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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">03504</article-id><article-id pub-id-type="doi">10.7554/eLife.03504</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Short report</subject></subj-group><subj-group subj-group-type="heading"><subject>Cell biology</subject></subj-group><subj-group subj-group-type="heading"><subject>Developmental biology and stem cells</subject></subj-group></article-categories><title-group><article-title>Mother-daughter asymmetry of pH underlies aging and rejuvenation in yeast</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-14526"><name><surname>Henderson</surname><given-names>Kiersten A</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-2295-1232</contrib-id><xref ref-type="aff" rid="aff1"/><xref ref-type="other" rid="par-4"/><xref ref-type="other" rid="par-5"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-14728"><name><surname>Hughes</surname><given-names>Adam L</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-7095-3793</contrib-id><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="other" rid="par-3"/><xref ref-type="other" rid="par-6"/><xref ref-type="other" rid="par-7"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-2629"><name><surname>Gottschling</surname><given-names>Daniel E</given-names></name><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0002-7303-6552</contrib-id><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="con3"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution content-type="dept">Division of Basic Sciences</institution>, <institution>Fred Hutchinson Cancer Research Center</institution>, <addr-line><named-content content-type="city">Seattle</named-content></addr-line>, <country>United States</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Weis</surname><given-names>Karsten</given-names></name><role>Reviewing editor</role><aff><institution>ETH Zürich</institution>, <country>Switzerland</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>dgottsch@fhcrc.org</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>Department of Biochemistry, University of Utah, Salt Lake City, United States</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>04</day><month>09</month><year>2014</year></pub-date><pub-date pub-type="collection"><year>2014</year></pub-date><volume>3</volume><elocation-id>e03504</elocation-id><history><date date-type="received"><day>28</day><month>05</month><year>2014</year></date><date date-type="accepted"><day>03</day><month>09</month><year>2014</year></date></history><permissions><copyright-statement>© 2014, Henderson et al</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Henderson et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">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="elife03504.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.03504.001</object-id><p>Replicative aging in yeast is asymmetric–mother cells age but their daughter cells are rejuvenated. Here we identify an asymmetry in pH between mother and daughter cells that underlies aging and rejuvenation. Cytosolic pH increases in aging mother cells, but is more acidic in daughter cells. This is due to the asymmetric distribution of the major regulator of cytosolic pH, the plasma membrane proton ATPase (Pma1). Pma1 accumulates in aging mother cells, but is largely absent from nascent daughter cells. We previously found that acidity of the vacuole declines in aging mother cells and limits lifespan, but that daughter cell vacuoles re-acidify. We find that Pma1 activity antagonizes mother cell vacuole acidity by reducing cytosolic protons. However, the inherent asymmetry of Pma1 increases cytosolic proton availability in daughter cells and facilitates vacuole re-acidification and rejuvenation.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.001">http://dx.doi.org/10.7554/eLife.03504.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.03504.002</object-id><title>eLife digest</title><p>Aging is a part of life—but its biological basis and, in particular, how aged cells give rise to young offspring (or progeny) has not been clearly established for any organism.</p><p>Budding yeast is a microorganism that is a valuable model to understand aging in more complex organisms like humans. Budding yeast cells undergo a process called ‘replicative aging’, meaning that each yeast mother cell produces a set number of daughter cells in her lifetime. However, when daughter cells arise from an aging mother cell, the daughter's age is ‘reset to zero’. How mother cells age, and how their daughters are rejuvenated, are questions that have been studied for decades.</p><p>Previously, researchers discovered that a mother cell's vacuole (an acidic compartment that stores important molecules that can become toxic) becomes less acidic as the mother cell ages. Daughter cells, on the other hand, have very acidic vacuoles, which was linked to their renewed lifespans. However, the mechanism behind this difference in the acidity of the vacuole between mother and daughter cells was unknown.</p><p>Now, Henderson et al. have found that a protein (called Pma1), which is found at the cell surface and pumps protons out of the cell, is present in mother cells but not in their newly-formed daughter cells. Furthermore, the Pma1 protein also accumulates as mother cells age. By pumping protons out of the cell, Pma1 effectively reduces the number of protons available to acidify the vacuole in the mother cell. However, because at first the daughter does not have Pma1, there are still plenty of protons inside the cell to acidify the vacuole.</p><p>When Henderson et al. reduced the activity of Pma1 in mother cells, the entire cell became more acidic, and so did their vacuoles. Conversely daughter cells engineered to have more Pma1 were less acidic and had less acidic vacuoles than normal.</p><p>Henderson et al. next asked whether reducing Pma1 activity to create a more acidic cell, could extend the lifespan of cells, and found that indeed cells with less Pma1 activity lived longer. As such, these findings indicate that an asymmetry in the acidity of the cell—caused by unequal levels of the Pma1 protein—contributes to reducing the lifespan of the mother cell and to rejuvenating the daughter cell. Thus Henderson et al. have identified one of the earliest events in the cellular aging process in budding yeast. Their findings suggest that an imbalance in an activity that is normally essential for cell survival (in this case, the activity of Pma1) can have long-term consequences for the cell that lead to aging.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.002">http://dx.doi.org/10.7554/eLife.03504.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>aging</kwd><kwd>asymmetry</kwd><kwd>pH</kwd><kwd>rejuvenation</kwd><kwd>H+-ATPase</kwd><kwd>vacuole</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd><italic>S. cerevisiae</italic></kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AG023779</award-id><principal-award-recipient><name><surname>Gottschling</surname><given-names>Daniel E</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100001642</institution-id><institution>Glenn Foundation for Medical Research</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Gottschling</surname><given-names>Daniel E</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100005237</institution-id><institution>Helen Hay Whitney Foundation</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Hughes</surname><given-names>Adam L</given-names></name></principal-award-recipient></award-group><award-group id="par-4"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100005189</institution-id><institution>Leukemia and Lymphoma Society</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Henderson</surname><given-names>Kiersten A</given-names></name></principal-award-recipient></award-group><award-group id="par-5"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AC965722</award-id><principal-award-recipient><name><surname>Henderson</surname><given-names>Kiersten A</given-names></name></principal-award-recipient></award-group><award-group id="par-6"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AG000057</award-id><principal-award-recipient><name><surname>Hughes</surname><given-names>Adam L</given-names></name></principal-award-recipient></award-group><award-group id="par-7"><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/100000002</institution-id><institution>National Institutes of Health</institution></institution-wrap></funding-source><award-id>AG043095</award-id><principal-award-recipient><name><surname>Hughes</surname><given-names>Adam L</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>Protons are pumped out of mother cells by a protein that accumulates over time and this leads to cellular aging, but this protein is largely absent from daughter cells, which mediates rejuvenation.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1"><title>Main text</title><p>During replicative aging in budding yeast, mother cells produce a finite number of daughter cells before arresting (<xref ref-type="bibr" rid="bib33">Mortimer and Johnston, 1959</xref>). Because replicative aging is asymmetric, the process of aging occurs in mother cells but is absent in daughter cells (<xref ref-type="bibr" rid="bib10">Egilmez and Jazwinski, 1989</xref>; <xref ref-type="bibr" rid="bib21">Kennedy et al., 1994</xref>). Several asymmetric phenotypes have been identified and proposed to contribute to mother cell decline (<xref ref-type="bibr" rid="bib45">Sinclair and Guarente, 1997</xref>; <xref ref-type="bibr" rid="bib23">Lai et al., 2002</xref>; <xref ref-type="bibr" rid="bib1">Aguilaniu et al., 2003</xref>; <xref ref-type="bibr" rid="bib12">Erjavec et al., 2007</xref>; <xref ref-type="bibr" rid="bib11">Eldakak et al., 2010</xref>; <xref ref-type="bibr" rid="bib30">McFaline-Figueroa et al., 2011</xref>; <xref ref-type="bibr" rid="bib35">Nystrom and Liu, 2014</xref>). We recently found that the acidity of the yeast lysosome-like vacuole is asymmetric between mother and daughter cells. Vacuole acidity declines in mother cells in early age and limits lifespan, but daughter cells have acidic vacuoles (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>). To identify what reduces vacuole acidity and how vacuole acidity is regenerated in daughter cells, we further characterized vacuole pH asymmetry. Cells were aged using a genetic system (<xref ref-type="bibr" rid="bib25">Lindstrom and Gottschling, 2009</xref>) and vacuole acidity was monitored by staining cells with quinacrine, a fluorescent dye that accumulates in the acidic vacuole (<xref ref-type="bibr" rid="bib50">Weisman et al., 1987</xref>). We observed bright vacuolar quinacrine staining indicative of acidic pH in a high percentage of buds (nascent daughter cells) regardless of mother cell age, whereas staining was diminished or undetectable in mother cell vacuoles (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>) (<xref ref-type="fig" rid="fig1">Figure 1A</xref>). Thus, throughout their lifespan mother cells produce daughter cells capable of regenerating vacuole acidity.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.03504.003</object-id><label>Figure 1.</label><caption><title>Vacuole acidity regenerates in daughter cells throughout mother cell aging and reacidification occurs prior to cytokinesis.</title><p>(<bold>A</bold> and <bold>B</bold>) Age (# of cell divisions) is shown in the second row and represents exact age determined by calcofluor staining of bud scars. Representative images are shown. n ≥30 cells per timepoint. Arrowheads indicate the daughter cell. DIC, differential interference contrast. (<bold>A</bold>) Vacuole acidity indicated by quinacrine staining of aged cells expressing Vph1-mCherry (vacuole membrane marker). (<bold>B</bold>) Vacuole acidity of cells expressing Vph1-mCherry and arrested prior to cytokinesis by nocodazole treatment. (<bold>C</bold>) Cells with septin morphology indicated by Cdc10-mCherry were quinacrine stained and vacuole acidity was examined before or after cytokinesis (one septin ring or two rings).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.003">http://dx.doi.org/10.7554/eLife.03504.003</ext-link></p></caption><graphic xlink:href="elife03504f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03504.