the effect of indicated drugs on ERGIC and Golgi.</title><p><italic>Atg5</italic> KO MEFs were treated with indicated drugs or drug combinations as depicted in <xref ref-type="fig" rid="fig9">Figure 9A,B</xref>. Cells were fixed for immunofluorescence with the indicated antibodies. Bar, 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.019">http://dx.doi.org/10.7554/eLife.00947.019</ext-link></p></caption><graphic xlink:href="elife00947fs008"/></fig></fig-group></p><p>Retrograde transport to the ER requires microtubules. Disruption of microtubules by nocodazole inhibited the retrograde transport of ERGIC53 towards ER induced by H89 or clofibrate, preserving its punctate localization (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). Correspondingly, the inhibitory effects of H89 and clofibrate were reversed in respect to the cell-free lipidation activity (<xref ref-type="fig" rid="fig9">Figure 9C</xref>). To examine the reversibility of these effects, we treated cells with BFA and H89 for 20 min and then washed the cells into fresh medium for periods up to 40 min (<xref ref-type="bibr" rid="bib56">Puri and Linstedt, 2003</xref>). ERGIC53 puncta began to reappear within 2 min and the normal localization was fully restored by 20 min. Cis-Golgi regeneration, indicated by the perinuclear accumulation of GM130, was slower and was completed by about 30 min (<xref ref-type="fig" rid="fig9">Figure 9D,F</xref>). Aliquots examined in the cell-free LC3 lipidation assay displayed a rapid return of activity correlating with the kinetics of ERGIC recovery (<xref ref-type="fig" rid="fig9">Figure 9E,F</xref>). These results support our membrane fractionation results concerning the role of the ERGIC in lipidation of LC3.</p></sec><sec id="s2-4"><title>ERGIC is required for autophagosome biogenesis</title><p>To test the role of ERGIC in autophagosome formation, we starved cells in the presence or absence of ERGIC-depleting drugs H89 and clofibrate (<xref ref-type="fig" rid="fig10">Figure 10A</xref>). We then monitored autophagosome biogenesis by immunofluorescence analysis of LC3 puncta formation (<xref ref-type="bibr" rid="bib32">Klionsky et al., 2012</xref>). Under normal conditions, LC3 is dispersed in the cell, indicating a low level of autophagy (<xref ref-type="fig" rid="fig10">Figure 10A</xref>). Starvation-induced LC3 puncta formation was abolished by H89 (100 µM) and clofibrate (<xref ref-type="fig" rid="fig10">Figure 10A,B</xref>). Depressed formation of LC3 puncta was not due to enhanced autophagosome turnover because chloroquine, which blocks a late step in autophagy (<xref ref-type="bibr" rid="bib32">Klionsky et al., 2012</xref>), did not mitigate the effect of H89 (100 µM) or clofibrate (<xref ref-type="fig" rid="fig10">Figure 10A,B</xref>). Puncta formation of another phagophore marker, ATG16 (<xref ref-type="bibr" rid="bib11">Fujita et al., 2008</xref>), was also blocked by ERGIC depletion (<xref ref-type="fig" rid="fig10s1">Figure 10—figure supplement 1</xref>).<fig-group><fig id="fig10" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.020</object-id><label>Figure 10.</label><caption><title>ERGIC is required for starvation-induced LC3 puncta formation.</title><p>(<bold>A</bold>) Drugs that disrupt ERGIC inhibit LC3 puncta formation. MEFs were transfected with plasmids encoding Myc-LC3. After transfection (24 hr), the cells were either non-starved (NT) or starved (ST) in the absence or presence of the indicated drugs followed by immunofluorescence using anti-Myc antibody. Bar, 10 µm. (<bold>B</bold>) Quantification of the cells shown in (<bold>A</bold>). Error bars represent standard deviations of three experiments. (<bold>C</bold>) Genetically disrupting ERGIC inhibits LC3 puncta formation. MEF cells were co-transfected with plasmids encoding Myc-LC3 and the indicated SAR1A variants. After transfection (24 hr), the cells were starved in the absence or presence of chloroquine followed by immunofluorescence using anti-Myc antibody. Bar, 10 µm. (<bold>D</bold>) Quantification of the cells shown in (<bold>C</bold>). Error bars represent standard deviations of three experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.020">http://dx.doi.org/10.7554/eLife.00947.020</ext-link></p></caption><graphic xlink:href="elife00947f010"/></fig><fig id="fig10s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.021</object-id><label>Figure 10—figure supplement 1.</label><caption><title>Drugs that disrupt ERGIC inhibit starvation-induced ATG16 puncta formation.</title><p>(<bold>A</bold>) MEFs were transfected with plasmids encoding ATG16-Myc. After transfection (24 hr), the cells were either non-starved (NT) or starved (ST) in the absence or presence of the indicated drugs followed by immunofluorescence using anti-Myc antibody. Bar, 10 µm (<bold>B</bold>) Quantification of the cells shown in (<bold>A</bold>). Error bars represent standard deviations of three experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.021">http://dx.doi.org/10.7554/eLife.00947.021</ext-link></p></caption><graphic xlink:href="elife00947fs009"/></fig><fig id="fig10s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.022</object-id><label>Figure 10—figure supplement 2.</label><caption><title>Effects of SAR1A variants on ERGIC.</title><p>MEFs were transfected with plasmids encoding the indicated SAR1A-DsRed variants or control DsRed. After transfection (24 hr), immunofluorescence with indicated antibodies was performed. Bar, 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.022">http://dx.doi.org/10.7554/eLife.00947.022</ext-link></p></caption><graphic xlink:href="elife00947fs010"/></fig></fig-group></p><p>In addition to inhibiting vesicle traffic from the ER, H89 is a potent inhibitor of PKA. In order to determine whether the observed decrease in the number of LC3 puncta could be due to PKA inhibition and not loss of ERGIC, we tested a low concentration of H89 that inhibits PKA but has no effect on the ERGIC (<xref ref-type="fig" rid="fig9s1">Figure 9—figure supplement 1</xref>). In contrast to the effect of a high concentration of H89 (100 µM), cells treated with the low concentration (10 µM) showed a moderate increase in LC3 puncta with or without chloroquine (<xref ref-type="fig" rid="fig10">Figure 10A,B</xref>). A recent study also reported that PKA inhibition promotes autophagy (<xref ref-type="bibr" rid="bib7">Cherra et al., 2010</xref>), thus PKA inhibition appears not to be the basis of the effect of H89 on autophagy.