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Mol Biol Cell. Dec 1, 2010; 21(23): 4173–4183.
PMCID: PMC2993746
A Highlights from MBoC Selection
Microautophagy of the Nucleus Coincides with a Vacuolar Diffusion Barrier at Nuclear–Vacuolar Junctions
Rosie Dawaliby and Andreas Mayercorresponding author
Département de Biochimie, Université de Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland
Akihiko Nakano, Monitoring Editor
corresponding authorCorresponding author.
Address correspondence to: Andreas Mayer (Andreas.Mayer/at/unil.ch).
Received September 11, 2009; Revised August 16, 2009; Accepted October 4, 2010.
Nuclei bind yeast vacuoles via nucleus-vacuole (NV) junctions. Under nutrient restriction, NV junctions invaginate and release vesicles filled with nuclear material into vacuoles, resulting in piecemeal microautophagy of the nucleus (PMN). We show that the electrochemical gradient across the vacuolar membrane promotes invagination of NV junctions. Existing invaginations persist independently of the gradient, but final release of PMN vesicles requires again V-ATPase activity. We find that NV junctions form a diffusion barrier on the vacuolar membrane that excludes V-ATPase but is enriched in the VTC complex and accessible to other membrane-integral proteins. V-ATPase exclusion depends on the NV junction proteins Nvj1p,Vac8p, and the electrochemical gradient. It also depends on factors of lipid metabolism, such as the oxysterol binding protein Osh1p and the enoyl-CoA reductase Tsc13p, which are enriched in NV junctions, and on Lag1p and Fen1p. Our observations suggest that NV junctions form in two separable steps: Nvj1p and Vac8p suffice to establish contact between the two membranes. The electrochemical potential and lipid-modifying enzymes are needed to establish the vacuolar diffusion barrier, invaginate NV junctions, and form PMN vesicles.
PMN is a selective autophagic process in yeast cells that targets parts of the nucleus for degradation. Like other autophagic processes PMN is induced by nitrogen or carbon starvation (Roberts et al., 2003 blue right-pointing triangle; Levine and Klionsky, 2004 blue right-pointing triangle). The response to starvation is regulated by the rapamycin-sensitive TORC1 complex (De Virgilio and Loewith, 2006 blue right-pointing triangle). Because rapamycin treatment reproduces many characteristic features of starved cells, such as growth arrest and autophagy, this drug is frequently used to study autophagic processes and also PMN.
Nuclei and vacuoles closely associate via nucleus-vacuole (NV) junctions which involve interactions between the outer nuclear membrane protein Nvj1p and the vacuolar membrane protein Vac8p (Pan et al., 2000 blue right-pointing triangle; Roberts et al., 2003 blue right-pointing triangle). NV junctions invaginate toward the vacuolar lumen and evolve into a tear drop–like bleb which releases a vesicle into the vacuolar lumen. This vesicle contains nuclear material and is degraded inside vacuoles (Roberts et al., 2003 blue right-pointing triangle). Using vacuolar degradation of an Nvj1-GFP fusion as a criterion, PMN was found to depend on numerous other proteins. Among these are core components of the autophagic and CVT machineries (Atg genes), phosphatidyl-inositol-3-kinase complex I, and components of the vacuolar fusion machinery (Krick et al., 2008 blue right-pointing triangle). Deletion of Atg18 does not disrupt formation of NV junctions, suggesting that the Atg machinery might affect PMN at later stages. They might assist in the closure of the PMN vesicle, similarly as shown for their role in microautophagy of peroxisomes, where they are concentrated in the micropexophagy-specific membrane apparatus (Mukaiyama et al., 2004 blue right-pointing triangle; Sakai et al., 2006 blue right-pointing triangle).
NV junctions contain proteins involved in lipid biosynthesis and trafficking. One of these, the enoyl-CoA reductase Tsc13p, is an essential ER membrane protein that catalyzes the terminal step of very-long-chain fatty acid biosynthesis (Kohlwein et al., 2001 blue right-pointing triangle). Pharmacological inhibition of fatty acid synthesis and elongation, as well as mutations in TSC13, decrease the size of PMN blebs and vesicles (Kvam et al., 2005 blue right-pointing triangle). Osh1p is another protein related to lipid metabolism that is recruited to the ER and outer nuclear membrane by Nvj1p (Kvam and Goldfarb, 2004 blue right-pointing triangle). Osh1p belongs to the oxysterol-binding protein (OSBP) family which has seven members in yeast (Beh et al., 2001 blue right-pointing triangle; Levine and Munro, 2001 blue right-pointing triangle). OSBPs are thought to mediate nonvesicular lipid trafficking, and some have been implicated in cell signaling (Levine, 2004 blue right-pointing triangle). Yeast mutants lacking certain combinations of Osh proteins exhibit defects in endocytosis, vesicular transport, and altered vacuolar structure (Beh and Rine, 2004 blue right-pointing triangle).