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>Subunits of the V-ATPase are not asymmetric between mother cells and buds.</title><p>(<bold>A</bold>) Cells in their first division expressing Vph1-GFP (V0 domain) or (<bold>B</bold>) Vma2-GFP (V1 domain).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.004">http://dx.doi.org/10.7554/eLife.03504.004</ext-link></p></caption><graphic xlink:href="elife03504fs001"/></fig></fig-group></p><p>To further characterize vacuole pH asymmetry, the timing of re-acidification of the bud vacuole was examined. Vacuole acidity was asymmetric in cells treated with nocodazole (<xref ref-type="fig" rid="fig1">Figure 1B</xref>), suggesting that the bud vacuole re-acidifies prior to cytokinesis. We confirmed that re-acidification occurred before cytokinesis by examining cells containing the septin marker Cdc10-mCherry (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). A single septin ring at the bud neck transitions to two rings during cytokinesis (<xref ref-type="bibr" rid="bib26">Lippincott et al., 2001</xref>). We observed high vacuole acidity in buds when there was a single septin ring, further supporting that vacuole acidity regenerates prior to cytokinesis. Thus, throughout their lifespan, mother cells produce daughter cells that regenerate vacuole acidity prior to cytokinesis, when mother and daughter cells share a common cytosol. Thus, whatever causes the asymmetry of vacuole pH must also be asymmetric between mother and daughter cells prior to cytokinesis.</p><p>One of the major points of regulation of vacuole acidity is assembly of the vacuolar proton ATPase (V-ATPase), a multi-subunit complex that pumps protons from the cytosol into the vacuole. The V-ATPase consists of the integral membrane V<sub>0</sub> complex and the V<sub>1</sub> complex that associates with the V<sub>0</sub> (<xref ref-type="bibr" rid="bib24">Li and Kane, 2009</xref>). We examined whether there was a difference in vacuole-associated V<sub>1</sub> or V<sub>0</sub> between mother and daughter cells by visualizing green fluorescent protein (GFP) tagged subunits of each domain and found no evidence of asymmetry (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1</xref>). Thus, no obvious difference in V-ATPase assembly can account for vacuole pH asymmetry between mother and bud.</p><p>In a screen to identify proteins asymmetrically retained in mother cells throughout aging, we identified the plasma membrane proton ATPase, Pma1 (<xref ref-type="bibr" rid="bib48">Thayer et al., 2014</xref>). Pma1 is the major regulator of cytosolic pH (<xref ref-type="bibr" rid="bib14">Ferreira et al., 2001</xref>; <xref ref-type="bibr" rid="bib41">Serrano et al., 1986</xref>), and has similar activity to the V-ATPase, in that they both translocate cytosolic protons across membranes. Pma1 pumps protons from the cytosol out of the cell, whereas the V-ATPase pumps cytosolic protons into the vacuole (<xref ref-type="bibr" rid="bib37">Orij et al., 2011</xref>). Because Pma1 regulates cytosolic pH, we hypothesized that it could antagonize vacuole acidity during aging and underlie vacuole pH asymmetry.</p><p>As a first step in testing our hypothesis, we analyzed Pma1 protein localization. There are conflicting reports on Pma1 asymmetry (<xref ref-type="bibr" rid="bib47">Smardon et al., 2013</xref>; <xref ref-type="bibr" rid="bib22">Khmelinskii et al., 2012</xref>; <xref ref-type="bibr" rid="bib28">Malínská et al., 2003</xref>), however we found that Pma1 was asymmetric between mother and daughter cells. Pma1 levels at the plasma membrane were higher in mother cells than daughter cells as indicated by indirect immunofluorescence with antibody to Pma1 (<xref ref-type="fig" rid="fig2">Figure 2A</xref>). Similarly, Pma1 was more abundant in mother cells than buds when visualized with either Pma1-GFP (<xref ref-type="fig" rid="fig2">Figure 2A</xref>) or Pma1-mCherry fusion protein (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). We also detected mCherry and GFP fluorescence in the vacuole, which likely represents misfolded protein directed to the vacuole for degradation (<xref ref-type="bibr" rid="bib7">Chang and Fink, 1995</xref>). Importantly, we found that asymmetry of Pma1 at the plasma membrane was maintained through at least 18 mother cell divisions and that Pma1 was asymmetric prior to cytokinesis (<xref ref-type="fig" rid="fig2">Figure 2B</xref>), paralleling the asymmetry of vacuole pH.<fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.03504.005</object-id><label>Figure 2.</label><caption><title>Plasma membrane Pma1 levels are asymmetric between mother cells and buds, accumulate with age, and are inversely correlated with vacuole acidity.</title><p>(<bold>A</bold>) Top panel: Indirect immunofluorescence imaging of Pma1 with anti-Pma1 antibody in untagged young cell. Bottom panel: Pma1-GFP localization in young cell. (<bold>B</bold>) Newborn daughter cells and aged mother cells expressing Pma1-mCherry were quinacrine stained. Arrowheads indicate the vacuoles of interest.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.005">http://dx.doi.org/10.7554/eLife.03504.005</ext-link></p></caption><graphic xlink:href="elife03504f002"/></fig></p><p>When we examined Pma1 distribution during mother cell aging, we found that Pma1 increased at the plasma membrane in early age (<xref ref-type="fig" rid="fig2">Figure 2B</xref>). Pma1 levels were very low in newborn daughter cells, increased as daughter cells became mothers, and continued to increase over the first three mother cell divisions. This pattern of Pma1 abundance in daughters and aging mother cells inversely correlated with vacuole acidity. When Pma1 was very low in buds and newborn cells, the vacuole was acidic. In contrast, vacuole acidity was reduced in mother cells that have high Pma1 levels.</p><p>The inverse correlation between Pma1 levels and vacuole acidity suggested that Pma1 could cause vacuole pH asymmetry by antagonizing V-ATPase activity in mother cells. We first tested whether high levels of Pma1 could reduce vacuole acidity by overexpressing an extra copy of <italic>PMA1</italic> in newborn daughter cells from an inducible promoter (<xref ref-type="bibr" rid="bib15">Gao and Pinkham, 2000</xref>; <xref ref-type="bibr" rid="bib49">Veatch et al., 2009</xref>). Overexpression of <italic>PMA1-mCherry</italic> increased Pma1 at the plasma membrane of newborn cells (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1</xref>). Without excess Pma1, 87% of newborn cells had highly acidic vacuoles, whereas vacuole acidity was only high in 13% of cells upon <italic>PMA1</italic> overexpression (<xref ref-type="fig" rid="fig3">Figure 3A</xref>). To further test whether Pma1 antagonized vacuole acidity, we reduced Pma1 activity and examined vacuole acidity in aging mother cells. <italic>PMA1</italic> is an essential gene and cannot be deleted (<xref ref-type="bibr" rid="bib41">Serrano et al., 1986</xref>), so we reduced its activity by 65% using the <italic>pma1-105</italic> allele that has a mutation in the catalytic domain (<xref ref-type="bibr" rid="bib29">McCusker et al., 1987</xref>; <xref ref-type="bibr" rid="bib38">Perlin et al., 1989</xref>). In contrast to wild-type cells where vacuole acidity was reduced in more than 80% of cells in the third and subsequent mother cell divisions (<xref ref-type="fig" rid="fig3">Figure 3B</xref>), <italic>pma1-105</italic> cells retained high vacuole acidity after 3 divisions and up to at least 18 divisions (84% and 79% respectively, <xref ref-type="fig" rid="fig3">Figure 3B</xref>). These results suggest that Pma1 activity antagonizes vacuole acidification and, combined with the expression pattern of Pma1, support the idea that increased Pma1 in aged mother cells causes the reduction of vacuole acidity.<fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.03504.006</object-id><label>Figure 3.</label><caption><title>Pma1 antagonizes vacuole acidity and its absence facilitates regeneration of vacuole acidity in buds.</title><p>(<bold>A</bold>) <italic>PMA1</italic> was overexpressed in newborn daughter cells expressing Vph1-mCherry using a β-estradiol inducible system where a GAL4-Estrogen binding domain-VP16 (GEV) fusion protein drives <italic>GAL1</italic> promoter expression of an extra copy of <italic>PMA1</italic>. (n ≥ 30 cells per condition). (<bold>B</bold>) Wild-type and <italic>pma1-105</italic> cells expressing Vph1-mCherry were aged and quinacrine stained (n ≥ 30 cells per timepoint). White arrowheads indicate mother cell vacuoles with reduced acidity. Orange arrowheads indicate acidic mother-cell vacuoles. (<bold>C</bold>) Replicative lifespan of wild-type, <italic>pma1-105, vma2</italic>, and <italic>vma2 pma1-105</italic> cells by micromanipulation. Median lifespan is indicated. For the difference between wild-type and <italic>pma1-105,</italic> p < 0.0001, one-tailed logrank test. (n = 114 cells for <italic>PMA1</italic>, n = 119 for <italic>pma1-105</italic>, n = 36 for <italic>vma2</italic>, and n = 39 for <italic>vma2 pma1-105</italic>). (<bold>D</bold>) <italic>PMA1-mCherry</italic> was overexpressed in cells undergoing their first division that expressed endogenous Pma1-mCherry and that were treated with β-estradiol and then with β-estradiol plus nocodazole (Noc). (<bold>E</bold>) As in <bold>D</bold>, cells that expressed Vph1-mCherry were induced to overexpress <italic>PMA1</italic> and were quinacrine stained. (n ≥ 30 cells per condition). Arrowheads indicate the vacuoles of interest.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.006">http://dx.doi.org/10.7554/eLife.03504.006</ext-link></p></caption><graphic xlink:href="elife03504f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03504.007</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Overexpression increases Pma1 levels at the plasma membrane.</title><p><italic>PMA1-mCherry</italic> was overexpressed in newborn daughter cells using a β-estradiol inducible system where a GAL4-Estrogen binding domain-VP16 (GEV) fusion protein drives <italic>GAL1</italic> promoter expression of an extra copy of <italic>PMA1-mCherry</italic> in cells that also expressed endogenous Pma1-mCherry.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.007">http://dx.doi.org/10.7554/eLife.03504.007</ext-link></p></caption><graphic xlink:href="elife03504fs002"/></fig></fig-group></p><p>We previously found that delaying the reduction of vacuole acidity during aging by increasing V-ATPase levels extends replicative lifespan (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>). Given the evidence presented above that Pma1 levels antagonize vacuolar acidity, we asked whether reduced Pma1 activity also affected lifespan. Indeed, the <italic>pma1-105</italic> allele increased median replicative lifespan by ∼30% (<xref ref-type="fig" rid="fig3">Figure 3C</xref>), comparable to well-characterized lifespan-extending mutations (<xref ref-type="bibr" rid="bib9">Delaney et al., 2011</xref>). The slope of the <italic>pma1-105</italic> lifespan curve is similar to the slope of the wild-type curve. This suggests that instead of influencing the rate of aging throughout lifespan, the <italic>pma1-105</italic> allele delays the onset of the normal aging process. To ascertain whether lifespan extension by the <italic>pma1-105</italic> allele occurred entirely via increased vacuolar acidity, we examined the lifespan of <italic>pma1-105</italic> cells that lacked V-ATPase function. Cells lacking the V-ATPase subunit Vma2 had a short median lifespan of 2 divisions, as previously reported (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>). The lifespan of cells that had reduced Pma1 activity and that were devoid of V-ATPase function (<italic>vma2</italic>Δ, <italic>pma1-105</italic>) was much shorter than wild-type lifespan (median 7 divisions), and more similar to cells lacking V-ATPase function. This suggests that most of the lifespan extension imparted by the <italic>pma1-105</italic> allele requires V-ATPase function, but that the mechanism of lifespan extension is not limited to increased vacuolar acidification. Taken together these results support the idea that high Pma1 levels on mother cells impair vacuole acidification and limit lifespan.