</p><p>As an additional test of the role of ERGIC in autophagosome formation, we introduced mutant forms of the SAR1A GTPase to inhibit the generation of COPII vesicles. A GTP-bound mutant of SAR1A (H79G) locks COPII membrane cargos on the ERES and a GDP-bound mutant, SAR1A T34N, completely blocks COPII coat formation (<xref ref-type="bibr" rid="bib71">Ward et al., 2001</xref>). Overexpression of either SAR1A H79G or T34N led to dispersed ERGIC53 localization (<xref ref-type="fig" rid="fig10s2">Figure 10—figure supplement 2</xref>) and inhibited starvation-induced LC3 puncta formation in both control and chloroquine-treated cells (<xref ref-type="fig" rid="fig10">Figure 10C,D</xref>). We conclude that the ERGIC is a precursor of or contributes to the formation of the autophagosome.</p></sec><sec id="s2-5"><title>ERGIC recruits ATG14 and DFCP1, two early markers of autophagosome formation</title><p>ATG14 is the key mediator bridging upstream cytosolic signals and the autophagic membrane reorganization response. Upon starvation, ATG14 is recruited to a membrane along with the rest of the class III PI3K complex to generate PI3P (<xref ref-type="bibr" rid="bib42">Matsunaga et al., 2009</xref>; <xref ref-type="bibr" rid="bib67">Sun et al., 2008</xref>; <xref ref-type="bibr" rid="bib81">Zhong et al., 2009</xref>; <xref ref-type="bibr" rid="bib41">Matsunaga et al., 2010</xref>). These events can be visualized by the localization of ATG14 and DFCP1 to puncta in starved cells (<xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>; <xref ref-type="bibr" rid="bib41">Matsunaga et al., 2010</xref>). To test the role of ERGIC in this pathway, we treated starved cells with H89. As shown in <xref ref-type="fig" rid="fig11">Figure 11</xref>, 100 µM H89 but not 10 µM H89 prevented the formation of both ATG14 and DFCP1 puncta (<xref ref-type="fig" rid="fig11">Figure 11A,B</xref>). Similar inhibition was also observed in starved cells expressing the two SAR1A mutants, H79G and T34N (<xref ref-type="fig" rid="fig11">Figure 11C,D</xref>).<fig id="fig11" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.023</object-id><label>Figure 11.</label><caption><title>ERGIC is required for the starvation-induced localization of ATG14 and DFCP1 to puncta.</title><p>(<bold>A</bold>) H89 inhibits ATG14 and DFCP1 puncta formation. MEF cells were transfected with plasmids encoding EGFP-tagged ATG14 or DFCP1. After transfection (24 hr), cells were starved in the absence or presence of the indicated concentrations of H89 followed by fixation and direct visualization of the EGFP signal. Bar, 10 µm. (<bold>B</bold>) Quantification of the cells shown in (<bold>A</bold>). Error bars represent standard deviations of three experiments. (<bold>C</bold>) Expression of SAR1A mutants inhibits the formation of puncta that contain ATG14 and DFCP1. MEF cells were co-transfected with plasmids encoding EGFP-tagged ATG14 or DFCP1 and indicated SAR1A-DsRed variants. After transfection (24 hr), cells were starved followed by fixation and direct visualization of EGFP signal. Bar, 10 µm. (<bold>D</bold>) Quantification of the cells shown in (<bold>C</bold>). Error bars represent standard deviations of three experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.023">http://dx.doi.org/10.7554/eLife.00947.023</ext-link></p></caption><graphic xlink:href="elife00947f011"/></fig></p><p>We developed a cell-free approach to measure the recruitment of ATG14 and DFCP1 to ERGIC membranes. <italic>Atg5</italic> KO MEFs were treated with or without H89 (100 µM). Cells were lysed and membranes from a post-nuclear supernatant fraction were mixed with cytosol from starved HEK293 cells transfected with tagged forms of ATG14 and DFCP1. The mixes were incubated in the presence of ATP and a regeneration system. Membranes were resolved on an OptiPrep buoyant density step gradient. We observed recruitment of tagged ATG14 and DFCP1 to a buoyant membrane fraction from untreated but much less from H89-treated cells (<xref ref-type="fig" rid="fig12">Figure 12</xref>). Recruitment was dependent on membranes, and stimulated by starvation (<xref ref-type="fig" rid="fig12s1">Figure 12—figure supplement 1</xref>). In starved cells, ATG14 acts upstream of PI3K activity whereas DFCP1 puncta formation requires PI3P generation (<xref ref-type="bibr" rid="bib27">Itakura and Mizushima, 2010</xref>). Correspondingly, membranes from cells treated with PI3K inhibitors recruited ATG14 but not DFCP1 (<xref ref-type="fig" rid="fig12s1">Figure 12—figure supplement 1B and C</xref>). In addition to the membrane recruitment result, immunofluorescence studies showed colocalization of ATG14 and DFCP1 with ERGIC53 after starvation (<xref ref-type="fig" rid="fig12s2">Figure 12—figure supplement 2</xref>). We conclude that the ERGIC membrane is an early site for the assembly of proteins responsible for the formation of the phagophore membrane.<fig-group><fig id="fig12" position="float"><object-id pub-id-type="doi">10.7554/eLife.00947.024</object-id><label>Figure 12.</label><caption><title>ERGIC is required for membrane recruitment of ATG14 and DFCP1.