The vacuolar membrane contains a proton-pumping V-ATPase which is composed of the peripheral V1 sector (subunits A–H) and the membrane-integral V0 sector (subunits a, c, c′, c″, d, and e). ATP-hydrolysis by the V1 sector drives proton translocation through V0 (Nishi and Forgac, 2002 blue right-pointing triangle). This acidifies the vacuolar lumen and generates an electrochemical potential over the vacuolar membrane (Kane, 2006 blue right-pointing triangle). The low pH stimulates the activity of vacuolar hydrolases that are required to efficiently degrade macromolecules which are transferred into the vacuolar lumen via endocytosis, autophagy, and other trafficking routes. Besides their crucial role in intracellular pH regulation, V-ATPases have been implicated in numerous vesicular trafficking steps, such as vacuole fusion in yeasts (Peters et al., 2001 blue right-pointing triangle; Bayer et al., 2003 blue right-pointing triangle), exocytosis of multivesicular bodies in worms (Liegeois et al., 2006 blue right-pointing triangle), regulated fusion of synaptic vesicles in flies (Hiesinger et al., 2005 blue right-pointing triangle), insulin secretion in mammalian cells (Sun-Wada et al., 2006 blue right-pointing triangle), and phagosome-lysosome fusion (Peri and Nusslein-Volhard, 2008 blue right-pointing triangle). In all these systems, membrane fusion requires physical presence of the V-ATPase complex, but not its proton translocation activity. Whereas V-ATPase pump activity is not essential for vacuole fusion, it is necessary for the opposing reaction of vacuole fragmentation. Therefore, it influences vacuole size and number that depend on the vacuolar fusion-fission equilibrium (Baars et al., 2007 blue right-pointing triangle). The proton pump activity of V-ATPases can be inhibited by bafilomycin A and concanamycin A. These inhibitors bind to V0 and are effective at nanomolar concentrations (Bowman and Bowman, 2002 blue right-pointing triangle; Huss et al., 2002 blue right-pointing triangle). The physical role of V-ATPases or V-ATPase subunits in vacuole fusion is not abolished by low concentrations of these inhibitors which suffice to completely suppress proton pump activity (Peters et al., 2001 blue right-pointing triangle; Bayer et al., 2003 blue right-pointing triangle; Muller et al., 2003 blue right-pointing triangle).
We performed a screen for factors necessary for PMN (Dawaliby et al., in preparation) which identified numerous subunits of the V-ATPase, seven of the eight V1 sector subunits (A, C, D, E, F, G, and H) and the V0 sector subunits Vph1 (subunit a) and Vma3 (subunit c). Therefore, we explored the role of the V-ATPase in PMN in detail. We analyzed which steps of PMN depend on V-ATPase activity and, in testing V-ATPase localization during PMN, we discovered a novel diffusion barrier at NV junction sites.
Yeast Strains and Plasmids
Yeast strains used are listed in Table 2, and the primers used for generating mutations are listed in Table 3. BJ3505 is a deletion strain for Pep4 and Prb1 (Jones et al., 1982 blue right-pointing triangle). Nvj1 was deleted in BJ3505 by one step PCR-mediated gene disruption with natNT2 cassette from the pFA6a-natNT2 plasmid (Janke et al., 2004 blue right-pointing triangle). Vph1-(Gly)6-eGFP (kanMX) was generated by genomic integration of a PCR fragment coding for EGFP and the auxotrophic marker using the pYM27 plasmid (Janke et al., 2004 blue right-pointing triangle). N-terminal tagging of Nvj1 was performed similarly using pYM-N16 and pYM-N17 plasmids, respectively (Janke et al., 2004 blue right-pointing triangle), creating BJ3505 pGPD-eGFP-Nvj1 (natNT2) and BJ 3505 pGPD-HA-Nvj1 (natNT2). Nvj1-eCFP and DsRed-Nop1 strains were obtained by transformation of yeast cells with p416-pADH-Nvj1-eCFP and pRS314-pNopDsRed-Nop1 (Gadal et al., 2001 blue right-pointing triangle).
Table 2.
Table 2.
Yeast strains used in the study
Table 3.
Table 3.
Primers used in the study
BJ3505 pGPD-HA-Nvj1 Vph1-GFP and BJ3505 pGPD-eGFP-Nvj1 Vph1-GFP resulted from transformation of p416-pVph1-GFP into the corresponding strains. Strains BY4741 and BY4741 Δvph1 were purchased from Euroscarf. BY4741 GFP-Nop1, BY4741 Δvph1 GFP-Nop1 and BY4741 Δvph1 GFP-Nop1 were obtained by transforming BY4741 and BY4741 Δvph1 with pRS315-pNop-GFP-Nop1.
Plasmid p416-pADH-Nvj1-eCFP was obtained by amplifying the Nvj1-eCFP cassette from genomic DNA and inserting it into p416-ADH using BamHI and HindIII restriction sites.
Plasmid p416-pVph1-Vph1-GFP was generated first by integrating the endogenous Vph1 promoter between XbaI and SacI restriction sites into pRS416. Genomically tagged Vph1-GFP was amplified by PCR and inserted into the resulting p416pVph1 plasmid using SpeI and SmaI restriction sites. pRS314-pNop-DsRedNop1 and pRS315-pNop-GFP-Nop1 were kindly provided by Ed Hurt.
General Methods
Yeast strains were grown (30°C, 150 rpm) in HC medium or in HC dropout medium lacking an amino acid or uracil corresponding to the auxotrophic marker of the plasmid carried by the strain. Cells were harvested by centrifugation at an OD600 of 0.4–0.8. Precultures were grown under the same conditions to saturation.
Reagents
Rapamaycin, concanamycin A, bafilomycin A1, and oligomycin were purchased from Alexis Biochemicals (Plymouth Meeting, PA), PMSF from Roche (Indianapolis, IN), FM4-64 (Synaptored TMC2) from VWR (Darmstadt, Germany), ClonNAT (nourseothricin) from Werner BioAgents (Jena, Germany), G418 sulfate from Calbiochem (La Jolla, CA) and low melting point agarose from Sigma-Aldrich (St. Louis, MO).
PMN Induction and Addition of Inhibitors
Cells were shaken overnight at 30°C in logarithmic phase (OD600 < 1) in HC or HC dropout medium lacking an amino acid or uracil, corresponding to the auxotrophic marker of the plasmid carried by the strain. Cultures were adjusted to OD600 = 0.5 and 0.2 μΜ rapamycin was added from a 20 μΜ stock in DMSO. Cells were incubated 3h at 30°C. For strains that did not carry a PEP4 deletion, 1 mM PMSF was added to the culture from a freshly prepared 200 mM stock in 70% ethanol every hour. Where indicated, 1 μΜ concanamycin A, bafilomycin A1, or 5 μΜ oligomycin were added after 2h of rapamycin treatment and the cells incubated for one more hour. Alternatively, rapamycin and one of the other inhibitors were added simultaneously and the cells incubated for 3 h.