</p><p>In addition to Pma1 antagonizing mother cell vacuole acidity with age, we also hypothesized that the inherent asymmetry of Pma1, and thus low levels on buds, allows for re-acidification of the vacuole in buds. To test this idea, we asked whether expressing Pma1 in buds reduced vacuole acidity. We induced overexpression of <italic>PMA1-mCherry</italic> in cells arrested prior to cytokinesis with nocodazole and in untreated cells (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). In untreated cells, overexpression increased mother cell Pma1 levels but maintained mother-bud asymmetry. However, in nocodazole-arrested cells, <italic>PMA1-mCherry</italic> became equivalently high in mother cells and buds. At least 80% of buds had acidic vacuoles without <italic>PMA1</italic> induction or when <italic>PMA1</italic> was induced in the absence of nocodazole (<xref ref-type="fig" rid="fig3">Figure 3E</xref>). In contrast, only 13% of buds had acidic vacuoles when Pma1 levels were high in buds. Because increased Pma1 levels in buds impaired re-acidification of the vacuole, we conclude that the inherent asymmetry of Pma1 is required for regeneration of vacuole acidity prior to cytokinesis. We speculate that regeneration of vacuole acidity is required for daughter cell rejuvenation and that if high levels of Pma1 were induced in the buds of aging mother cells, daughter cells would not rejuvenate.</p><p>We wondered how Pma1 antagonizes vacuole acidity and how low Pma1 levels in buds permit vacuole reacidification. Given that Pma1 pumps protons out of the cell, we hypothesized that increased Pma1 activity antagonizes vacuole acidity by reducing cytosolic protons available to the V-ATPase. A prediction of this hypothesis is that cytosolic pH may become more basic with age, and may differ between mother and daughter cells. To test this hypothesis, we examined cytosolic pH with ratiometric pHluorin (a pH-sensitive GFP) (<xref ref-type="bibr" rid="bib31">Miesenbock et al., 1998</xref>) fused to a plasma membrane targeting sequence (residues 1–28 of the Psr1 protein) (<xref ref-type="bibr" rid="bib46">Siniossoglou et al., 2000</xref>). With this reagent, cytosolic pH at the cell cortex was visualized (<xref ref-type="fig" rid="fig4">Figure 4A</xref>) and quantified in mother cells of varying ages and in their buds. Newborn daughter cells or mother cells that had undergone 1 or 2 divisions had a mean cytosolic pH of ∼7.1, similar to previous measurements of bulk log phase cultures (<xref ref-type="bibr" rid="bib36">Orij et al., 2009</xref>) (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). However, as mother cells aged, cortical cytosolic pH increased as much as ∼0.5 pH units (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Moreover, when we examined mother cells (on average 3 or 18 divisions old) and their attached buds, cortical pH was ∼0.2 or ∼0.1 pH units lower in buds than mother cells (<xref ref-type="fig" rid="fig4">Figure 4C</xref>). All together, these results indicate that cortical cytosolic pH increases during replicative aging and is asymmetric between mother and daughter cells. Mother-daughter asymmetry of cytosolic pH might be surprising given the rapid diffusion of protons (<xref ref-type="bibr" rid="bib52">Wraight, 2006</xref>). However, local cytosolic pH differences have been observed in tumor cell invadopodia (<xref ref-type="bibr" rid="bib27">Magalhaes et al., 2011</xref>) and cytosolic pH gradients can form during polarized growth (<xref ref-type="bibr" rid="bib13">Feijo et al., 1999</xref>; <xref ref-type="bibr" rid="bib16">Gibbon and Kropf, 1994</xref>).<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.03504.008</object-id><label>Figure 4.</label><caption><title>Cortex-proximal cytosolic pH increases with age and is asymmetric between mother cells and buds.</title><p>(<bold>A</bold>) Visualization of cortical pH of newborn and aged mother cells and their buds as indicated with a plasma membrane-anchored ratiometric pHluorin using its bimodal excitation spectrum. Age is indicated by wheatgerm agglutinin-Alexa 594 staining of budscars, which also detects birth scars on newborn cells. (<bold>B</bold>) As in <bold>A</bold>, measurement of cortical pH of newborn and aged mother cells was made at the plasma membrane. n ≥13 cells per timepoint. Mean cortical pH is significantly increased in mother cells undergoing their third division and thereafter compared to newborn cells (p ≤ 0.014, one-tailed unpaired <italic>t</italic> test). Error bars represent SEM. (<bold>C</bold>) Difference of the cortical pH of mother cells and their buds. Bud pH was lower (more acidic) than mother cell pH (p = 0.003, n = 17 cells at 3 divisions and p = 0.04, n = 16 cells at 18 divisions, one-tailed paired <italic>t</italic> tests) and was subtracted from mother cell pH. (<bold>D</bold>) Model of the effect of Pma1 asymmetry and increased Pma1 levels during aging on the magnitude of proton translocation out of the cytosol and into the vacuole.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.008">http://dx.doi.org/10.7554/eLife.03504.008</ext-link></p></caption><graphic xlink:href="elife03504f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.03504.009</object-id><label>Figure 4—figure supplement 1.</label><caption><title>Pma1 asymmetry mediates cortical cytosolic pH asymmetry.</title><p>(<bold>A</bold>) <italic>PMA1</italic> was overexpressed as in <xref ref-type="fig" rid="fig3">Figure 3</xref> and cortical pH measured in mother cells undergoing their first division and their buds as in <xref ref-type="fig" rid="fig4">Figure 4</xref>. Cells were treated with β-estradiol for 2 hr to induce <italic>PMA1</italic> and with Nocodazole (Noc) and β-estradiol for an additional 1.5 hr. For all mother-bud pairs, bud pH was significantly more acidic than mother pH (p < 0.05, one-tailed paired <italic>t</italic> test, n ≥ 14 cell per condition), except when Pma1 levels in buds were high due to the combination of <italic>PMA1</italic> overexpression and nocodazole treatment. The difference between mother and bud pH was significantly greater when Pma1 was asymmetric (<italic>PMA1</italic> induced without nocodazole) than when Pma1 was symmetric (<italic>PMA1</italic> induced plus nocodazole) (p < 0.0001, one-tailed unpaired <italic>t</italic> test). Mean cortical pH is indicated, error bars represent SEM. (<bold>B</bold>) Cortical pH of wild-type and <italic>pma1-105</italic> mother cells in their first cell division (p < 0.0005, <italic>t</italic> test, n = 12 cells for <italic>PMA1</italic> and n = 13 for <italic>pma1-105</italic>).</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.009">http://dx.doi.org/10.7554/eLife.03504.009</ext-link></p></caption><graphic xlink:href="elife03504fs003"/></fig></fig-group></p><p>To test whether Pma1 asymmetry was required for cortical cytosolic pH asymmetry, we modulated Pma1 levels and activity in mother cells and buds and monitored cytosolic pH. We overexpressed <italic>PMA1</italic>, which increased mother cell levels but maintained mother-bud asymmetry (as in <xref ref-type="fig" rid="fig3">Figure 3D</xref>), and we overexpressed <italic>PMA1</italic> in nocodazole treated cells to generate equivalent high levels of Pma1 in mother cells and buds. Overexpression of <italic>PMA1</italic> in mother cells alone elevated mother cell cytosolic pH by ∼0.7 pH units and increased the mother-bud difference in cytosolic pH by ∼0.5 pH units (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>). However, when Pma1 levels in buds were elevated to mother cell levels, bud pH increased, abrogating cytosolic pH asymmetry. Moreover, decreasing Pma1 activity with the <italic>pma1-105</italic> allele decreased cortical cytosolic pH by ∼0.7 pH units (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>). Taken together our results suggest that the inherent asymmetry of Pma1 creates mother-daughter cytosolic pH asymmetry prior to cytokinesis.</p><p>Our findings support the idea that Pma1 activity antagonizes vacuole acidification via a competition with the V-ATPase for limited cytosolic protons (at pH 7 there are ∼3000 free protons per yeast cell, ∼10<sup>6</sup> Pma1 molecules and ∼10<sup>5</sup> V-ATPase V<sub>0</sub> subunits) (<xref ref-type="bibr" rid="bib37">Orij et al., 2011</xref>; <xref ref-type="bibr" rid="bib20">Huh et al., 2003</xref>). While other modes of regulation may also be involved, our results can be explained by high Pma1 activity in aged mother cells translocating a sufficient number of protons out of the cytosol to restrict proton availability for the V-ATPase and reduce vacuole acidity (<xref ref-type="fig" rid="fig4">Figure 4D</xref>). Conversely, lower Pma1 activity in buds leads to more cytosolic protons and higher vacuole acidification.</p><p>Our findings identify increased cytosolic pH as an early contributing step to the aging process in budding yeast and suggest that cytosolic pH asymmetry facilitates daughter cell rejuvenation. Interestingly, this same discontinuity of cytosolic pH in plant and algae cells (<xref ref-type="bibr" rid="bib13">Feijo et al., 1999</xref>; <xref ref-type="bibr" rid="bib16">Gibbon and Kropf, 1994</xref>) and of Pma1 orthologs in fission yeast and pollen tubes (<xref ref-type="bibr" rid="bib32">Minc and Chang, 2010</xref>; <xref ref-type="bibr" rid="bib6">Certal et al., 2008</xref>) is conserved during polarized cell growth. We speculate that non-uniform cytosolic pH is generally important in polarized growth and has been co-opted in cell types that undergo asymmetric divisions to regenerate full cellular capacity.</p></sec><sec id="s2" sec-type="materials|methods"><title>Materials and methods</title><sec id="s2-1"><title>Strains</title><p>Yeast strains are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>. All strains are derivatives of <italic>Saccharomyces cerevisiae</italic> S288c (BY) (<xref ref-type="bibr" rid="bib4">Brachmann et al., 1998</xref>). One-step PCR-mediated gene replacement and epitope tagging were performed using standard techniques, with template plasmids pRS306, pRS400, pKT127 and pKTmCherryKanMX (<xref ref-type="bibr" rid="bib43">Sheff and Thorn, 2004</xref>; <xref ref-type="bibr" rid="bib44">Sikorski and Hieter, 1989</xref>). Oligonucleotides for gene replacement, tagging, and cloning are listed in <xref ref-type="supplementary-material" rid="SD1-data">Supplementary file 1</xref>.</p><p>Strains expressing Vma2–GFP were derived from the yeast GFP collection (<xref ref-type="bibr" rid="bib20">Huh et al., 2003</xref>). The Gal4-Estrogen binding domain-VP16 (GEV) fusion protein (<xref ref-type="bibr" rid="bib49">Veatch et al., 2009</xref>) was integrated into the <italic>leu2Δ0</italic> allele by transforming PmeI-linearized pAGL plasmid. Strains expressing <italic>PMA1</italic> or <italic>PMA1-mCherry</italic> from a <italic>GAL</italic> promoter were constructed by transformation of GEV yeast strains with NotI-digested pAG306-GAL-PMA1 chr1 or pAG306-GAL-PMA1-mCherry chr1, which integrated them into an empty region of chromosome 1 (199456–199457) (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>).</p><p>The <italic>pma1-105</italic> mutant was derived from the heterozygous yeast deletion collection strain, <italic>pma1Δ::KanMX4/PMA1</italic> (<xref ref-type="bibr" rid="bib51">Winzeler et al., 1999</xref>). The strain was transformed with a linear fragment (derived from NotI and SacII digestion of pma1-105-URA3 plasmids) containing <italic>pma1-105</italic> marked with URA3. We chose transformants that replaced the <italic>pma1Δ::KanMX4</italic> allele similar to a previously described strategy (<xref ref-type="bibr" rid="bib17">Harris et al., 1994</xref>). This heterozygote was sporulated to obtain <italic>pma1-105</italic> haploids, which were sequenced to verify the mutation. The <italic>PMA1 URA3</italic> variant was created from the <italic>pma1Δ::KanMX4/PMA1</italic> strain; the <italic>ura3Δ0</italic> mutation was converted to <italic>URA3</italic> via PCR amplification of <italic>URA3</italic> from pRS306 and transformation. This resulting diploid strain was sporulated to obtain a <italic>PMA1 URA3</italic> haploid. All <italic>PMA1</italic> or <italic>pma1-105</italic> strains that expressed other markers were created by backcrossing to these original haploids.