</title><p>(<bold>A</bold>) Disruption of ERGIC inhibits membrane recruitment of ATG14 and DFCP1. <italic>Atg5</italic> KO MEFs were either untreated or treated with H89. Membranes were collected and incubated with cytosol of HEK293T cells expressing ATG14-HA and EGFP-DFCP1. A buoyant density flotation assay was performed followed by immunoblot. (<bold>B</bold>) Quantification of the floated markers shown in (<bold>A</bold>). The quantification of samples from the buoyant density gradient was calculated as the ratio of chemiluminescence in the first two fractions to the sum of all fractions. Error bars represent standard deviations of three experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.024">http://dx.doi.org/10.7554/eLife.00947.024</ext-link></p></caption><graphic xlink:href="elife00947f012"/></fig><fig id="fig12s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.025</object-id><label>Figure 12—figure supplement 1.</label><caption><title>Establishment of the in vitro membrane recruitment assay.</title><p>(<bold>A</bold>) Membrane dependence for the flotation of ATG14 and DFCP1. Cytosol from HEK293T cells expressing ATG14-HA and EGFP-DFCP1 was incubated with or without membrane. A buoyant density gradient flotation assay was performed followed by SDS-PAGE and immunoblot. (<bold>B</bold>) Starvation and PI3K regulation of the membrane recruitment of ATG14 and DFCP1. <italic>Atg5</italic> KO MEF membranes were incubated with the cytosol from either non-starved or starved HEK293T cells expressing ATG14-HA and EGFP-DFCP1 in the presence of indicated drugs. A buoyant density flotation was performed and samples were evaluated by SDS-PAGE and immunoblot. (<bold>C</bold>) Quantification of the floated markers shown in (<bold>B</bold>). Error bars represent standard deviations of three experiments.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.025">http://dx.doi.org/10.7554/eLife.00947.025</ext-link></p></caption><graphic xlink:href="elife00947fs011"/></fig><fig id="fig12s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00947.026</object-id><label>Figure 12—figure supplement 2.</label><caption><title>Atg14L and DFCP1 puncta colocalize with ERGIC.</title><p>(<bold>A</bold> and <bold>B</bold>) MEF cells were transfected with plasmids encoding ATG14-EGFP (<bold>A</bold>) or EGFP-DFCP1 (<bold>B</bold>). 24 hr after transfection, the cells were starved for 20 min. Cells were then fixed and visualized by immunofluorescence with anti-ERGIC53 antibody. Insets show the magnified view of the boxed areas. Bar, 10 µm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00947.026">http://dx.doi.org/10.7554/eLife.00947.026</ext-link></p></caption><graphic xlink:href="elife00947fs012"/></fig></fig-group></p></sec></sec><sec id="s3" sec-type="discussion"><title>Discussion</title><p>In this study, we have identified the ERGIC as a key membrane determinant in the biogenesis of autophagosomes. We first developed a cell-free assay based on in vitro LC3 lipidation to measure autophagosome biogenesis (<xref ref-type="fig" rid="fig1 fig2 fig3">Figures 1–3</xref>). By combining this assay with membrane fractionation, we identified an ERGIC-enriched fraction as the most active membrane to trigger LC3 lipidation (<xref ref-type="fig" rid="fig4 fig5 fig6 fig7">Figures 4–7</xref>). Next we used organelle immuno-/inhibitor depletion and immunoisolation to demonstrate that the ERGIC is necessary and sufficient to support LC3 lipidation (<xref ref-type="fig" rid="fig8 fig9">Figures 8 and 9</xref>). Finally, we provided evidence that the ERGIC membrane acts by recruiting ATG14 to initiate PI3K activity, an early step essential for autophagosome biogenesis (<xref ref-type="fig" rid="fig10 fig11 fig12">Figures 10–12</xref>).</p><p>Numerous morphological studies have indicated several possible sources of the autophagosomal membrane (<xref ref-type="bibr" rid="bib5">Burman and Ktistakis, 2010</xref>; <xref ref-type="bibr" rid="bib40">Mari et al., 2011</xref>; <xref ref-type="bibr" rid="bib73">Weidberg et al., 2011</xref>; <xref ref-type="bibr" rid="bib60">Rubinsztein et al., 2012</xref>). Indeed, it is improbable that one organelle contributes all the membrane constituents that become part of a mature autophagosome. Nonetheless, it seems likely that one membrane responds to the signal that triggers autophagosome formation and the identity of that membrane has remained elusive. Our isolation of the ERGIC as the locus of LC3 lipidation is in line with morphological studies that describe an omegasome structure projecting directly from the ER membrane (<xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>; <xref ref-type="bibr" rid="bib23">Hayashi-Nishino et al., 2009</xref>). However, our results show clearly that the bulk ER membrane is not the site of lipidation, thus if the omegasome arises from the ER, it must become modified in some way to be active for LC3 lipidation.</p><p>Starvation induces the membrane localization of soluble oligomeric proteins including ATG14 and the PI3K complex, followed by the recruitment of DFCP1 to generate the omegasome (<xref ref-type="bibr" rid="bib3">Axe et al., 2008</xref>; <xref ref-type="bibr" rid="bib42">Matsunaga et al., 2009</xref>; <xref ref-type="bibr" rid="bib67">Sun et al., 2008</xref>; <xref ref-type="bibr" rid="bib81">Zhong et al., 2009</xref>; <xref ref-type="bibr" rid="bib41">Matsunaga et al., 2010</xref>). This process occurs upstream of phagophore generation (<xref ref-type="bibr" rid="bib27">Itakura and Mizushima, 2010</xref>). Our data show that in starved cells and in isolated membranes, the presence of ERGIC is required for the efficient membrane recruitment of ATG14 and DFCP1 (<xref ref-type="fig" rid="fig11 fig12">Figures 11 and 12</xref>). Thus the ERGIC may play a role in an early stage of phagophore formation by providing a platform to recruit the class III PI3K complex and provide precursor membranes for phagophore initiation, which may be further expanded in a special subdomain of ER.