FM4-64 Staining
To visualize vacuolar membranes in vivo, cells were stained with the lipophilic dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl)-pyridinium dibromide (FM4-64) as previously described (Müller et al., 2000). Cells were grown overnight at 30°C to logarithmic phase (OD600 < 1) in HC or selective medium. Cultures were adjusted to OD600 = 0.4, and 10 μM FM4-64 was added from a 10 mM stock solution in DMSO. Cells were incubated for 1 h at 30°C, harvested (1 min, 3000 × g, RT), washed twice in fresh medium, resuspended in medium to OD600 = 0.4, and shaken for 1 h at 30°C. Further treatment of the cells with inhibitors was begun after this chase period.
Assay of PMN
PMN was monitored via the transfer of nucleolar material to vacuoles. Cells expressing the nucleolar marker Nop1p-GFP were stained with FM4-64. Cells were pelleted (15 s, 8000 × g, room temperature), resuspended in medium to OD600 = 5, supplemented with 0.2% low melting point agarose from a 1% stock solution in water (kept liquid at 40°C), and immediately analyzed by confocal microscopy using an LSM 510 microscope (Zeiss) with a ×100 Zeiss Plan Apochromat objective (numerical aperture 1.4). Excitation wavelengths of 488 nm for GFP and of 543 nm for FM4-64 were used. Alternatively, a standard fluorescence microscope (Leica DMI6000) with a Hamamatsu Orca R2 camera was used, equipped with a ×100, 1.4 NA lens and driven by Volocity software. The number of cells carrying both PMN structures and GFP stained vesicles in their vacuolar lumen was counted.
Statistical Analysis
To quantify PMN, NV junctions, or exclusion sites, photos of at least 10 random fields were taken for each condition. At least 200 cells showing the respective markers were evaluated per condition and experiment. Data from three or more independent experiments was averaged, standard deviations were calculated and are shown as error bars.
Vph1 Exclusion Analysis
Cells were grown and analyzed as for the assay of PMN. Photos of at least 10 random fields were taken for each condition. The number of cells, of 200 cells per condition, presenting Vph1p exclusion sites in the vicinity of Nop1p and/or Nvj1p was counted. For random exclusion of Vph1p we scored 200 cells per condition for Vph1p exclusion sites that were not adjacent to Nop1p. Data from three or more independent experiments was averaged and the corresponding standard deviations calculated.
PMN Requires V-ATPase Pumping Activity
Screening of the yeast deletion collection for mutants defective in PMN has identified many V-ATPase subunits (Dawaliby et al., in preparation). Because an increasing number of V-ATPase activities become apparent that do not depend on the H+ pump function of the enzyme, we tested the effect of acute pharmacological inhibition of V-ATPase activity. We investigated the effect of concanamycin A and bafilomycin A, two potent and highly specific inhibitors that leave the V-ATPase physically intact yet abolish its pumping activity. PMN was measured by confocal fluorescence microscopy, using the transfer of the nucleolar marker GFP-Nop1p into vesicular or tubular structures inside vacuoles (Figure 1A). We quantified the fraction of cells whose vacuoles contained PMN vesicles or structures bearing the fluorescent nucleolar marker. Under normal growth conditions cells showed only basal PMN activity, whereas treatment with rapamycin induced PMN fivefold (Figure 1B) (Roberts et al., 2003 blue right-pointing triangle). When V-ATPase inhibitors were added to the medium at the time of induction of PMN, PMN was reduced to the levels observed in noninduced control cells (Figure 1, A and B). Oligomycin, an inhibitor of the mitochondrial F-ATPase applied in the same solvent, did not affect PMN but the protonophore FCCP inhibited PMN as potently as the V-ATPase inhibitors (Figure 1A). The same effects were observed when Nvj1p-GFP was used as a marker for PMN activity (not shown). Analysis of PMN in knockout mutants of the V1 sector subunit Vma1 confirmed these results. PMN was observed in only 15% of the induced Δvma1 cells but in more than 50% of the wild-type cells (Figure 2). This suggests that the pumping activity of the V-ATPase is necessary for efficient PMN.
Figure 1.
Figure 1.
The V-ATPase inhibitor concanamycin A reduces PMN. (A) Wild-type cells expressing GFP-Nop1 under its endogenous promotor were stained with FM4-64 in HC media to visualize vacuoles. Cells were then incubated for a total of 3 h with 0.2 μΜ (more ...)
Figure 2.
Figure 2.
V-ATPase cells are defective in PMN. (A) Wild-type or Δvma1 cells expressing GFP-Nop1p under its endogenous promotor were stained with FM4-64 in HC media to visualize vacuoles. Cells were then treated with 0.2 μΜ rapamycin, incubated (more ...)
V-ATPase Pump Activity Affects Two Steps of PMN
Because PMN can be microscopically subdivided into the stages of membrane association, membrane invagination, bleb formation, and final release of a vesicle (Kvam and Goldfarb, 2006b blue right-pointing triangle), we tried to determine which of these steps depends on V-ATPase activity. We quantified the influence of concanamycin A when added together with the induction of PMN by rapamycin and, for comparison, when added after an initial phase of induction (2h). At this time, most invaginations had already been formed and further incubation until the end of the 3h incubation period can then serve to test the persistence of these structures in the absence of V-ATPase pump activity. If V-ATPase activity were only required for an initial step of PMN, the process should acquire resistance to V-ATPase inhibition once this early step has been completed. To distinguish early and late phases of PMN we differentiated between cells showing PMN vesicles and cells showing only PMN structures (i.e., NV junctions and their invaginations into the vacuolar lumen). Use of concanamycin A from the beginning of the rapamycin treatment decreased the frequency of PMN structures and PMN vesicles equally (Figure 3). When added two hours after rapamycin, concanamycin A reduced the fraction of cells with PMN vesicles to the same low levels as observed in the samples that had received concanamycin A from the beginning (Figure 3B). By contrast, the frequency of PMN structures remained significantly higher, suggesting that PMN structures remained relatively stable after inactivation of V-ATPase. Thus, V-ATPase activity appears to contribute to two different steps of PMN. First, it is needed to form invaginations at NV junctions. Once formed, the PMN structures persist even after inhibition of V-ATPase. However, the scission of vesicles from these structures into the vacuolar lumen remains dependent on V-ATPase activity. This suggests that invagination and vesicle formation can be uncoupled.