</p><p>Strains carrying the cortical pHluorin were constructed by amplifying plasmid pADH1pr-PSR1-RMP with primers (URA3-tTA-intChr1F and R) that allowed insertion into a second empty region of chromosome 1 (17068–17161).</p></sec><sec id="s2-2"><title>Plasmids</title><p>pKTmCherryKanMX (a kind gift from W Shou) was obtained by digestion of pKT127 (<xref ref-type="bibr" rid="bib43">Sheff and Thorn, 2004</xref>) with PacI and BglII and insertion of the mCherry containing fragment from similarly digested pBS34. pBS34 was obtained from the Yeast Resource Center at the University of Washington with permission from R. Tsien (<xref ref-type="bibr" rid="bib42">Shaner et al., 2004</xref>).</p><p>The GEV plasmid was previously described (<xref ref-type="bibr" rid="bib49">Veatch et al., 2009</xref>). pAG306-GAL-PMA1chr1, was generated in two steps. First, we created pAG306-GAL-ccdBchr1, a plasmid for gene expression from the <italic>GAL</italic> promoter that can be integrated into chromosome 1 (199456–199457) after NotI digestion. We generated pAG306-GAL-ccdBchr1 by ligation of a SmaI-digested fusion PCR product that contained two ∼500-base-pair regions of chromosome 1 flanking a NotI site into AatII-digested pAG306-GAL-ccdB (Addgene plasmid 14139) (<xref ref-type="bibr" rid="bib2">Alberti et al., 2007</xref>). We generated the fusion PCR product using oligonucleotides ChrI PartB SmaI F and ChrI PartA SmaI R to amplify two templates generated by PCR of yeast genomic DNA using oligonucleotide pairs ChrI PartA NotI F and ChrI PartA SmaI R, and ChrI PartB SmaI F and ChrI PartB NotI R, respectively. Second, we inserted <italic>PMA1</italic> into pAG306-GAL-ccdBchr1 from donor Gateway plasmid pDONR221-PMA1 (Harvard Institute of Proteomics [HIP] accession ScCD00008895) (<xref ref-type="bibr" rid="bib18">Hu et al., 2007</xref>), using LR Clonase according to the manufacturer's instructions (Invitrogen, Carlsbad, CA).</p><p>pAG306-GAL-PMA1-mCherryChr1 was generated by Gibson Assembly according to the manufacturer's instructions (New England Biolabs, Ipswich, MA). First <italic>PMA1-mCherry</italic> was amplified from genomic DNA from strain UCC9645 using primers GibRXNpAGdest_Pma1ChryF and GibRXNpAGdest_Pma1ChryR and assembled with a PCR product amplified from pAG306-GAL-ccdBChr1 with primers GibsonRXNpAGdstnF-2 and R-2.</p><p>pADH1pr-PSR1-RMP was derived from pADH1pr-RMP using Quikchange Site-Directed Mutagenesis (Stratagene, La Jolla, CA) to insert the first 28 amino acids of <italic>PSR1</italic> between the <italic>ADH1</italic> promoter and the N-terminus of RMP (ratiometric pHluorin) using primers PSR1-28-RMpHluorinF and R. pADH1pr-RMP was generated in four steps. First, pKT127-RMP was created by removing GFP from pKT127 by restriction digestion with PacI-AscI and replacing it with similarly digested RMP generated by PCR of template plasmid pGM1 (<xref ref-type="bibr" rid="bib31">Miesenbock et al., 1998</xref>) using oligonucleotides SEP PacIF and SEP AscIR. pADH1pr-RMP was created when RMP and the ADH1 terminator were amplified with primers UPGFP/pHluorin F and R from pKT127-RMP, digested with EcoRI-EagI, and ligated into similarly digested backbone of COX4-dsREDURA3int. COX4-dsREDURA3int was created by ligating the XhoI-NotI fragment of pHS12 (<xref ref-type="bibr" rid="bib3">Bevis and Glick, 2002</xref>) containing the ADH1 promoter and COX4 mitochondrial presequence fused to dsRED.T4 into similarly digested pRG919. pRG919 was created when a SacI-SacII PCR fragment containing a <italic>URA3</italic> targeting construct was inserted between the SacI-SacII sites in pRS406 (<xref ref-type="bibr" rid="bib8">Christianson et al., 1992</xref>).</p><p>The PMA1-URA plasmid was created in two steps. First, the <italic>URA3</italic> gene was inserted downstream of the previously characterized <italic>PMA1</italic> transcriptional termination sites (<xref ref-type="bibr" rid="bib34">Nagalakshmi et al., 2008</xref>; <xref ref-type="bibr" rid="bib53">Yassour et al., 2009</xref>) on chrVII (479252–479253) with primers MRKdownPma1F and MRKdownPma1R to create UCC9656. Genomic DNA from this strain was amplified with primers Pma1_1kbupNotIF and Pma1_1kbdownSacIIR to acquire a fragment containing the entire <italic>PMA1</italic> locus plus 1 kb upstream and 1 kb downstream and <italic>URA3</italic>. This fragment was digested with NotI and SacII and ligated into similarly digested pBluescript SK+ (Stratagene). The pma1-105-URA plasmid was generated by Quikchange Site-Directed Mutagenesis (Stratagene) of the PMA1-URA plasmid using primers pma1-S368FF and pma1-S368FR.</p></sec><sec id="s2-3"><title>Media and cell culture</title><p>Cells were cultured in YEPD (1% yeast extract, 2% peptone, 2% glucose) and maintained in exponential growth for 15 hr to a maximum density of 5 × 10<sup>6</sup> cells ml<sup>−1</sup> before initiating experiments. Where indicated, cells were treated with nocodazole (Sigma, St. Louis, MO) at 10 μg ml<sup>−1</sup> for 1.5 hr or with 5 μM β-estradiol (Sigma) for 2 hr to induce <italic>PMA1</italic> or <italic>PMA1-mCherry</italic> overexpression.</p></sec><sec id="s2-4"><title>Culturing and purification of aged MEP cells</title><p>Cells were cultured, biotin labeled, aged, and purified for quinacrine staining as previously described (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>). For cortical pH analysis of aged cells, cell labeling and purification were performed as described (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>) except incubation with streptavidin-coated magnetic beads (MicroMACS, Miltenyi Biotec, Bergisch Gladbach, Germany) and purification took place in YEPD depleted of biotin. This was achieved by overnight incubation at 4°C of 45 ml YEP with 300 μl Avidin-Agarose beads (Sigma). Glucose was added to 2%. Cells were recovered for 1 hr in YEPD prior to imaging.</p></sec><sec id="s2-5"><title>Quinacrine staining, indirect immunofluorescence and fluorescent microscopy</title><p>Pma1 was detected by indirect immunofluorescence as described (<xref ref-type="bibr" rid="bib5">Burke et al., 2000</xref>) using the 40B7 monoclonal antibody (Abcam, Cambridge, England) followed by Alexa Fluor 488-conjugated goat anti-mouse secondary (Invitrogen).</p><p>Quinacrine (Sigma) staining was performed as previously described (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>). In most experiments, age was determined by calcofluor (Sigma) staining of bud scars by including 5 μg ml<sup>−1</sup> calcofluor in the last wash step before imaging. Calcoflour staining reveals bud scars (<xref ref-type="bibr" rid="bib40">Pringle, 1991</xref>) and facilitates identification of newborn cells and the replicative age of mother cells. Calcofluor also stains the mother cell birth scar (<xref ref-type="bibr" rid="bib40">Pringle, 1991</xref>) and allowed identification of the new mother cell during nocodazole arrest when mother cells and buds are similar in size.</p><p>Cells were imaged under ×60 oil magnification using a Nikon Eclipse E800 (Nikon, Tokyo, Japan) with the appropriate filter set: UV-2E/C DAPI for calcofluor; FITC-HYQ for quinacrine, GFP and Alexa Fluor 488; and G-2E/C TRITC for mCherry. Images were acquired with a CoolSNAP HQ<sup>2</sup> CCD camera (Photometrics, Tucson, AZ) and Metamorph version 7.1.1.0 imaging software (Molecular Devices, Sunnyvale, CA).</p></sec><sec id="s2-6"><title>Lifespan measurement by micromanipulation</title><p>Replicative lifespan was measured by micromanipulation as previously described (<xref ref-type="bibr" rid="bib19">Hughes and Gottschling, 2012</xref>).</p></sec><sec id="s2-7"><title>Single-cell analysis of cortical pH of mother cells and buds</title><p>Cells were cultured in YEPD, but transferred to low fluorescence medium (<xref ref-type="bibr" rid="bib36">Orij et al., 2009</xref>) for pH measurement after rinsing them in an equal volume of low fluorescence medium. Calibration curves were created as previously described (<xref ref-type="bibr" rid="bib36">Orij et al., 2009</xref>) except that that cells were permeabilized prior to pH equilibration by treatment in 5 μg ml<sup>−1</sup> digitonin (Sigma) in 1× PBS for 5 min. To quantify replicative age, 1 × 10<sup>7</sup> cells were stained in YEPD for 5 min with 10 μg ml<sup>−1</sup> Wheat Germ Agglutinin-Alexa fluor 594 conjugate (Molecular Probes, Eugene, OR), washed once with an equal volume of YEPD, once with low fluorescence medium, and transferred to low fluorescence medium for 20 min prior to imaging at a density of 1 × 10<sup>7</sup> cells ml<sup>−1</sup>.</p><p>To quantify cortical pH of live single cells and to generate pH calibration curves, cells were imaged with a Leica DMI6000 B under ×63 oil magnification. Images were acquired with a Leica DFC365 FX camera and Leica Application Suite Advanced Fluorescence software (Leica, Wetzlar, Germany). TRITC excitation and emission filters (Ex525/25, Em605/52) were used to image bud scars and the combination of FITC Excitation/FITC Emission (Ex490/20, Em525/36) and DAPI Excitation/FITC Emission (Ex402/15, Em525/36) filters were used to derive cortical pH using the bimodal excitation spectrum of ratiometric pHluorin (<xref ref-type="bibr" rid="bib31">Miesenbock et al., 1998</xref>) to calculate 402/490-nm excitation ratios. Image analysis was performed using ImageJ (Version 1.47m, NIH) to quantify mean pHluorin intensity from the identical regions of images acquired at 402 and 490 nm excitation with a 2 pixel-wide freehand line tool traced along the majority of the length of the mother cell or attached bud plasma membrane. Local background was calculated from a cell-free region one cell diameter away and subtracted from all membrane intensity measurements prior to calculation of 402/490-nm excitation ratios that were fitted to calibration curves to derive cortical pH. Statistical analyses were performed using GraphPad Prism version 4.0a software (GraphPad, La Jolla, CA).</p><p>We and others (<xref ref-type="bibr" rid="bib39">Pineda Rodo et al., 2012</xref>) note that repeated imaging differentially affected signal intensity captured at 402 and 490 nm excitation wavelengths, which altered the excitation ratios of sequential images in a pH-independent manner. Therefore we captured a single set of images per cell and never exposed cells to excitation wavelengths prior to pHluorin imaging.</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>We thank M Patel, J Roberts, and members of the Gottschling laboratory for reviewing the manuscript; A Merz for insightful discussions; G Smits, and R Orij for technical advice; G Miesenbock, J McCusker, and W Shou for reagents; and J Hsu, Z Tan, N Thayer, A Waite, and N Yazvenko for technical assistance.</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>The 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>KAH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>ALH, Drafting or revising the article, Contributed unpublished essential data or reagents</p></fn><fn fn-type="con" id="con3"><p>DEG, Conception and design, Analysis and interpretation of data, Drafting or revising the article</p></fn></fn-group></sec><sec sec-type="supplementary-material"><title>Additional files</title><supplementary-material id="SD1-data"><object-id pub-id-type="doi">10.7554/eLife.03504.010</object-id><label>Supplementary file 1.</label><caption><p>(<bold>A</bold>) Yeast Strains. (<bold>B</bold>) Oligonucleotides.