</p><p>How and why the ERGIC is used to trigger phagophore formation remains unclear. Perhaps the tubular and curved structure of the ERGIC (<xref ref-type="bibr" rid="bib1">Appenzeller-Herzog and Hauri, 2006</xref>) in mammalian cells favors recruitment of the ATG14 complex and subsequently of other components. ATG14 has been reported to sense membrane curvature via an amphipathic alpha helix located in a C-terminal ‘BATS’ domain (<xref ref-type="bibr" rid="bib10">Fan et al., 2011</xref>). In yeast, it has been shown that highly curved membranes positive for ATG9 are delivered to the PAS (<xref ref-type="bibr" rid="bib39">Mari et al., 2010</xref>; <xref ref-type="bibr" rid="bib49">Nair et al., 2011</xref>; <xref ref-type="bibr" rid="bib75">Yamamoto et al., 2012</xref>). Subsequently, the curvature sensing protein Atg1 recruits Atg13 and the Atg17–31–29 protein complex to initiate the formation of the phagophore (<xref ref-type="bibr" rid="bib57">Ragusa et al., 2012</xref>). The suggested requirement for a tubular membrane together with the possible existence of an integral membrane protein(s) that triggers ATG14 recruitment are now open for biochemical analysis.</p><p>The cell-free LC3 lipidation reaction responds to a starvation signal, likely originating in the cytosolic fraction. Fractionation of the cytosol should reveal the full range of biochemical requirements including regulatory components induced by starvation as well as the core proteins essential for LC3 lipidation. Furthermore, this approach could be exploited to evaluate the maturation of the phagophore through subsequent stages of morphological transformation including envelope closure and fusion with the lysosome.</p></sec><sec id="s4" sec-type="materials|methods"><title>Materials and methods</title><sec id="s4-1"><title>Materials, antibodies, and plasmids</title><p>We obtained horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG from Jackson ImmunoResearch Laboratories (West Grove, PA); fluorescent secondary antibodies and Earle’s Balanced Salt Solution (EBSS) from Invitrogen (Grand Island, NY); PIP Strips from Echelon (Salt Lake City, UT); 3-methyladenine (3-MA), wortmannin, rapamycin, H89 and clofibrate from Sigma (St. Louis, MO); ATG4B from Boston Biochem (Cambridge, MA); SEC22B antibody blocking peptides (sequence CG+HSEFDEQHGKKVPTVSRPYSFIEFDT) from David King (University of California, Berkeley); Torin 1 and kbNB142-70 from Tocris (Minneapolis, MN); Pitstop 2 from Abcam (Cambridge, MA); reagents for PE measurement as described by Hokazono et al. (<xref ref-type="bibr" rid="bib25">Hokazono et al., 2011</xref>) and other reagents from previously described sources (<xref ref-type="bibr" rid="bib14">Ge et al., 2008</xref>, <xref ref-type="bibr" rid="bib13">2011</xref>). Amine oxidase was kindly provided by Eisaku Hokazono (Kyushu University, Higashi-ku, Japan).</p><p>Mouse anti-GM130, transferrin receptor, PMP70 and rabbit anti-Prohibitin-1, SEC22B and Ribophorin 1 antibodies were described before (<xref ref-type="bibr" rid="bib14">Ge et al., 2008</xref>; <xref ref-type="bibr" rid="bib61">Schindler and Schekman, 2009</xref>; <xref ref-type="bibr" rid="bib13">Ge et al., 2011</xref>). We purchased mouse anti-Flag, rabbit anti-ERGIC53, anti-LC3, anti-LAMP2, anti-ULK1, anti-ATG14 and anti-BECN1 antibodies from Sigma (St. Louis, MO); mouse anti-T7 antibody from EMD (Billerica, MA); hamster anti-ATG9, mouse anti-tubulin, rabbit anti-FACL4 and HRP-labeled anti-GST antibodies from Abcam (Cambridge, MA); rabbit anti-Cathepsin D from Epitomics (Burlingame, CA); rabbit anti-TGN38 from Novus Biologicals (Littleton, CO); mouse anti-ATG7, anti-ATG3, anti-ATG5 and anti-ATG16 from MBL (Woburn, MA); goat anti-SEC12 antibody from R&D Systems (Minneapolis, MN); mouse anti-Myc antibodies from Cell Signaling (Boston, MA); mouse anti-GFP antibody from Santa Cruz (Dallas, Texas); rabbit anti-Histone H4 antibody from the Robert Tjian lab (University of California, Berkeley).</p><p>The plasmids encoding ATG14-EGFP, ATG14-HA, Myc-LC3 and ATG16-Myc were kindly provided by Qing Zhong lab (University of California, Berkeley). The EGFP-DFCP1 plasmid was kindly provided by Nicholas Ktistakis lab (Babraham Institute, UK). And the LAMP1-RFP-Flag plasmid was from Addgene (provided by the Sabatini lab, Whitehead Institute). The GST-FYVE, GST-FYVE(C/S) and T7-LC3 plasmids were constructed by subcloning the indicated inserts from the GFP-TM-FYVE, GFP-TM-FYVE(C/S) constructs (Nicholas Ktistakis, Babraham Institute, UK) and Myc-LC3 plasmids into pGEX4T1 and pET28a vectors. The encoded proteins were expressed in <italic>E. coli</italic> BL21 and affinity purified with glutathione or Ni Sepharose (GE Healthcare Life Sciences, Piscataway, NJ). The Flag-GFP-ER-TM plasmid was generated by PCR insertion of a Flag tag into the GFP-ER-TM plasmid (Nicholas Ktistakis, Babraham Institute, UK). The human Vangl2-myc plasmid was described before (<xref ref-type="bibr" rid="bib18">Guo et al., 2013</xref>). Inserts from the pGEX-Sar1As (<xref ref-type="bibr" rid="bib29">Kim et al., 2005</xref>) were subcloned into the DsRed-Monomer-N1 vector to generate DsRed-tagged SAR1A plasmids.</p></sec><sec id="s4-2"><title>Cytosol preparation</title><p>The cells were cultured to confluence and either untreated or starved in EBSS (for lipidation using endogenous LC3, MEF cells were starved for 30 min while HEK293T and COS-7 cells for 1 hr; for lipidation using T7-LC3, HEK293T cells were starved for 1.5 hr). Then the cells were harvested by scraping and centrifuging at 600×<italic>g</italic> for 5 min, washed with PBS followed by another 600×<italic>g</italic>-spin for 5 min and homogenized by passing through a 22 G needle in a 1.5× cell pellet volume of B88 buffer (20 mM HEPES-KOH, pH 7.2, 250 mM sorbitol, 150 mM potassium acetate and 5 mM magnesium acetate) plus cocktail protease inhibitors (Roche, Indianapolis, IN), phosphatase inhibitors (Roche) and 0.3 mM DTT. The cell homogenates were centrifuged at 160,000×<italic>g</italic> for 30 min, supernatant fractions were collected and the centrifugation was repeated three times to achieve a clarified fraction (approximately 6–10 mg/ml of protein) which was used in the lipidation reaction.