Figure 3.
Figure 3.
Effect of concanamycin A on PMN structures and vesicles, depending on the time of addition. (A) Cells expressing GFP-Nop1 under its endogenous promotor were stained with FM4-64 in HC and were then treated for a total of 3 h as follows: With 0.2 μΜ (more ...)
NV Junctions Form a Diffusion Barrier at the Vacuolar Membrane
To further investigate the role of the V-ATPase in PMN we analyzed the localization of a GFP-tagged version of the a-subunit (Vph1p) of the V-ATPase V0 sector (Peters et al., 2001 blue right-pointing triangle), the nucleolar marker DsRed-Nop1p, and Nvj1p-eCFP. The tagged proteins are functional because the cells were viable and showed normal growth and PMN activity. They did not display phenotypes associated with loss of V-ATPase activity, such as inviability at pH 7.5, diminished quinacrine accumulation, or defective vacuole fragmentation upon hypertonic shock (Peters et al., 2001 blue right-pointing triangle; Baars et al., 2007 blue right-pointing triangle; Ryan et al., 2008 blue right-pointing triangle). The cells were grown in rich Hartwell's synthetic complete (HC) medium and analyzed by fluorescence microscopy. Vph1p-GFP was homogeneously distributed over the vacuolar rim in >80% of the cells that showed Nvj1p-eCFP in the optical section. On PMN induction by rapamycin, the distribution of Vph1-GFP changed strikingly. The protein became excluded from sharply defined stretches of the vacuolar membrane. These sites faced the nucleolar marker DsRed-Nop1p and the NV junction marker Nvj1p (Figure 4). 50% of cells in which DsRed-Nop1 was visible in the optical section showed an exclusion of Vph1p-GFP from the adjacent vacuolar membrane. These zones of Vph1p exclusion corresponded in all cases to Nvj1p-eCFP localization. Experiments with a GFP-fusion of the V1 subunit B (Vma2) yielded similar results, suggesting that the effect concerns the entire V-ATPase rather than only a specific subunit (Figure 4B; Supplemental Figure 1). In Δnvj1 background, exclusion of Vph1p was observed with the same low frequency as in wild-type cells that were noninduced, treated with concanamycin A or FCCP (Figure 5). Thus, the induction of PMN and the increase in the number of NV junctions, which depend on Nvj1p, correlate to the exclusion of V-ATPase from vacuolar membrane patches facing the nucleus. This suggests that NV junctions might form a diffusion barrier that prevents access of V-ATPase to this region.
Figure 4.
Figure 4.
Vph1p exclusion is abolished by concanamycin A. (A) Wild-type yeast cells carrying Vph1p-GFP, DsRed-Nop1p, and Nvj1p-ECFP were treated in HC medium with 0.2 μΜ rapamycin and/or 1 μΜ concanamycin A for 3 h, or left untreated. (more ...)
Figure 5.
Figure 5.
Vph1p exclusion depends on Nvj1. (A) Wild-type and Δnvj1 mutants carrying Vph1p-GFP and DsRed-Nop1p were treated with 0.2 μΜ rapamycin for 3 h. Samples were analyzed by confocal microscopy. (B) Wild-type cells carrying Vph1p-GFP (more ...)
To test whether there is selectivity in the exclusion from NV junctions we analyzed GFP fusions of other vacuolar membrane proteins. As a small protein we chose alkaline phosphatase (Pho8p) which is entirely oriented toward the vacuolar lumen. The Pho8p fusion protein exposes only its GFP tag to the cytosol (Cowles et al., 1997 blue right-pointing triangle). As a complex that exposes large hydrophilic domains to the cytosol we chose the VTC complex. VTC is a vacuolar polyphosphate polymerase of approx. 500 kDa that is completely oriented toward the cytosol. It can be labeled by N-terminal tagging of Vtc4p or Vtc1p (Muller et al., 2003 blue right-pointing triangle; Uttenweiler et al., 2007 blue right-pointing triangle; Hothorn et al., 2009 blue right-pointing triangle). The cytoplasmic parts of VTC are of similar molecular mass as those of V-ATPase. In contrast to Vph1p-GFP, GFP-Pho8p was homogeneously distributed over the vacuoles in presence and absence of rapamycin (Figure 6). Likewise, GFP fusions of the vacuolar transporters Zrc1p and Ycf1p and of the vacuolar SNARE Vam3p were not excluded from NV junctions (Figure 6B; Supplemental Figure 2). The exclusion sites were also accessible to the fluorescent lipid dye FM4-64, which inserts into the vacuolar boundary membrane (Supplemental Figure 2). GFP-Vtc1p was even concentrated at NV junctions and PMN structures (Figure 6, A and B). 55% of the cells accumulated GFP-Vtc1p at NV junctions upon rapamycin treatment while only 24% of the untreated cells showed similar accumulation. GFP-Vtc4p fusions behaved similarly. Quantification with ImageJ revealed that the GFP-Vtc fluorescence intensity at NV junction sites was more than twofold higher than in other parts of the vacuolar membrane of rapamycin-treated cells. These observations suggest that the exclusion zone is continuous with the vacuolar membrane and that exclusion of proteins from PMN sites is not general but selective for Vph1p. It is unlikely to be a simple consequence of the size of the protein parts exposed to the cytosol because this parameter is similar for the VTC and V-ATPase complexes, yet only V-ATPase shows exclusion. In line with a previous proposal (Kvam et al., 2005 blue right-pointing triangle) we speculate that NV junctions might induce a phase separation in the plane of the vacuolar membrane, creating a membrane zone that favors enrichment of certain proteins while providing an unfavorable environment for others.