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.03504.010">http://dx.doi.org/10.7554/eLife.03504.010</ext-link></p></caption><media mime-subtype="docx" mimetype="application" xlink:href="elife03504s001.docx"/></supplementary-material></sec><ref-list><title>References</title><ref id="bib1"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Aguilaniu</surname><given-names>H</given-names></name><name><surname>Gustafsson</surname><given-names>L</given-names></name><name><surname>Rigoulet</surname><given-names>M</given-names></name><name><surname>Nystrom</surname><given-names>T</given-names></name></person-group><year>2003</year><article-title>Asymmetric inheritance of oxidatively damaged proteins during cytokinesis</article-title><source>Science</source><volume>299</volume><fpage>1751</fpage><lpage>1753</lpage><pub-id pub-id-type="doi">10.1126/science.1080418</pub-id></element-citation></ref><ref id="bib2"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Alberti</surname><given-names>S</given-names></name><name><surname>Gitler</surname><given-names>AD</given-names></name><name><surname>Lindquist</surname><given-names>S</given-names></name></person-group><year>2007</year><article-title>A suite of Gateway cloning vectors for high-throughput genetic analysis in <italic>Saccharomyces cerevisiae</italic></article-title><source>Yeast</source><volume>24</volume><fpage>913</fpage><lpage>919</lpage><pub-id pub-id-type="doi">10.1002/yea.1502</pub-id></element-citation></ref><ref id="bib3"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Bevis</surname><given-names>BJ</given-names></name><name><surname>Glick</surname><given-names>BS</given-names></name></person-group><year>2002</year><article-title>Rapidly maturing variants of the <italic>Discosoma</italic> red fluorescent protein (DsRed)</article-title><source>Nature Biotechnology</source><volume>20</volume><fpage>83</fpage><lpage>87</lpage><pub-id pub-id-type="doi">10.1038/nbt0102-83</pub-id></element-citation></ref><ref id="bib4"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Brachmann</surname><given-names>CB</given-names></name><name><surname>Davies</surname><given-names>A</given-names></name><name><surname>Cost</surname><given-names>GJ</given-names></name><name><surname>Caputo</surname><given-names>E</given-names></name><name><surname>Li</surname><given-names>J</given-names></name><name><surname>Hieter</surname><given-names>P</given-names></name><name><surname>Boeke</surname><given-names>JD</given-names></name></person-group><year>1998</year><article-title>Designer deletion strains derived from <italic>Saccharomyces cerevisiae</italic> S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications</article-title><source>Yeast</source><volume>14</volume><fpage>115</fpage><lpage>132</lpage><pub-id pub-id-type="doi">10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2</pub-id></element-citation></ref><ref id="bib5"><element-citation publication-type="book"><person-group person-group-type="author"><name><surname>Burke</surname><given-names>D</given-names></name><name><surname>Dawson</surname><given-names>D</given-names></name><name><surname>Stearns</surname><given-names>T</given-names></name></person-group><year>2000</year><source>Methods in yeast genetics. A Cold Spring harbor laboratory Course Manual</source><publisher-name>CSHL Press</publisher-name></element-citation></ref><ref id="bib6"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Certal</surname><given-names>AC</given-names></name><name><surname>Almeida</surname><given-names>RB</given-names></name><name><surname>Carvalho</surname><given-names>LM</given-names></name><name><surname>Wong</surname><given-names>E</given-names></name><name><surname>Moreno</surname><given-names>N</given-names></name><name><surname>Michard</surname><given-names>E</given-names></name><name><surname>Carneiro</surname><given-names>J</given-names></name><name><surname>Rodriguéz-Léon</surname><given-names>J</given-names></name><name><surname>Wu</surname><given-names>HM</given-names></name><name><surname>Cheung</surname><given-names>AY</given-names></name><name><surname>Feijó</surname><given-names>JA</given-names></name></person-group><year>2008</year><article-title>Exclusion of a proton ATPase from the apical membrane is associated with cell polarity and tip growth in <italic>Nicotiana tabacum</italic> pollen tubes</article-title><source>The Plant Cell</source><volume>20</volume><fpage>614</fpage><lpage>634</lpage><pub-id pub-id-type="doi">10.1105/tpc.106.047423</pub-id></element-citation></ref><ref id="bib7"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Chang</surname><given-names>A</given-names></name><name><surname>Fink</surname><given-names>GR</given-names></name></person-group><year>1995</year><article-title>Targeting of the yeast plasma membrane [H+]ATPase: a novel gene AST1 prevents mislocalization of mutant ATPase to the vacuole</article-title><source>The Journal of Cell Biology</source><volume>128</volume><fpage>39</fpage><lpage>49</lpage><pub-id pub-id-type="doi">10.1083/jcb.128.1.39</pub-id></element-citation></ref><ref id="bib8"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Christianson</surname><given-names>TW</given-names></name><name><surname>Sikorski</surname><given-names>RS</given-names></name><name><surname>Dante</surname><given-names>M</given-names></name><name><surname>Shero</surname><given-names>JH</given-names></name><name><surname>Hieter</surname><given-names>P</given-names></name></person-group><year>1992</year><article-title>Multifunctional yeast high-copy-number shuttle vectors</article-title><source>Gene</source><volume>110</volume><fpage>119</fpage><lpage>122</lpage><pub-id pub-id-type="doi">10.1016/0378-1119(92)90454-W</pub-id></element-citation></ref><ref id="bib9"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Delaney</surname><given-names>JR</given-names></name><name><surname>Murakami</surname><given-names>CJ</given-names></name><name><surname>Olsen</surname><given-names>B</given-names></name><name><surname>Kennedy</surname><given-names>BK</given-names></name><name><surname>Kaeberlein</surname><given-names>M</given-names></name></person-group><year>2011</year><article-title>Quantitative evidence for early life fitness defects from 32 longevity-associated alleles in yeast</article-title><source>Cell Cycle</source><volume>10</volume><fpage>156</fpage><lpage>165</lpage><pub-id pub-id-type="doi">10.4161/cc.10.1.14457</pub-id></element-citation></ref><ref id="bib10"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Egilmez</surname><given-names>NK</given-names></name><name><surname>Jazwinski</surname><given-names>SM</given-names></name></person-group><year>1989</year><article-title>Evidence for the involvement of a cytoplasmic factor in the aging of the yeast <italic>Saccharomyces cerevisiae</italic></article-title><source>Journal of Bacteriology</source><volume>171</volume><fpage>37</fpage><lpage>42</lpage></element-citation></ref><ref id="bib11"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Eldakak</surname><given-names>A</given-names></name><name><surname>Rancati</surname><given-names>G</given-names></name><name><surname>Rubinstein</surname><given-names>B</given-names></name><name><surname>Paul</surname><given-names>P</given-names></name><name><surname>Conaway</surname><given-names>V</given-names></name><name><surname>Li</surname><given-names>R</given-names></name></person-group><year>2010</year><article-title>Asymmetrically inherited multidrug resistance transporters are recessive determinants in cellular replicative ageing</article-title><source>Nature Cell Biology</source><volume>12</volume><fpage>799</fpage><lpage>805</lpage><pub-id pub-id-type="doi">10.1038/ncb2085</pub-id></element-citation></ref><ref id="bib12"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Erjavec</surname><given-names>N</given-names></name><name><surname>Larsson</surname><given-names>L</given-names></name><name><surname>Grantham</surname><given-names>J</given-names></name><name><surname>Nystrom</surname><given-names>T</given-names></name></person-group><year>2007</year><article-title>Accelerated aging and failure to segregate damaged proteins in Sir2 mutants can be suppressed by overproducing the protein aggregation-remodeling factor Hsp104p</article-title><source>Genes & Development</source><volume>21</volume><fpage>2410</fpage><lpage>2421</lpage><pub-id pub-id-type="doi">10.1101/gad.439307</pub-id></element-citation></ref><ref id="bib13"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Feijo</surname><given-names>JA</given-names></name><name><surname>Sainhas</surname><given-names>J</given-names></name><name><surname>Hackett</surname><given-names>GR</given-names></name><name><surname>Kunkel</surname><given-names>JG</given-names></name><name><surname>Hepler</surname><given-names>PK</given-names></name></person-group><year>1999</year><article-title>Growing pollen tubes possess a constitutive alkaline band in the clear zone and a growth-dependent acidic tip</article-title><source>The Journal of Cell Biology</source><volume>144</volume><fpage>483</fpage><lpage>496</lpage><pub-id pub-id-type="doi">10.1083/jcb.144.3.483</pub-id></element-citation></ref><ref id="bib14"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Ferreira</surname><given-names>T</given-names></name><name><surname>Mason</surname><given-names>AB</given-names></name><name><surname>Slayman</surname><given-names>CW</given-names></name></person-group><year>2001</year><article-title>The yeast Pma1 proton pump: a model for understanding the biogenesis of plasma membrane proteins</article-title><source>The Journal of Biological Chemistry</source><volume>276</volume><fpage>29613</fpage><lpage>29616</lpage><pub-id pub-id-type="doi">10.1074/jbc.R100022200</pub-id></element-citation></ref><ref id="bib15"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gao</surname><given-names>CY</given-names></name><name><surname>Pinkham</surname><given-names>JL</given-names></name></person-group><year>2000</year><article-title>Tightly regulated, beta-estradiol dose-dependent expression system for yeast</article-title><source>Biotechniques</source><volume>29</volume><fpage>1226</fpage><lpage>1231</lpage></element-citation></ref><ref id="bib16"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Gibbon</surname><given-names>BC</given-names></name><name><surname>Kropf</surname><given-names>DL</given-names></name></person-group><year>1994</year><article-title>Cytosolic pH gradients associated with tip growth</article-title><source>Science</source><volume>263</volume><fpage>1419</fpage><lpage>1421</lpage><pub-id pub-id-type="doi">10.1126/science.263.5152.1419</pub-id></element-citation></ref><ref id="bib17"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Harris</surname><given-names>SL</given-names></name><name><surname>Na</surname><given-names>S</given-names></name><name><surname>Zhu</surname><given-names>X</given-names></name><name><surname>Seto-Young</surname><given-names>D</given-names></name><name><surname>Perlin</surname><given-names>DS</given-names></name><name><surname>Teem</surname><given-names>JH</given-names></name><name><surname>Haber</surname><given-names>JE</given-names></name></person-group><year>1994</year><article-title>Dominant lethal mutations in the plasma membrane H(+)-ATPase gene of <italic>Saccharomyces cerevisiae</italic></article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>91</volume><fpage>10531</fpage><lpage>10535</lpage><pub-id pub-id-type="doi">10.1073/pnas.91.22.10531</pub-id></element-citation></ref><ref id="bib18"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hu</surname><given-names>Y</given-names></name><name><surname>Rolfs</surname><given-names>A</given-names></name><name><surname>Bhullar</surname><given-names>B</given-names></name><name><surname>Murthy</surname><given-names>TV</given-names></name><name><surname>Zhu</surname><given-names>C</given-names></name><name><surname>Berger</surname><given-names>MF</given-names></name><name><surname>Camargo</surname><given-names>AA</given-names></name><name><surname>Kelley</surname><given-names>F</given-names></name><name><surname>McCarron</surname><given-names>S</given-names></name><name><surname>Jepson</surname><given-names>D</given-names></name><name><surname>Richardson</surname><given-names>A</given-names></name><name><surname>Raphael</surname><given-names>J</given-names></name><name><surname>Moreira</surname><given-names>D</given-names></name><name><surname>Taycher</surname><given-names>E</given-names></name><name><surname>Zuo</surname><given-names>D</given-names></name><name><surname>Mohr</surname><given-names>S</given-names></name><name><surname>Kane</surname><given-names>MF</given-names></name><name><surname>Williamson</surname><given-names>J</given-names></name><name><surname>Simpson</surname><given-names>A</given-names></name><name><surname>Bulyk</surname><given-names>ML</given-names></name><name><surname>Harlow</surname><given-names>E</given-names></name><name><surname>Marsischky</surname><given-names>G</given-names></name><name><surname>Kolodner</surname><given-names>RD</given-names></name><name><surname>LaBaer</surname><given-names>J</given-names></name></person-group><year>2007</year><article-title>Approaching