</p></sec><sec id="s4-3"><title>Purification of HisT7-LC3 and HisT7-LC3 (G/A)</title><p><italic>E. coli</italic> BL21 cells with the indicated expression plasmids were cultured at 37°C overnight. The overnight culture was inoculated at 1:50 dilution to a one liter volume and shaken at 37°C to an OD600 of 0.6–0.8. Protein expression was induced with 100 µM IPTG at 23°C for 5 hr and the cells were collected by centrifuging at 10,000×<italic>g</italic> for 10 min. The pellet was washed with 0.1 M PBS and suspended with 20 ml 0.2 M PBS (pH 7.4) with 15 mM imidazole and 1x protease inhibitors (Roche). Lysozyme was added to the cells at a concentration of 0.5 mg/ml and the digestion was performed on ice for 30 min after which Triton X-100 was added to a concentration of 0.5%. The cell suspension was sonicated with five to seven 15 s bursts until the solution was not viscous and the lysate was centrifuged at 100,000×<italic>g</italic> for 30 min. The supernatant was collected and incubated with 1 ml Ni Sepharose (packed beads) at 4°C for 2 hr. Then the beads were collected and washed with 70 vol of cold 0.2 M PBS with 25 mM imidazole and 0.2% Tween-20 followed by 10 vol of 0.2 M PBS with 25 mM imidazole. The bound proteins were eluted with 0.2 M PBS with 250 mM imidazole, buffer exchanged to 0.1 M PBS (pH 7.4) and stored at −80°C. Thrombin digestion was performed in the presence of 1 U/ml of thrombin (Roche) at room temperature for 1 hr followed by adding 1 mg/ml AEBSF (Santa Cruz) to deactivate thrombin.</p></sec><sec id="s4-4"><title>In vitro lipidation assay</title><p>For each reaction, cytosol (2 mg/ml final concentration), ATP regeneration system (40 mM creatine phosphate, 0.2 mg/ml creatine phosphokinase, and 1 mM ATP), GTP (0.15 mM) (<xref ref-type="bibr" rid="bib29">Kim et al., 2005</xref>), 0.2 µg HisT7-LC3 (1-120) or T7-LC3 (1-120) generated by thrombin digestion and different membrane fractions (0.2 mg/ml PC content final concentration) were incubated in a final volume of 30 µl. The reactions were performed at 30°C for the indicated times. Where indicated, compounds or proteins were added to the reactions.</p></sec><sec id="s4-5"><title>Membrane fractionation</title><p>Cells (ten 15-cm dishes) were cultured to confluence, harvested and homogenized in a 2.7× cell pellet volume of buffer containing 20 mM HEPES-KOH, pH 7.2, 400 mM sucrose and 1 mM EDTA by passing through a 22 G needle until ∼85% lysis analyzed by Trypan Blue staining. Homogenates were either centrifuged at 100,000×<italic>g</italic> for 45 min to collect total membranes or subjected to sequential differential centrifugation at 1,000×<italic>g</italic> (10 min), 3,000×<italic>g</italic> (10 min), 25,000×<italic>g</italic> (20 min) and 100,000×<italic>g</italic> (30 min, TLA100.3 rotor, Beckman) to collect the membranes sedimented at each speed. The PC content of each fraction was measured as described before (<xref ref-type="bibr" rid="bib13">Ge et al., 2011</xref>). Membrane fractions containing equal amounts of PC were used to test LC3 lipidation activity. The 25,000×<italic>g</italic> membrane pellet, which contained the highest activity, was suspended in 0.75 ml 1.25 M sucrose buffer and overlayed with 0.5 ml 1.1 M and 0.5 ml 0.25 M sucrose buffer (Golgi isolation kit; Sigma). Centrifugation was performed at 120,000×<italic>g</italic> for 2 hr (TLS 55 rotor, Beckman), after which two fractions, one at the interface between 0.25 M and 1.1 M sucrose (L fraction) and the pellet on the bottom (P fraction), were separated. Activities of the two fractions were then tested as described above, and the L fraction was selected and suspended in 1 ml 19% OptiPrep for a step gradient containing 0.5 ml 22.5%, 1 ml 19% (sample), 0.9 ml 16%, 0.9 ml 12%, 1 ml 8%, 0.5 ml 5% and 0.2 ml 0% OptiPrep each. Each density of OptiPrep was prepared by diluting 50% OptiPrep (20 mM Tricine-KOH, pH 7.4, 42 mM sucrose and 1 mM EDTA) with a buffer containing 20 mM Tricine-KOH, pH 7.4, 250 mM sucrose and 1 mM EDTA. The OptiPrep gradient was centrifuged at 150,000×<italic>g</italic> for 3 hr (SW 55 Ti rotor, Beckman) and subsequently ten fractions, 0.5 ml each, were collected from the top. Fractions were diluted with B88 buffer and membranes were collected by centrifugation at 100,000×<italic>g</italic> for 1 hr. The activity of each fraction was determined and the distribution of the activity was compared with that of each membrane marker. Membranes containing an equal amount of PC from each fraction were also measured for PE content using an enzymatic assay (<xref ref-type="bibr" rid="bib25">Hokazono et al., 2011</xref>).</p></sec><sec id="s4-6"><title>Immunoisolation</title><p>Cells (ten 15-cm dishes) expressing indicated protein markers were cultured to confluence and harvested as indicated in the ‘Membrane fractionation’ section. Membranes from either the 25,000×<italic>g</italic> membrane pellet (for Flag-GFP-ER-TM or LAMP1-RFP-Flag immunoisolation) or the L fraction (for SEC22B, KDELR-Flag or Vangl2-myc immunoisolation) were collected, suspended in immunoisolation buffer containing 25 mM HEPES, pH 7.4, 140 mM potassium chloride, 5 mM sodium chloride, 2.5 mM magnesium acetate, 50 mM sucrose and 2 mM EGTA (<xref ref-type="bibr" rid="bib82">Zoncu et al., 2011</xref>), and diluted to a PC content of 0.2 mg/ml. Anti-Flag (100 µl, packed volume) or anti-Myc agarose (Sigma) was added to a 1 ml membrane suspension with or without 0.2 mg/ml blocking peptides (3xFlag peptide and Myc peptide; Sigma) and mixed by rotation at 4°C overnight. For immunoisolation of endogenous SEC22B vesicles, 20 µl rabbit anti-SEC22B antibody was added to a 1 ml L fraction membrane suspension with or without 0.2 mg/ml SEC22B antibody blocking peptide and incubated for 3 hr at 4°C followed by addition of 100 µl (packed volume) Protein A Sepharose for overnight incubation at 4°C. Beads with the associated membranes were washed with 1 ml immunoisolation buffer three times and membranes bound to the beads were eluted by incubating with 0.5 mg/ml of the indicated competing peptides for 0.5 hr at room temperature. The eluted membranes were collected by ultracentrifugation. The sedimented activities were determined and compared to input membrane of equal PC content.