Figure 6.
Figure 6.
Presence of vacuolar proteins at PMN sites. (A) Wild-type cells expressing DsRed-Nop1 and GFP-Vtc1p or GFP-Vtc4p were incubated in HC medium with or without 0.2 μΜ rapamycin for 3 h before being analyzed by confocal microscopy. Only GFP-Vtc1p (more ...)
Vph1p Exclusion from NV Junctions Depends on the Electrochemical Potential of the Vacuole
Because V-ATPase activity promotes PMN, we tested whether H+ pumping influenced the Vph1p diffusion barrier. We investigated the effect of concanamycin A on NV junction frequency and Vph1p-GFP exclusion in cells carrying DsRed-Nop1p and Nvj1p-eCFP (Figure 7). In rapamycin-treated cells, concanamycin A decreased the percentage of cells presenting Vph1p exclusion and the percentage of cells with PMN structures to values seen in the absence of PMN induction. By contrast, Nvj1p patch frequency was only slightly affected. These data suggest that NV junctions form a diffusion barrier in the vacuolar membrane that is promoted by the electrochemical potential of the vacuole.
Figure 7.
Figure 7.
Concanamycin A inhibits Vph1p exclusion but not formation of Nvj1p patches. Wild-type cells carrying Vph1p-GFP and Nvj1p-CFP had been stained with FM4-64 in HC and were then treated for a total of 3 h as follows: With 0.2 μΜ rapamycin (more ...)
V-ATPase Exclusion Is Independent of PMN and Recruitment of the Nucleolus to NV Junctions
In the course of a screening project (Dawaliby et al., manuscript in preparation) we noted that the nucleolus is preferentially degraded by PMN, consistent with electron microscopic observations that suggested that the nucleolus is often present in PMN structures (Roberts et al., 2003 blue right-pointing triangle).We hence tested whether some characteristics of Vph1p exclusion are distinct from those of PMN. We analyzed whether the nucleolus needs to be present at a NV junction and whether components of the Atg machinery, which are also involved in PMN (Krick et al., 2008 blue right-pointing triangle), are required for Vph1p exclusion. Deletion mutants in core components of the autophagy machinery, Δatg12 and Δatg7, showed normal levels of Vph1p exclusion but strong defects in PMN (Table 1; Supplemental Figure 4). To test whether the nucleolus or other nuclear material needs to be positioned next to the NV junction to induce Vph1p exclusion we exploited the observation that N-terminal tagging and overexpression of Nvj1p creates Nvj1p patches not only at NV junctions but also along ER-like cytoplasmic membranes. This produces a surplus of ectopic patches of Nvj1p which do not face the nucleus. (Kvam and Goldfarb, 2006b blue right-pointing triangle). We tested Vph1p-GFP exclusion relative to the nucleolus in cells that carried the nucleolar marker Nop1p-DsRed and overexpressed Nvj1p from the strong GPD promoter (Figure 8, A and B). We separately scored Vph1p exclusion at sites distant from NV junctions. We refer to those as random exclusion sites, in contrast to restricted exclusion sites that are adjacent to the nucleus. Restricted exclusion of Vph1p did not increase by overexpression of Nvj1p. Upon rapamycin-induction, however, their frequency increased up to threefold, confirming that Nvj1p overexpression alone does not determine the full onset of PMN (Figure 8B). Nvj1 overexpression increased the frequency of random exclusion from 4 to 20% in rapamycin-treated cells, indicating that an exclusion site can form in the absence of nuclear material. Also this increase was rapamycin-sensitive, suggesting that both restricted and random exclusion sites may be under the control of TORC1 signaling. In a similar experiment we tested the morphological relationship of ectopic Nvj1p patches to the exclusion sites in strain that carried Vph1p-CFP and overexpressed GFP-Nvj1p (Figure 8, C and D). The random exclusion sites were always adjacent to a patch of GFP-Nvj1p. In sum, these experiments suggest that ectopic patches of Nvj1p suffice to induce phase separation on the vacuolar membrane. Thus, Vph1 exclusion is a membrane differentiation that arises already from an association of the ER and the vacuolar membrane. It is independent of the recruitment of nuclear or nucleolar material to this site and can be distinguished from PMN by its independence of the autophagy core machinery.
Table 1.
Table 1.
Vph1p exclusion in various strains (% of Nop1-DsRed positive cells showing Vph1 exclusion next to the nucleolus)
Figure 8.
Figure 8.
Random exclusion of Vph1p upon overexpression of Nvj1p. (A) Cells overexpressing HA-Nvj1 from the GPD promoter and carrying Vph1p-GFP and DsRed-Nop1p were treated in HC medium for 3 h with 0.2 μΜ rapamycin and 1 μM concanamycin (more ...)
Diffusion Barrier Formation at NV Junctions Depends on Lipid Metabolism
Two observations link PMN to lipid metabolism. First, oxysterol-binding (OSH) proteins are necessary for PMN (Kvam and Goldfarb, 2004 blue right-pointing triangle) and are enriched at NV junctions. Second, Tsc13p, which is involved in the synthesis of very long chain fatty acids and sphingolipids, is found in PMN blebs and influences the size of PMN vesicles (Kvam et al., 2005 blue right-pointing triangle). This suggests that NV junctions might coincide with and depend on a special lipid composition. Because such lateral heterogeneity of the vacuolar membrane might underlie the exclusion of Vph1p from NV junctions we tested several mutants of lipid metabolism for their effect on Vph1p exclusion (Table 1). Δosh1 and Δosh2 mutants showed a phenotype very similar to the one observed in Δnvj1 mutants (i.e., strongly reduced Vph1p-GFP exclusion in rapamycin-treated cells) (Supplemental Figure 3). Because Tsc13 is essential and a conditional mutant was not available, we tested nonlethal mutants in the same metabolic pathway, such as fat1, scs7, fen1, and sur4 (Mitchell and Martin, 1997 blue right-pointing triangle; Oh et al., 1997 blue right-pointing triangle; Watkins et al., 1998 blue right-pointing triangle). Fen1 and Sur4 interact genetically and physically with Tsc13 (Kohlwein et al., 2001 blue right-pointing triangle; Miller et al., 2005 blue right-pointing triangle). Δfen1 and Δsur4 cells showed >50% defect in PMN activity and Vph1p exclusion was reduced to a similar degree (Table 1). This result suggests a requirement for very long chain fatty acids in the formation of NV junctions and supports a role of Tsc13 partners in this process.