a complete repository of sequence-verified protein-encoding clones for <italic>Saccharomyces cerevisiae</italic></article-title><source>Genome Research</source><volume>17</volume><fpage>536</fpage><lpage>543</lpage><pub-id pub-id-type="doi">10.1101/gr.6037607</pub-id></element-citation></ref><ref id="bib19"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Hughes</surname><given-names>AL</given-names></name><name><surname>Gottschling</surname><given-names>DE</given-names></name></person-group><year>2012</year><article-title>An early age increase in vacuolar pH limits mitochondrial function and lifespan in yeast</article-title><source>Nature</source><volume>492</volume><fpage>261</fpage><lpage>265</lpage><pub-id pub-id-type="doi">10.1038/nature11654</pub-id></element-citation></ref><ref id="bib20"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Huh</surname><given-names>WK</given-names></name><name><surname>Falvo</surname><given-names>JV</given-names></name><name><surname>Gerke</surname><given-names>LC</given-names></name><name><surname>Carroll</surname><given-names>AS</given-names></name><name><surname>Howson</surname><given-names>RW</given-names></name><name><surname>Weissman</surname><given-names>JS</given-names></name><name><surname>O'Shea</surname><given-names>EK</given-names></name></person-group><year>2003</year><article-title>Global analysis of protein localization in budding yeast</article-title><source>Nature</source><volume>425</volume><fpage>686</fpage><lpage>691</lpage><pub-id pub-id-type="doi">10.1038/nature02026</pub-id></element-citation></ref><ref id="bib21"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Kennedy</surname><given-names>BK</given-names></name><name><surname>Austriaco</surname><given-names>NR</given-names><suffix>Jnr</suffix></name><name><surname>Guarente</surname><given-names>L</given-names></name></person-group><year>1994</year><article-title>Daughter cells of <italic>Saccharomyces cerevisiae</italic> from old mothers display a reduced life span</article-title><source>The Journal of Cell Biology</source><volume>127</volume><fpage>1985</fpage><lpage>1993</lpage><pub-id pub-id-type="doi">10.1083/jcb.127.6.1985</pub-id></element-citation></ref><ref id="bib22"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Khmelinskii</surname><given-names>A</given-names></name><name><surname>Keller</surname><given-names>PJ</given-names></name><name><surname>Bartosik</surname><given-names>A</given-names></name><name><surname>Meurer</surname><given-names>M</given-names></name><name><surname>Barry</surname><given-names>JD</given-names></name><name><surname>Mardin</surname><given-names>BR</given-names></name><name><surname>Kaufmann</surname><given-names>A</given-names></name><name><surname>Trautmann</surname><given-names>S</given-names></name><name><surname>Wachsmuth</surname><given-names>M</given-names></name><name><surname>Pereira</surname><given-names>G</given-names></name><name><surname>Huber</surname><given-names>W</given-names></name><name><surname>Schiebel</surname><given-names>E</given-names></name><name><surname>Knop</surname><given-names>M</given-names></name></person-group><year>2012</year><article-title>Tandem fluorescent protein timers for in vivo analysis of protein dynamics</article-title><source>Nature Biotechnology</source><volume>30</volume><fpage>708</fpage><lpage>714</lpage><pub-id pub-id-type="doi">10.1038/nbt.2281</pub-id></element-citation></ref><ref id="bib23"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lai</surname><given-names>CY</given-names></name><name><surname>Jaruga</surname><given-names>E</given-names></name><name><surname>Borghouts</surname><given-names>C</given-names></name><name><surname>Jazwinski</surname><given-names>SM</given-names></name></person-group><year>2002</year><article-title>A mutation in the ATP2 gene abrogates the age asymmetry between mother and daughter cells of the yeast <italic>Saccharomyces cerevisiae</italic></article-title><source>Genetics</source><volume>162</volume><fpage>73</fpage><lpage>87</lpage></element-citation></ref><ref id="bib24"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Li</surname><given-names>SC</given-names></name><name><surname>Kane</surname><given-names>PM</given-names></name></person-group><year>2009</year><article-title>The yeast lysosome-like vacuole: endpoint and crossroads</article-title><source>Biochimica et Biophysica Acta</source><volume>1793</volume><fpage>650</fpage><lpage>663</lpage><pub-id pub-id-type="doi">10.1016/j.bbamcr.2008.08.003</pub-id></element-citation></ref><ref id="bib25"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lindstrom</surname><given-names>DL</given-names></name><name><surname>Gottschling</surname><given-names>DE</given-names></name></person-group><year>2009</year><article-title>The mother enrichment program: a genetic system for facile replicative life span analysis in <italic>Saccharomyces cerevisiae</italic></article-title><source>Genetics</source><volume>183</volume><fpage>413</fpage><lpage>422</lpage><pub-id pub-id-type="doi">10.1534/genetics.109.106229</pub-id></element-citation></ref><ref id="bib26"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Lippincott</surname><given-names>J</given-names></name><name><surname>Shannon</surname><given-names>KB</given-names></name><name><surname>Shou</surname><given-names>W</given-names></name><name><surname>Deshaies</surname><given-names>RJ</given-names></name><name><surname>Li</surname><given-names>R</given-names></name></person-group><year>2001</year><article-title>The Tem1 small GTPase controls actomyosin and septin dynamics during cytokinesis</article-title><source>Journal of Cell Science</source><volume>114</volume><fpage>1379</fpage><lpage>1386</lpage></element-citation></ref><ref id="bib27"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Magalhaes</surname><given-names>MA</given-names></name><name><surname>Larson</surname><given-names>DR</given-names></name><name><surname>Mader</surname><given-names>CC</given-names></name><name><surname>Bravo-Cordero</surname><given-names>JJ</given-names></name><name><surname>Gil-Henn</surname><given-names>H</given-names></name><name><surname>Oser</surname><given-names>M</given-names></name><name><surname>Chen</surname><given-names>X</given-names></name><name><surname>Koleske</surname><given-names>AJ</given-names></name><name><surname>Condeelis</surname><given-names>J</given-names></name></person-group><year>2011</year><article-title>Cortactin phosphorylation regulates cell invasion through a pH-dependent pathway</article-title><source>The Journal of Cell Biology</source><volume>195</volume><fpage>903</fpage><lpage>920</lpage><pub-id pub-id-type="doi">10.1083/jcb.201103045</pub-id></element-citation></ref><ref id="bib28"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Malínská</surname><given-names>K</given-names></name><name><surname>Malínsky</surname><given-names>J</given-names></name><name><surname>Opekarová</surname><given-names>M</given-names></name><name><surname>Tanner</surname><given-names>W</given-names></name></person-group><year>2003</year><article-title>Visualization of protein compartments within the plasma membrane of living cells</article-title><source>Molecular Biology of the Cell</source><volume>14</volume><fpage>4427</fpage><lpage>4436</lpage><pub-id pub-id-type="doi">10.1091/mbc.E03-04-0221</pub-id></element-citation></ref><ref id="bib29"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McCusker</surname><given-names>JH</given-names></name><name><surname>Perlin</surname><given-names>DS</given-names></name><name><surname>Haber</surname><given-names>JE</given-names></name></person-group><year>1987</year><article-title>Pleiotropic plasma membrane ATPase mutations of <italic>Saccharomyces cerevisiae</italic></article-title><source>Molecular and Cellular Biology</source><volume>7</volume><fpage>4082</fpage><lpage>4088</lpage></element-citation></ref><ref id="bib30"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>McFaline-Figueroa</surname><given-names>JR</given-names></name><name><surname>Vevea</surname><given-names>J</given-names></name><name><surname>Swayne</surname><given-names>TC</given-names></name><name><surname>Zhou</surname><given-names>C</given-names></name><name><surname>Liu</surname><given-names>C</given-names></name><name><surname>Leung</surname><given-names>G</given-names></name><name><surname>Boldogh</surname><given-names>IR</given-names></name><name><surname>Pon</surname><given-names>LA</given-names></name></person-group><year>2011</year><article-title>Mitochondrial quality control during inheritance is associated with lifespan and mother-daughter age asymmetry in budding yeast</article-title><source>Aging Cell</source><volume>10</volume><fpage>885</fpage><lpage>895</lpage><pub-id pub-id-type="doi">10.1111/j.1474-9726.2011.00731.x</pub-id></element-citation></ref><ref id="bib31"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Miesenbock</surname><given-names>G</given-names></name><name><surname>De Angelis</surname><given-names>DA</given-names></name><name><surname>Rothman</surname><given-names>JE</given-names></name></person-group><year>1998</year><article-title>Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins</article-title><source>Nature</source><volume>394</volume><fpage>192</fpage><lpage>195</lpage><pub-id pub-id-type="doi">10.1038/28190</pub-id></element-citation></ref><ref id="bib32"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Minc</surname><given-names>N</given-names></name><name><surname>Chang</surname><given-names>F</given-names></name></person-group><year>2010</year><article-title>Electrical control of cell polarization in the fission yeast <italic>Schizosaccharomyces pombe</italic></article-title><source>Current Biology</source><volume>20</volume><fpage>710</fpage><lpage>716</lpage><pub-id pub-id-type="doi">10.1016/j.cub.2010.02.047</pub-id></element-citation></ref><ref id="bib33"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Mortimer</surname><given-names>RK</given-names></name><name><surname>Johnston</surname><given-names>JR</given-names></name></person-group><year>1959</year><article-title>Life span of individual yeast cells</article-title><source>Nature</source><volume>183</volume><fpage>1751</fpage><lpage>1752</lpage><pub-id pub-id-type="doi">10.1038/1831751a0</pub-id></element-citation></ref><ref id="bib34"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nagalakshmi</surname><given-names>U</given-names></name><name><surname>Wang</surname><given-names>Z</given-names></name><name><surname>Waern</surname><given-names>K</given-names></name><name><surname>Shou</surname><given-names>C</given-names></name><name><surname>Raha</surname><given-names>D</given-names></name><name><surname>Gerstein</surname><given-names>M</given-names></name><name><surname>Snyder</surname><given-names>M</given-names></name></person-group><year>2008</year><article-title>The transcriptional landscape of the yeast genome defined by RNA sequencing</article-title><source>Science</source><volume>320</volume><fpage>1344</fpage><lpage>1349</lpage><pub-id pub-id-type="doi">10.1126/science.1158441</pub-id></element-citation></ref><ref id="bib35"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Nystrom</surname><given-names>T</given-names></name><name><surname>Liu</surname><given-names>B</given-names></name></person-group><year>2014</year><article-title>The mystery of aging and rejuvenation-a budding topic</article-title><source>Current Opinion in Microbiology</source><volume>18</volume><fpage>61</fpage><lpage>67</lpage><pub-id pub-id-type="doi">10.1016/j.mib.2014.02.003</pub-id></element-citation></ref><ref id="bib37"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Orij</surname><given-names>R</given-names></name><name><surname>Brul</surname><given-names>S</given-names></name><name><surname>Smits</surname><given-names>GJ</given-names></name></person-group><year>2011</year><article-title>Intracellular pH is a tightly controlled signal in yeast</article-title><source>Biochimica et Biophysica Acta</source><volume>1810</volume><fpage>933</fpage><lpage>944</lpage><pub-id pub-id-type="doi">10.1016/j.bbagen.2011.03.011</pub-id></element-citation></ref><ref