</p></sec><sec id="s4-7"><title>Membrane recruitment assay</title><p>For cytosol preparation, HEK293T cells were transfected with plasmids encoding the genes for the indicated proteins by X-tremeGene HP (Roche). At 48 hr post-transfection, the cytosols were harvested as described above.</p><p>For membrane preparation, <italic>Atg5</italic> KO MEF cells were treated with indicated compounds and the cells were lysed. After a 1,000×<italic>g</italic> centrifugation, the total membranes from the supernatant were sedimented at 100,000×<italic>g</italic> for 1 hr. Similar reactions containing the cytosols, ATP regeneration system, GTP and the total membranes from different treatments were carried out in a final volume of 50 µl at 30°C for 1 hr.</p><p>After the reaction, a membrane flotation experiment was performed. OptiPrep (200 µl of 50%) diluted in B88 was added to the reaction mixture to make a 40% solution which was overlayed with 200 µl 35% OptiPrep and 50 µl B88. The gradient was centrifuged at 150,000×<italic>g</italic> for 1.5 hr. Seven fractions, 80 µl each, were collected from the top. The bottom fractions no. 5 to 7 were combined and evaluated by SDS-PAGE and immunoblot to examine the distribution of indicated protein markers.</p></sec><sec id="s4-8"><title>Immunofluorescence microscopy, immunoblot and dot blot</title><p>Immunofluorescence was performed as previously described (<xref ref-type="bibr" rid="bib14">Ge et al., 2008</xref>, <xref ref-type="bibr" rid="bib13">2011</xref>). Images were acquired with a Zeiss LSM 710 laser confocal scanning microscope. ERGIC recovery quantification was described previously (<xref ref-type="bibr" rid="bib56">Puri and Linstedt, 2003</xref>). Golgi recovery was quantified by manually counting the percent of cells displaying a perinuclear location of GM130. For each sample, more than 100 cells were counted. Immunoblot was performed as previously described (<xref ref-type="bibr" rid="bib14">Ge et al., 2008</xref>, <xref ref-type="bibr" rid="bib13">2011</xref>) and dot blot was carried out according to the PIP Strip user manual (Echelon). Images were acquired and bands were quantified with Chemidoc MP Imaging System (Bio-Rad).</p></sec><sec id="s4-9"><title>Cell culture</title><p>HEK293T cells were grown in a tissue culture facility. <italic>Atg5</italic> KO and control MEFs (<xref ref-type="bibr" rid="bib34">Kuma et al., 2004</xref>), <italic>Atg3</italic> KO, <italic>Atg7</italic> KO and control MEFs (<xref ref-type="bibr" rid="bib33">Komatsu et al., 2005</xref>; <xref ref-type="bibr" rid="bib65">Sou et al., 2008</xref>), and <italic>Ulk1</italic> KO and control MEFs (<xref ref-type="bibr" rid="bib35">Kundu et al., 2008</xref>) were generously provided by Noboru Mizushima (Tokyo Medical and Dental University, Japan), Masaaki Komatsu (Tokyo Metropolitan Institute of Medical Science, Japan) and Kundu Mondira (St. Jude Children’s Research Hospital). The cells were grown in monolayer at 37°C in 5% CO<sub>2</sub> and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. For starvation, the cells were incubated in EBSS for the indicated times in the absence or presence of the drugs indicated in the manuscript.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank Qing Zhong, Nicholas Ktistakis, Noboru Mizushima, Masaaki Komatsu, David King, Kundu Mondira, Eisaku Hokazono, and Jennifer Lippincott-Schwartz (NICHD) for reagents; Bob Lesch, Ann Fisher, Xiaozhu Zhang and Amita Gorur (University of California, Berkeley) for technical assistance; Qing Zhong, Jeremy Thorner (University of California, Berkeley), Ta-Yuan Chang (Dartmouth Medical School), David Sabatini, Roberto Zoncu (Whitehead Institute) and Xuejun Jiang (Memorial Sloan-Kettering Cancer Center) for helpful information and advice on the study; and Daniel Klionsky (University of Michigan) and Livy Wilz (University of California, Berkeley) for manuscript editing. LG was supported by a fellowship from the Human Frontier Science Program (HFSP) and the Jane Coffin Childs Fund (JCCF). DM and MZ are HHMI Associates. RS is an Investigator of the HHMI and a Senior Fellow of the University of California, Berkeley Miller Institute.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>RS: Editor-in-Chief, <italic>eLife</italic>.</p></fn><fn fn-type="conflict" id="conf2"><p>The other authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>LG, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con2"><p>DM, Acquisition of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con3"><p>MZ, Acquisition of data, Contributed unpublished essential data or reagents</p></fn><fn 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contrib-type="editor"><name><surname>Levine</surname><given-names>Beth</given-names></name><role>Reviewing editor</role><aff><institution>University of Texas Southwestern Medical School</institution>, <country>United States</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “Lipidation of LC3 identifies the ER-Golgi intermediate compartment as a precursor of the mammalian autophagosome” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor, a Reviewing editor, and two reviewers.</p><p>The Reviewing editor and the two reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.