Because very-long chain fatty acids are incorporated into phytoceramide, we also tested mutants in biosynthesis of this sphingolipid (Δypc1, Δlag1, Δlcb3) (D'Mello N et al., 1994 blue right-pointing triangle; Qie et al., 1997 blue right-pointing triangle; Mao et al., 2000 blue right-pointing triangle). These mutations significantly decreased PMN activity and Vph1p exclusion under rapamycin treatment (Table 1). The strongest phenotype was observed in the Δlag1 mutant. Even after rapamycin induction, Vph1p exclusion was reduced to the basal level observed with untreated wild-type cells. Because sphingolipids and sterols are associated and coordinated in yeast cells (Guan et al., 2009 blue right-pointing triangle), we also tested a mutant in ergosterol biosynthesis (Δerg5) (Ashman et al., 1991 blue right-pointing triangle; Kelly et al., 1997 blue right-pointing triangle). The C-22 sterol desaturase mutant Δerg5 showed severely reduced PMN activity and Vph1p exclusion upon rapamycin treatment. A caveat for the evaluation of this mutant is that the vacuoles in Δerg5 were fragmented, rendering quantification difficult.
The NV-junction is a zone of contact between vacuoles and the nuclear envelope. It affects membrane organization on the nuclear side because nuclear pore complexes are not found in vicinity of the vacuolar membrane (Severs et al., 1976 blue right-pointing triangle; Pan et al., 2000 blue right-pointing triangle). Here we show that Vph1p-GFP is excluded from the vacuolar membrane sections that form an NV junction. This barrier is specific for V-ATPase and the exclusion does not affect the VTC complex, which has a cytosolic domain of similar size. It has been speculated that NV junctions might have a special lipid composition (Kvam and Goldfarb, 2006a blue right-pointing triangle). The results we obtained with the mutants in Osh genes and in ceramide metabolism support this notion and suggest a role of lipids in NV junction formation and function. An observation that strengthens this view is that the accumulation of the VTC complex at NV junctions is abolished by inhibition of phosphatidylinositol-3-kinase (C. Dangelmayr, R. Dawaliby et al., unpublished results). NV junctions might hence form a specialized lipid zone on the vacuolar membrane that excludes certain vacuolar proteins by providing an unsuitable membrane environment for them. In that sense they resemble lipid rafts, specialized membrane microdomains that have been extensively characterized. Rafts are rich in cholesterol and sphingolipids (Simons and Ehehalt, 2002 blue right-pointing triangle; Korade and Kenworthy, 2008 blue right-pointing triangle) and serve as organizing centers for the assembly of signaling molecules. They influence membrane fluidity and membrane protein trafficking and regulate neurotransmission and receptor trafficking (Simons and Ehehalt, 2002 blue right-pointing triangle; Pichler and Riezman, 2004 blue right-pointing triangle). Similarly, NV junctions recruit selected proteins such as Osh1p, Tsc13p, and VTC and exclude others.
V-ATPase exclusion is reverted by concanamycin A. This suggests that NV junctions form a barrier that requires the electrochemical potential of the vacuolar membrane. This potential depends to a large extent on the proton translocation activity of the V-ATPase which acidifies vacuoles (Forgac, 2007 blue right-pointing triangle). Numerous studies demonstrated roles of the V-ATPase in membrane homeostasis and vesicular traffic. Only some of these depend on proton pump activity, such as autophagosome-lysosome fusion, passage through endosomes, or the fragmentation of yeast vacuoles (Schmid et al., 1989 blue right-pointing triangle; Yamamoto et al., 1998 blue right-pointing triangle; Liu et al., 2005 blue right-pointing triangle; Baars et al., 2007 blue right-pointing triangle; Kawai et al., 2007 blue right-pointing triangle). An example for a function independent of proton-translocation is the physical role of the V0 sector during membrane fusion at various membrane trafficking steps (Peters et al., 2001 blue right-pointing triangle; Bayer et al., 2003 blue right-pointing triangle; Hiesinger et al., 2005 blue right-pointing triangle; Liegeois et al., 2006 blue right-pointing triangle; Sun-Wada et al., 2006 blue right-pointing triangle; Peri and Nusslein-Volhard, 2008 blue right-pointing triangle). A purely physical role of the V-ATPase in formation of PMN vesicles is unlikely because the process is sensitive to concanamycin A and because V-ATPase is excluded from PMN structures and NV junctions that give rise to these vesicles. Thus, one should rather consider effects via the membrane potential. Vesicle formation and fission can depend on lateral inhomogeneities within membranes, induced either by spontaneous phase separations of lipids (Julicher and Lipowsky, 1993 blue right-pointing triangle; Sackmann and Feder, 1995 blue right-pointing triangle; Julicher and Lipowsky, 1996 blue right-pointing triangle; Munn et al., 1999 blue right-pointing triangle; Proszynski et al., 2005 blue right-pointing triangle; Falguieres et al., 2009 blue right-pointing triangle), or by the concentration of membrane-apposed or membrane-integral proteins (Wenk and De Camilli, 2004 blue right-pointing triangle; Lee et al., 2005 blue right-pointing triangle; Ramos et al., 2006 blue right-pointing triangle). Such lateral phase separations can be strongly influenced by the membrane potential, as exemplified by the massive compartmentation of the yeast plasma membrane into two clearly distinct zones which are occupied either by the plasma membrane ATPase Pma1p or the arginine symporter Can1p (Malinska et al., 2003 blue right-pointing triangle). These two zones mix when the membrane is depolarized (Grossmann et al., 2007 blue right-pointing triangle). Also, phase separation in synthetic membrane systems can depend on the membrane potential (Herman et al., 2004 blue right-pointing triangle; Schaffer and Thiele, 2004 blue right-pointing triangle). Thus, our observations and those from previous studies are consistent with the hypothesis that the full differentiation of an NV junction may comprise lipid phase separations depending on the electrochemical potential across the vacuolar membrane.