id="bib36"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Orij</surname><given-names>R</given-names></name><name><surname>Postmus</surname><given-names>J</given-names></name><name><surname>Ter Beek</surname><given-names>A</given-names></name><name><surname>Brul</surname><given-names>S</given-names></name><name><surname>Smits</surname><given-names>GJ</given-names></name></person-group><year>2009</year><article-title>In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in <italic>Saccharomyces cerevisiae</italic> reveals a relation between intracellular pH and growth</article-title><source>Microbiology</source><volume>155</volume><fpage>268</fpage><lpage>278</lpage><pub-id pub-id-type="doi">10.1099/mic.0.022038-0</pub-id></element-citation></ref><ref id="bib38"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Perlin</surname><given-names>DS</given-names></name><name><surname>Harris</surname><given-names>SL</given-names></name><name><surname>Seto-Young</surname><given-names>D</given-names></name><name><surname>Haber</surname><given-names>JE</given-names></name></person-group><year>1989</year><article-title>Defective H(+)-ATPase of hygromycin B-resistant pma1 mutants from <italic>Saccharomyces cerevisiae</italic></article-title><source>The Journal of Biological Chemistry</source><volume>264</volume><fpage>21857</fpage><lpage>21864</lpage></element-citation></ref><ref id="bib39"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pineda Rodo</surname><given-names>A</given-names></name><name><surname>Vachova</surname><given-names>L</given-names></name><name><surname>Palkova</surname><given-names>Z</given-names></name></person-group><year>2012</year><article-title>In vivo determination of organellar pH using a universal wavelength-based confocal microscopy approach</article-title><source>PLOS ONE</source><volume>7</volume><fpage>e33229</fpage><pub-id pub-id-type="doi">10.1371/journal.pone.0033229</pub-id></element-citation></ref><ref id="bib40"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Pringle</surname><given-names>JR</given-names></name></person-group><year>1991</year><article-title>Staining of bud scars and other cell wall chitin with calcofluor</article-title><source>Methods in Enzymology</source><volume>194</volume><fpage>732</fpage><lpage>735</lpage><pub-id pub-id-type="doi">10.1016/0076-6879(91)94055-H</pub-id></element-citation></ref><ref id="bib41"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Serrano</surname><given-names>R</given-names></name><name><surname>Kielland-Brandt</surname><given-names>MC</given-names></name><name><surname>Fink</surname><given-names>GR</given-names></name></person-group><year>1986</year><article-title>Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases</article-title><source>Nature</source><volume>319</volume><fpage>689</fpage><lpage>693</lpage><pub-id pub-id-type="doi">10.1038/319689a0</pub-id></element-citation></ref><ref id="bib42"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Shaner</surname><given-names>NC</given-names></name><name><surname>Campbell</surname><given-names>RE</given-names></name><name><surname>Steinbach</surname><given-names>PA</given-names></name><name><surname>Giepmans</surname><given-names>BN</given-names></name><name><surname>Palmer</surname><given-names>AE</given-names></name><name><surname>Tsien</surname><given-names>RY</given-names></name></person-group><year>2004</year><article-title>Improved monomeric red, orange and yellow fluorescent proteins derived from <italic>Discosoma sp.</italic> red fluorescent protein</article-title><source>Nature Biotechnology</source><volume>22</volume><fpage>1567</fpage><lpage>1572</lpage><pub-id pub-id-type="doi">10.1038/nbt1037</pub-id></element-citation></ref><ref id="bib43"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sheff</surname><given-names>MA</given-names></name><name><surname>Thorn</surname><given-names>KS</given-names></name></person-group><year>2004</year><article-title>Optimized cassettes for fluorescent protein tagging in <italic>Saccharomyces cerevisiae</italic></article-title><source>Yeast</source><volume>21</volume><fpage>661</fpage><lpage>670</lpage><pub-id pub-id-type="doi">10.1002/yea.1130</pub-id></element-citation></ref><ref id="bib44"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sikorski</surname><given-names>RS</given-names></name><name><surname>Hieter</surname><given-names>P</given-names></name></person-group><year>1989</year><article-title>A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in <italic>Saccharomyces cerevisiae</italic></article-title><source>Genetics</source><volume>122</volume><fpage>19</fpage><lpage>27</lpage></element-citation></ref><ref id="bib45"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Sinclair</surname><given-names>DA</given-names></name><name><surname>Guarente</surname><given-names>L</given-names></name></person-group><year>1997</year><article-title>Extrachromosomal rDNA circles–a cause of aging in yeast</article-title><source>Cell</source><volume>91</volume><fpage>1033</fpage><lpage>1042</lpage><pub-id pub-id-type="doi">10.1016/S0092-8674(00)80493-6</pub-id></element-citation></ref><ref id="bib46"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Siniossoglou</surname><given-names>S</given-names></name><name><surname>Hurt</surname><given-names>EC</given-names></name><name><surname>Pelham</surname><given-names>HR</given-names></name></person-group><year>2000</year><article-title>Psr1p/Psr2p, two plasma membrane phosphatases with an essential DXDX(T/V) motif required for sodium stress response in yeast</article-title><source>The Journal of Biological Chemistry</source><volume>275</volume><fpage>19352</fpage><lpage>19360</lpage><pub-id pub-id-type="doi">10.1074/jbc.M001314200</pub-id></element-citation></ref><ref id="bib47"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Smardon</surname><given-names>AM</given-names></name><name><surname>Diab</surname><given-names>HI</given-names></name><name><surname>Tarsio</surname><given-names>M</given-names></name><name><surname>Diakov</surname><given-names>TT</given-names></name><name><surname>Nasab</surname><given-names>ND</given-names></name><name><surname>West</surname><given-names>RW</given-names></name><name><surname>Kane</surname><given-names>PM</given-names></name></person-group><year>2013</year><article-title>The RAVE complex is an isoform-specific V-ATPase assembly factor in yeast</article-title><source>Molecular Biology of the Cell</source><volume>25</volume><fpage>356</fpage><lpage>367</lpage><pub-id pub-id-type="doi">10.1091/mbc.E13-05-0231</pub-id></element-citation></ref><ref id="bib48"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Thayer</surname><given-names>N</given-names></name><name><surname>Leverich</surname><given-names>C</given-names></name><name><surname>Fitzgibbon</surname><given-names>M</given-names></name><name><surname>Nelson</surname><given-names>Z</given-names></name><name><surname>Henderson</surname><given-names>KA</given-names></name><name><surname>Gafken</surname><given-names>PR</given-names></name><name><surname>Hsu</surname><given-names>JJ</given-names></name><name><surname>Gottschling</surname><given-names>DE</given-names></name></person-group><year>2014</year><article-title>Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions</article-title><source>Proceedings of the National Academy of Sciences of USA</source><comment>[Epub ahead of print]</comment><pub-id pub-id-type="doi">10.1073/pnas.1416079111</pub-id></element-citation></ref><ref id="bib49"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Veatch</surname><given-names>JR</given-names></name><name><surname>McMurray</surname><given-names>MA</given-names></name><name><surname>Nelson</surname><given-names>ZW</given-names></name><name><surname>Gottschling</surname><given-names>DE</given-names></name></person-group><year>2009</year><article-title>Mitochondrial dysfunction leads to nuclear genome instability via an iron-sulfur cluster defect</article-title><source>Cell</source><volume>137</volume><fpage>1247</fpage><lpage>1258</lpage><pub-id pub-id-type="doi">10.1016/j.cell.2009.04.014</pub-id></element-citation></ref><ref id="bib50"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Weisman</surname><given-names>LS</given-names></name><name><surname>Bacallao</surname><given-names>R</given-names></name><name><surname>Wickner</surname><given-names>W</given-names></name></person-group><year>1987</year><article-title>Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle</article-title><source>The Journal of Cell Biology</source><volume>105</volume><fpage>1539</fpage><lpage>1547</lpage><pub-id pub-id-type="doi">10.1083/jcb.105.4.1539</pub-id></element-citation></ref><ref id="bib51"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Winzeler</surname><given-names>EA</given-names></name><name><surname>Shoemaker</surname><given-names>DD</given-names></name><name><surname>Astromoff</surname><given-names>A</given-names></name><name><surname>Liang</surname><given-names>H</given-names></name><name><surname>Anderson</surname><given-names>K</given-names></name><name><surname>Andre</surname><given-names>B</given-names></name><name><surname>Bangham</surname><given-names>R</given-names></name><name><surname>Benito</surname><given-names>R</given-names></name><name><surname>Boeke</surname><given-names>JD</given-names></name><name><surname>Bussey</surname><given-names>H</given-names></name><name><surname>Chu</surname><given-names>AM</given-names></name><name><surname>Connelly</surname><given-names>C</given-names></name><name><surname>Davis</surname><given-names>K</given-names></name><name><surname>Dietrich</surname><given-names>F</given-names></name><name><surname>Dow</surname><given-names>SW</given-names></name><name><surname>El Bakkoury</surname><given-names>M</given-names></name><name><surname>Foury</surname><given-names>F</given-names></name><name><surname>Friend</surname><given-names>SH</given-names></name><name><surname>Gentalen</surname><given-names>E</given-names></name><name><surname>Giaever</surname><given-names>G</given-names></name><name><surname>Hegemann</surname><given-names>JH</given-names></name><name><surname>Jones</surname><given-names>T</given-names></name><name><surname>Laub</surname><given-names>M</given-names></name><name><surname>Liao</surname><given-names>H</given-names></name><name><surname>Liebundguth</surname><given-names>N</given-names></name><name><surname>Lockhart</surname><given-names>DJ</given-names></name><name><surname>Lucau-Danila</surname><given-names>A</given-names></name><name><surname>Lussier</surname><given-names>M</given-names></name><name><surname>M'Rabet</surname><given-names>N</given-names></name><name><surname>Menard</surname><given-names>P</given-names></name><name><surname>Mittmann</surname><given-names>M</given-names></name><name><surname>Pai</surname><given-names>C</given-names></name><name><surname>Rebischung</surname><given-names>C</given-names></name><name><surname>Revuelta</surname><given-names>JL</given-names></name><name><surname>Riles</surname><given-names>L</given-names></name><name><surname>Roberts</surname><given-names>CJ</given-names></name><name><surname>Ross-MacDonald</surname><given-names>P</given-names></name><name><surname>Scherens</surname><given-names>B</given-names></name><name><surname>Snyder</surname><given-names>M</given-names></name><name><surname>Sookhai-Mahadeo</surname><given-names>S</given-names></name><name><surname>Storms</surname><given-names>RK</given-names></name><name><surname>Véronneau</surname><given-names>S</given-names></name><name><surname>Voet</surname><given-names>M</given-names></name><name><surname>Volckaert</surname><given-names>G</given-names></name><name><surname>Ward</surname><given-names>TR</given-names></name><name><surname>Wysocki</surname><given-names>R</given-names></name><name><surname>Yen</surname><given-names>GS</given-names></name><name><surname>Yu</surname><given-names>K</given-names></name><name><surname>Zimmermann</surname><given-names>K</given-names></name><name><surname>Philippsen</surname><given-names>P</given-names></name><name><surname>Johnston</surname><given-names>M</given-names></name><name><surname>Davis</surname><given-names>RW</given-names></name></person-group><year>1999</year><article-title>Functional characterization of the <italic>S. cerevisiae</italic> genome by gene deletion and parallel