</p><p>We agree that your manuscript represents an elegantly designed and carefully performed study that provides an important new tool to the field (i.e., the development of an in vitro assay system to analyze LC3 lipidation using components of mammalian cells) and increased insight into the mechanism of autophagosome biogenesis (i.e., further clarification of the role of the ERGIC as a membrane determinant of the site of LC3 lipidation).</p><p>The reviewers have suggested some studies that would either (a) further increase the impact of your findings or (b) provide additional controls to further strengthen the findings. Here, we provide a summary of the suggestions. In our collective view, the most crucial point is to provide additional data such as EM analysis (or another approach) to determine whether your in vitro system for studying LC3 lipidation results in actual autophagosome reconstitution.</p><p><italic>Suggestions to increase the impact/significance of the findings:</italic></p><p>1) As mentioned above, further characterization of the cell-free system would be important. Specifically, does the reconstitution system allow for the formation of mature autophagosomes and autolysosomes? Protease protection assays, EM analysis, and analysis of the effects of lysosomal inhibitors should be considered to strengthen this aspect of the study.</p><p>2) EM analysis of the tissue culture experiments would help determine whether the ERGIC compartment directly contributes to autophagosome formation.</p><p><italic>Suggestions for additional controls to further strengthen your data:</italic></p><p>1) The lipidation of LC3 requires the Atg12–5–16 complex. Using gel filtration of the cytosol, we suggest that you demonstrate that this complex is formed or maintained in your reconstitution systems using 1) ERGIC membrane fractions, and 2) a membrane fraction you have ruled out as being a membrane source (see <xref ref-type="fig" rid="fig8">Figure 8</xref>), such as ER, late endosomes and plasma membrane. This would be an important control to show the integrity of the E3 activity in the cytosol even though the lipidation is reduced, and eliminate the possibility that the membrane fractions or assay conditions can deplete or contribute inhibitory factors, which may be altering the lipidation activity of the Atg12–5–16 complex.</p><p>2) In light of the recent data about the MAM and autophagosome biogenesis (<xref ref-type="bibr" rid="bib20">Hamasaki, et al. 2013</xref>), you should consider validating your MAM fractionation for Atg14L by Western blot.</p><p>3) One of the most important experiments is the recruitment of Atg14 and DFCP1 to the ERGIC in <xref ref-type="fig" rid="fig12">Figure 12</xref>. These data are not as robust as the rest of the manuscript and it would be important to show that a transfected negative control (e.g., GFP alone) does not float up on the ERGIC membranes. In addition, you might consider testing GFP–LC3–II recruitment as an additional further validation in this assay.</p><p>[Editors’ note: before acceptance, the following revisions were also requested.]</p><p>There is one remaining issue that needs to be addressed before acceptance, as outlined below:</p><p>We agree that the formation of a closed concentric membrane sphere (autophagosome) is likely to be quite complex, and we are willing to accept your argument that the in vitro reconstitution of this process is beyond the scope of the present paper. Nonetheless, in order for this paper to have the proper impact in the field and provide as useful a tool as possible for future researchers, we think that it is important to provide as much characterization as you can in the text of what your system is reconstituting in vitro. Accordingly, we would like you to mention (and discuss as you deem appropriate) the results of LC3-II protease protection experiments in the text. As you point out, the absence of complete reconstitution of a closed membrane sphere does not detract from the overall importance of your work; however, we feel that it is important for readers to know this limitation of your in vitro LC3 lipidation system.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00947.028</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p>[Suggestions to increase the impact/significance of the findings:]</p><p><italic>1) As mentioned above, further characterization of the cell-free system would be important. Specifically, does the reconstitution system allow for the formation of mature autophagosomes and autolysosomes? Protease protection assays, EM analysis, and analysis of the effects of lysosomal inhibitors should be considered to strengthen this aspect of the study</italic>.</p><p>We too would like to have our cell-free reaction recapitulate the entire pathway of authophagosome biogenesis, but this goal may be a bit much for a first report. Nonetheless, we have performed a protease protection assay hoping to observe the progressive enclosure of lipidated LC3 within a sealed compartment as might be expected when the autophagosome precursor membrane closes to segregate cytosolic components. Unfortunately, on repeated tests in prolonged incubations, we find little evidence for reproducible protease protection of the LC3-II species. We are not discouraged by this because the overall pathway is likely to be quite complex, including the fusion of additional membranes carrying such proteins as ATG9 (which is missing from our enriched ERGIC membrane), as well as a substantial morphological transformation from a membrane sheet into a closed concentric membrane sphere. An alternative strategy could be to start with membranes from an <italic>Atg</italic> null cell line blocked at a distinctly later step in the pathway. We hope the reviewers will appreciate that this is beyond the scope of this paper, which we believe is already quite complex.</p><p><italic>2) EM analysis of the tissue culture experiments would help determine whether the ERGIC compartment directly contributes to autophagosome formation</italic>.