A comprehensive study systematically tested vacuole- and autophagy-related genes for a role in PMN (Krick et al., 2008 blue right-pointing triangle). This confirmed numerous known PMN factors and identified a novel requirement for Atg genes in PMN. Whereas our microscopic observations on atg mutants agree with this study, there is a discrepancy concerning the V-ATPase which Krick et al. (2008 blue right-pointing triangle) found not to be required for PMN. This discrepancy could be due to the different assays used. We visualized transfer of the nucleolar protein Nop1p-GFP into vacuoles microscopically. This microscopic assay is independent of the actual degradation of the PMN vesicles or the reporter. In their tests of V-ATPase mutants Krick et al. (2008 blue right-pointing triangle) used the proteolytic degradation of a GFP-Osh1p fusion protein as a measure for PMN. The assay is based on the fact that GFP is more protease-resistant than the linker between GFP and Osh1p and hence accumulates in vacuoles. The parameters influencing this assay are complex. On induction of PMN, the GFP-Osh1p fusion is transferred into vacuoles, the PMN vesicle membranes must be degraded, and then the vacuolar proteases cleave GFP-Osh1p and GFP. Although the GFP domain is more protease-resistant than the GFP-Osh1p fusion, it is nevertheless degradable by vacuoles, which can easily be visualized by pulse-chase experiments in which expression of new GFP is prevented by Gal-shut-off experiments (our unpublished results). The relative protease-resistance of GFP leads to its accumulation inside vacuoles to a certain level. This level, however, depends on multiple parameters, such as the protease and lipase activities inside the vacuoles, the time that the fusion protein is exposed to vacuolar hydrolases, the kinetics with which the GFP-Osh1p linker and the released GFP domain are degraded, and the level of PMN activity. Mutations can reduce the activities of vacuolar lipases and proteases to varying degrees (Kane, 2006 blue right-pointing triangle; Forgac, 2007 blue right-pointing triangle), which can influence the half-lives of GFP-Osh1p and its GFP fragment differently. V-ATPase mutants still show some proteolytic activity in their vacuoles but at much lower levels than wild-type cells. Thus, V-ATPase mutants might still be able to cleave the sensitive GFP-Osh1p linker but degrade the more resistant GFP more slowly than wild types. This can hide a defect in PMN since even if less GFP-Osh1p is transferred into vacuoles delayed degradation of its GFP fragment could lead to its overproportional accumulation. The data of Krick et al. (Figure 5C) are consistent with this notion because they show two important differences between V-ATPase mutants and the wild-type: Wild-type cells contain virtually no fragment before induction of PMN. Induction dramatically enhances the transfer of GFP-Osh1p into wild-type cells, producing the fragment. In contrast, the V-ATPase knockout Δvma2 shows high levels of GFP fragment already before induction of PMN and these remain unaltered upon PMN induction. Furthermore, Δvph1 mutants, which retain limited vacuolar acidification due to the presence of the Stv1p-containing V-ATPase complexes (Manolson et al., 1994 blue right-pointing triangle), show low levels of GFP fragment before induction. On induction of PMN, GFP fragment accumulates but with a significant delay relative to wild-type cells. The interpretation of the proteolysis-based assay of PMN is hence not straightforward for mutants influencing the hydrolytic capacity of the vacuoles. In these cases the assay must be validated by determining the half-lives of the GFP-fusion and the GFP-fragment in each mutant.
Cells overexpressing N-terminally tagged Nvj1 excluded Vph1p from vacuolar sites that were not adjacent to the nucleus. These random exclusion sites were next to Nvj1p patches but far from the nucleus. This confirms that contact with nuclear material is dispensable for making contacts between vacuoles and the ER or outer nuclear membrane (Roberts et al., 2003 blue right-pointing triangle). Furthermore, it shows that differentiation of the contact sites into “NV” junctions (or their ER-equivalents) is independent of the nucleus. That this “differentiation” can be regarded as a separate step in the establishment of NV junctions is supported by the fact that inhibition of V-ATPase pump activity by concanamycin A barely reduces the frequency of nuclear-vacuolar contacts but strongly reduces Vph1p exclusion from these sites (Figure 7).
Integrating our results with those of earlier studies (Kvam and Goldfarb, 2007 blue right-pointing triangle; Krick et al., 2008 blue right-pointing triangle), we can hence extend the model of NV junction formation and PMN by a new step. Whereas V-ATPase activity is not required for forming nuclear-vacuolar contact sites, further evolution into NV junctions that generate a diffusion barrier on the vacuolar side does depend on it. This diffusion barrier likely depends on a specific lipidic environment. The deformation of NV junctions into PMN sites and the subsequent formation of vesicles from them requires the electrochemical potential and completion of the process depends on Atg genes.
Supplementary Material
[Supplemental Materials]
ACKNOWLEDGMENTS
We thank Nicole Gas and Claudio de Virgilio for strains and Lydie Michaillat for critical reading of the manuscript.
Footnotes
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E09-09-0782) on October 13, 2010.