analysis</article-title><source>Science</source><volume>285</volume><fpage>901</fpage><lpage>906</lpage><pub-id pub-id-type="doi">10.1126/science.285.5429.901</pub-id></element-citation></ref><ref id="bib52"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Wraight</surname><given-names>CA</given-names></name></person-group><year>2006</year><article-title>Chance and design-proton transfer in water, channels and bioenergetic proteins</article-title><source>Biochimica et Biophysica Acta</source><volume>1757</volume><fpage>886</fpage><lpage>912</lpage><pub-id pub-id-type="doi">10.1016/j.bbabio.2006.06.017</pub-id></element-citation></ref><ref id="bib53"><element-citation publication-type="journal"><person-group person-group-type="author"><name><surname>Yassour</surname><given-names>M</given-names></name><name><surname>Kaplan</surname><given-names>T</given-names></name><name><surname>Fraser</surname><given-names>HB</given-names></name><name><surname>Levin</surname><given-names>JZ</given-names></name><name><surname>Pfiffner</surname><given-names>J</given-names></name><name><surname>Adiconis</surname><given-names>X</given-names></name><name><surname>Schroth</surname><given-names>G</given-names></name><name><surname>Luo</surname><given-names>S</given-names></name><name><surname>Khrebtukova</surname><given-names>I</given-names></name><name><surname>Gnirke</surname><given-names>A</given-names></name><name><surname>Nusbaum</surname><given-names>C</given-names></name><name><surname>Thompson</surname><given-names>DA</given-names></name><name><surname>Friedman</surname><given-names>N</given-names></name><name><surname>Regev</surname><given-names>A</given-names></name></person-group><year>2009</year><article-title>Ab initio construction of a eukaryotic transcriptome by massively parallel mRNA sequencing</article-title><source>Proceedings of the National Academy of Sciences of USA</source><volume>106</volume><fpage>3264</fpage><lpage>3269</lpage><pub-id pub-id-type="doi">10.1073/pnas.0812841106</pub-id></element-citation></ref></ref-list></back><sub-article article-type="article-commentary" id="SA1"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03504.011</article-id><title-group><article-title>Decision letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Weis</surname><given-names>Karsten</given-names></name><role>Reviewing editor</role><aff><institution>ETH Zürich</institution>, <country>Switzerland</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://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 “Mother-Daughter Asymmetry of pH Underlies Aging and Rejuvenation in Yeast” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by Randy Schekman (Senior editor) and 3 reviewers, one of whom, Karsten Weis, is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other 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>In this manuscript, Henderson, Hughes, and Gottschling report on the asymmetric localization of the plasma membrane ATPase Pma1, the major proton pump in the plasma membrane of budding yeast. They show that Pma1 plays a role in establishing the asymmetry in vacuolar pH between yeast mother and daughter cells that was previously identified by the authors. They demonstrate that Pma1 is enriched at the plasma membrane of mother cells and absent from buds. This correlates with an increase in the cortical pH in the mother cell compared to the daughter, and a less acidic vacuole in the mother than in the bud. Upon over-expression of Pma1 in cells arrested in mitosis, the asymmetry of Pma1 localization is lost, and the pH asymmetry between mother and bud are reduced or lost both at the cortex and in the vacuole. Likewise, reducing the activity of Pma1 restores the lower pH in the vacuole of mother cells and extends their lifespan. Thus, the authors conclude that asymmetric distribution of Pma1 contributes to the ageing of mother cells and the formation of young daughter cells.</p><p>The referees agreed that the data are interesting and overall of good technical quality. However, there was also a consensus that some additional experiments are needed to solidify the results and the conclusion of the authors:</p><p>Major points:</p><p>1) There was a concern about the low number of cells that were used in the ageing experiments (both wt and pma1-1) and the experiments seem to have been done only once. Higher numbers will be needed to evaluate the mortality of the cells as a function of age (see also point 3).</p><p>2) PMA1 mutants are long-lived and the model indicates that this is because the pH balance is altered as fewer protons are being competed for with V-ATPase to acidify the vacuole. If so, does loss of V-ATPase suppress the long lifespan of pma1 mutant yeast? Does overexpression of V-ATPase result in a long lifespan?</p><p>3) Another issue that was discussed by the reviewers concerns the interpretation of the results. Does the higher pH of the mother cell's vacuole contribute directly to ageing, or does it simply create a burden to the cell that impacts longevity? By definition ageing causes mortality (frequency of death at a certain time point) to increase with age. Thus, when the log of mortality is plotted as a function of age, the curve should have a positive linear slope. Furthermore, a process contributing to ageing should increase the slope of the curve. A rapid evaluation of the curves presented in <xref ref-type="fig" rid="fig3">Figure 3</xref> suggests that in the pma1 mutant the curve is only shifted relative to the wild type and that the slope is not affected. Thus, it appears as if the pH of the vacuole modulates the time at which ageing “starts” without affecting that process itself. Furthermore, if low pH resets age in the bud, mutants with high Pma1 activity in the bud should show a shorter lifespan that decreases proportionally to the age of their mothers at the time of cytokinesis (i.e. they inherit age and cannot reset it). Alternatively, the cells might not show any difference to their mothers (everybody restarts with the same slight deficit due to the fact that ageing starts earlier for all). While the reviewers agreed that experiments addressing some of these points are laborious (but doable) these issues need to be at least discussed. Furthermore, an increase in the number of cells evaluated (see point 1) will help to get a better picture of how mortality changes with age.</p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.03504.012</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>Major points:</italic></p><p><italic>1) There was a concern about the low number of cells that were used in the ageing experiments (both wt and pma1-1) and the experiments seem to have been done only once. Higher numbers will be needed to evaluate the mortality of the cells as a function of age (see also point 3)</italic>.</p><p>In response to reviewers’ suggestion, we tripled the number of wild-type and <italic>pma1-105</italic> cells whose lifespan we analyzed (now 114 and 119 cells, respectively). The additional data support our previous conclusion and are included in <xref ref-type="fig" rid="fig3">Figure 3C</xref>.</p><p><italic>2) PMA1 mutants are long-lived and the model indicates that this is because the pH balance is altered as fewer protons are being competed for with V-ATPase to acidify the vacuole. If so, does loss of V-ATPase suppress the long lifespan of pma1 mutant yeast?</italic></p><p>To address the reviewers’ suggestion, we have added lifespan analysis of strains lacking V-ATPase activity (via deletion of <italic>vma2</italic>) in the wild-type <italic>PMA1</italic> or <italic>pma1-105</italic> background and these data are now included in <xref ref-type="fig" rid="fig3">Figure 3C</xref>. The median lifespan of the <italic>vma2Δ, pma1-105</italic> strain (7 divisions) is much shorter than wild-type (38 divisions) and more similar to the <italic>vma2Δ</italic> lifespan (2 divisions). Thus, the <italic>pma1-105</italic> allele no longer extends lifespan in the absence of V-ATPase activity, suggesting that the ability to acidify the vacuole is required for the majority of the effect of the <italic>pma1-105</italic> allele on lifespan. However, the <italic>vma2Δ, pma1-105</italic> lifespan is extended (7 divisions) compared to <italic>vma2Δ</italic> lifespan (2 divisions), suggesting that the <italic>pma1-105</italic> allele contributes additional benefits besides increased vacuole acidity. We have also added a discussion of these results to the text.</p><p><italic>Does overexpression of V-ATPase result in a long lifespan?</italic></p><p>We previously found the overexpression of <italic>VMA1</italic> (a component of the V-ATPase) extends median lifespan by 40% (Hughes and Gottschling, Nature, 2012) and refer to this in the text.</p><p><italic>3) Another issue that was discussed by the reviewers concerns the interpretation of the results. Does the higher pH of the mother cell's vacuole contribute directly to ageing, or does it simply create a burden to the cell that impacts longevity? By definition ageing causes mortality (frequency of death at a certain time point) to increase with age. Thus, when the log of mortality is plotted as a function of age, the curve should have a positive linear slope. Furthermore, a process contributing to ageing should increase the slope of the curve. A rapid evaluation of the curves presented in</italic> <xref ref-type="fig" rid="fig3"><italic>Figure 3</italic></xref> <italic>suggests that in the pma1 mutant the curve is only shifted relative to the wild type and that the slope is not affected. Thus, it appears as if the pH of the vacuole modulates the time at which ageing “starts” without affecting that process itself</italic>.</p><p>We agree with the reviewers’ comment that while the median lifespan of the <italic>pma1-105</italic> strain is extended by 30%, the slope of the <italic>pma1-105</italic> lifespan curve is similar to the wild-type curve and instead appears shifted compared to wild-type. We have increased the number of cells evaluated as suggested, and the slope of the wild-type and <italic>pma1-105</italic> lifespan curves remain similar. We agree with the reviewers’ interpretation of the data that the <italic>pma1-105</italic> allele likely delays the initiation of the events that normally occur during aging (when aging starts), rather than directly affecting the later events of aging.</p><p>We previously published that when vacuole acidity is increased during aging by <italic>VMA1</italic> overexpression, median lifespan increases by 40% and the slope of the lifespan curve is visibly decreased compared to wild-type (Hughes and Gottschling, 2012). These findings suggest that reduced vacuole acidity contributes to aging throughout lifespan, and that increasing vacuole acidity does not simply delay the onset of the normal events of aging. The work we present here suggests that Pma1 activity influences lifespan by antagonizing vacuole acidity during aging. We therefore suspect that the <italic>pma1-105</italic> allele simply delays the division at which vacuole acidity is reduced and that once vacuole acidity is reduced, the normal events of aging that follow occur at the normal rate. The shape of the <italic>pma1-105</italic> lifespan curve is thus consistent with our conclusion that Pma1 activity initiates aging by reducing vacuole acidity and we have added a discussion of this idea.</p><p><italic>Furthermore, if low pH resets age in the bud, mutants with high Pma1 activity in the bud should show a shorter lifespan that decreases proportionally to the age of their mothers at the time of cytokinesis (i.e. they inherit age and cannot reset it). Alternatively, the cells might not show any difference to their mothers (everybody restarts with the same slight deficit due to the fact that ageing starts earlier for all). While the reviewers agreed that experiments addressing some of these points are laborious (but doable) these issues need to be at least discussed. Furthermore, an increase in the number of cells evaluated (see point 1) will help to get a better picture of how mortality changes with age</italic>.</p><p>As suggested by the reviewers, we have added a discussion of the expectation that increasing Pma1 activity in buds should decrease rejuvenation.</p></body></sub-article></article> |