</p><p>We thank the reviewers for the suggestion. Our antibodies are not good enough for immunogold labeling in EM analysis. Instead, we performed confocal microscopy to analyze the colocalization of ERGIC with ATG14 and DFCP1, two early autophagy markers as indicated in the manuscript. We found that the starvation-induced puncta of ATG14 partially overlaps with ERGIC (<xref ref-type="fig" rid="fig12s2">Figure 12—figure supplement 2A</xref>). DFCP1, the autophagy-related PI3P binding protein, colocalizes with ERGIC shortly after starvation (<xref ref-type="fig" rid="fig12s2">Figure 12—figure supplement 2B</xref>). These data are consistent with our membrane recruitment assay in <xref ref-type="fig" rid="fig12">Figure 12</xref>. Together, the results suggest that ATG14 may transiently associate with ERGIC to generate PI3P, and subsequently be recognized by DFCP1 to initiate autophagosome biogenesis.</p><p>[Suggestions for additional controls to further strengthen your data:]</p><p><italic>1) The lipidation of LC3 requires the Atg12-5-16 complex. Using gel filtration of the cytosol, we suggest that you demonstrate that this complex is formed or maintained in your reconstitution systems using 1) ERGIC membrane fractions, and 2) a membrane fraction you have ruled out as being a membrane source (see <xref ref-type="fig" rid="fig8">Figure 8</xref>), such as ER, late endosomes and plasma membrane. This would be an important control to show the integrity of the E3 activity in the cytosol even though the lipidation is reduced, and eliminate the possibility that the membrane fractions or assay conditions can deplete or contribute inhibitory factors, which may be altering the lipidation activity of the Atg12–5–16 complex</italic>.</p><p>We thank the reviewers for the suggestion. We did the lipidation assay with ERGIC membrane fractions and the P fraction enriched in ER. After the reaction, we sedimented the membranes and collected the supernatant fractions followed by chromatography on a Superpose 6 column. Fractions were collected and immunoblots were performed to examine the distribution of ATG5–12 and ATG16. We found that the ATG5–12–16 complex is formed and maintained in both conditions (<xref ref-type="fig" rid="fig7s1">Figure 7—figure supplement 1B</xref>). Therefore, the distinct lipidation activity observed with different membrane fractions is not due to an alteration of ATG5–12–16 complex.</p><p><italic>2) In light of the recent data about the MAM and autophagosome biogenesis (<xref ref-type="bibr" rid="bib20">Hamasaki, et al. 2013</xref>), you should consider validating your MAM fractionation for Atg14L by western blot</italic>.</p><p>We thank the reviewers for the suggestion. We performed an immunoblot with ATG14 antibody and detected a tiny amount of ATG14 in the MAM fraction under normal conditions (<xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1A</xref>). However, after starvation, we observed an increase of ATG14 in the MAM faction (<xref ref-type="fig" rid="fig8s1">Figure 8—figure supplement 1B</xref>). The observation is consistent with <xref ref-type="bibr" rid="bib20">Hamasaki, et al. 2013</xref>.</p><p><italic>3) One of the most important experiments is the recruitment of Atg14 and DFCP1 to the ERGIC in <xref ref-type="fig" rid="fig12">Figure 12</xref>. These data are not as robust as the rest of the manuscript and it would be important to show that a transfected negative control (e.g., GFP alone) does not float up on the ERGIC membranes. In addition, you might consider testing GFP–LC3–II recruitment as an addition further validation in this assay</italic>.</p><p>We thank the reviewers for the suggestion. In <xref ref-type="fig" rid="fig12s1">Figure 12—figure supplement 1</xref>, we validated the assay by showing that the recruitment of ATG14 and DFCP1 is regulated similarly to cellular autophagy, including starvation stimulation and PI3K dependency. We also showed that tubulin, an abundant cytosolic protein, was barely recruited to the membrane. For the LC3-II recruitment, we took the reviewer’s suggestion and added the T7-tagged LC3-I in the in vitro recruitment reaction. We found that the lipidated T7-LC3 was recruited to the membrane fraction while the soluble form LC3-I was not. Moreover, depletion of ERGIC mitigated the generation of LC3-II as indicated by the reduced signal in the top membrane fraction (<xref ref-type="fig" rid="fig12">Figure 12</xref>).</p><p><italic>[Editors’ note: before acceptance, the following revisions were also requested.</italic>]</p><p><italic>There is one remaining issue that needs to be addressed before acceptance, as outlined below</italic>:</p><p><italic>We agree that the formation of a closed concentric membrane sphere (autophagosome) is likely to be quite complex, and we are willing to accept your argument that the in vitro reconstitution of this process is beyond the scope of the present paper. Nonetheless, in order for this paper to have the proper impact in the field and provide as useful a tool as possible for future researchers, we think that it is important to provide as much characterization as you can in the text of what your system is reconstituting in vitro. Accordingly, we would like you to mention (and discuss as you deem appropriate) the results of LC3-II protease protection experiments in the text. As you point out, the absence of complete reconstitution of a closed membrane sphere does not detract from the overall importance of your work; however, we feel that it is important for readers to know this limitation of your in vitro LC3 lipidation system</italic>.</p><p>We thank the reviewers for appreciating the complexity of a mature autophagosome formation in vitro, which is beyond the scope of this paper. We have pointed out the limitation of our system in the Results and Discussion sections.</p></body></sub-article></article> |