  • Ashman W. H., Barbuch R. J., Ulbright C. E., Jarrett H. W., Bard M. Cloning and disruption of the yeast C-8 sterol isomerase gene. Lipids. 1991;26:628–632. [PubMed]
  • Baars T. L., Petri S., Peters C., Mayer A. Role of the V-ATPase in regulation of the vacuolar fission-fusion equilibrium. Mol. Biol. Cell. 2007;18:3873–3882. [PMC free article] [PubMed]
  • Bayer M. J., Reese C., Buhler S., Peters C., Mayer A. Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel. J. Cell Biol. 2003;162:211–222. [PMC free article] [PubMed]
  • Beh C. T., Cool L., Phillips J., Rine J. Overlapping functions of the yeast oxysterol-binding protein homologues. Genetics. 2001;157:1117–1140. [PubMed]
  • Beh C. T., Rine J. A role for yeast oxysterol-binding protein homologs in endocytosis and in the maintenance of intracellular sterol-lipid distribution. J. Cell Sci. 2004;117:2983–2996. [PubMed]
  • Bowman B. J., Bowman E. J. Mutations in subunit C of the vacuolar ATPase confer resistance to bafilomycin and identify a conserved antibiotic binding site. J. Biol. Chem. 2002;277:3965–3972. [PubMed]
  • Cowles C. R., Snyder W. B., Burd C. G., Emr S. D. Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component. EMBO J. 1997;16:2769–2782. [PubMed]
  • D'Mello N.P., Childress A. M., Franklin D. S., Kale S. P., Pinswasdi C., Jazwinski S. M. Cloning and characterization of LAG1, a longevity-assurance gene in yeast. J. Biol. Chem. 1994;269:15451–15459. [PubMed]
  • De Virgilio C., Loewith R. The TOR signalling network from yeast to man. Int J Biochem. Cell Biol. 2006;38:1476–1481. [PubMed]
  • Falguieres T., Luyet P. P., Gruenberg J. Molecular assemblies and membrane domains in multivesicular endosome dynamics. Exp. Cell Res. 2009;315:1567–1573. [PubMed]
  • Forgac M. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat. Rev. Mol. Cell Biol. 2007;8:917–929. [PubMed]
  • Gadal O., Strauss D., Kessl J., Trumpower B., Tollervey D., Hurt E. Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Mol. Cell. Biol. 2001;21:3405–3415. [PMC free article] [PubMed]
  • Grossmann G., Opekarova M., Malinsky J., Weig-Meckl I., Tanner W. Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast. EMBO J. 2007;26:1–8. [PubMed]
  • Guan X. L., Souza C. M., Pichler H., Dewhurst G., Schaad O., Kajiwara K., Wakabayashi H., Ivanova T., Castillon G. A., Piccolis M., Abe F., Loewith R., Funato K., Wenk M. R., Riezman H. Functional interactions between sphingolipids and sterols in biological membranes regulating cell physiology. Mol. Biol. Cell. 2009;20:2083–2095. [PMC free article] [PubMed]
  • Herman P., Malinsky J., Plasek J., Vecer J. Pseudo real-time method for monitoring of the limiting anisotropy in membranes. J. Fluoresc. 2004;14:79–85. [PubMed]
  • Hiesinger P. R., Fayyazuddin A., Mehta S. Q., Rosenmund T., Schulze K. L., Zhai R. G., Verstreken P., Cao Y., Zhou Y., Kunz J., Bellen H. J. The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell. 2005;121:607–620. [PMC free article] [PubMed]
  • Hothorn M., Neumann H., Lenherr E. D., Wehner M., Rybin V., Hassa P. O., Uttenweiler A., Reinhardt M., Schmidt A., Seiler J., Ladurner A. G., Herrmann C., Scheffzek K., Mayer A. Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase. Science. 2009;324:513–516. [PubMed]
  • Huss M., Ingenhorst G., Konig S., Gassel M., Drose S., Zeeck A., Altendorf K., Wieczorek H. Concanamycin A, the specific inhibitor of V-ATPases, binds to the V(o) subunit c. J. Biol. Chem. 2002;277:40544–40548. [PubMed]
  • Janke C., Magiera M. M., Rathfelder N., Taxis C., Reber S., Maekawa H., Moreno-Borchart A., Doenges G., Schwob E., Schiebel E., Knop M. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004;21:947–962. [PubMed]
  • Jones E. W., Zubenko G. S., Parker R. R. PEP4 gene function is required for expression of several vacuolar hydrolases in Saccharomyces cerevisiae. Genetics. 1982;102:665–677. [PubMed]
  • Julicher F., Lipowsky R. Domain-induced budding of vesicles. Phys Rev Lett. 1993;70:2964–2967. [PubMed]
  • Julicher F., Lipowsky R. Shape transformations of vesicles with intramembrane domains. Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics. 1996;53:2670–2683. [PubMed]
  • Kane P. M. The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase. Microbiol. Mol. Biol. Rev. 2006;70:177–191. [PMC free article] [PubMed]
  • Kawai A., Uchiyama H., Takano S., Nakamura N., Ohkuma S. Autophagosome-lysosome fusion depends on the pH in acidic compartments in CHO cells. Autophagy. 2007;3:154–157. [PubMed]
  • Kelly S. L., Lamb D. C., Baldwin B. C., Corran A. J., Kelly D. E. Characterization of Saccharomyces cerevisiae CYP61, sterol delta22-desaturase, and inhibition by azole antifungal agents. J. Biol. Chem. 1997;272:9986–9988. [PubMed]
  • Kohlwein S. D., Eder S., Oh C. S., Martin C. E., Gable K., Bacikova D., Dunn T. Tsc13p is required for fatty acid elongation and localizes to a novel structure at the nuclear-vacuolar interface in Saccharomyces cerevisiae. Mol. Cell. Biol. 2001;21:109–125. [PMC free article] [PubMed]
  • Korade Z., Kenworthy A. K. Lipid rafts, cholesterol, and the brain. Neuropharmacology. 2008;55:1265–1273. [PMC free article] [PubMed]
  • Krick R., Muehe Y., Prick T., Bremer S., Schlotterhose P., Eskelinen E. L., Millen J., Goldfarb D. S., Thumm M. Piecemeal microautophagy of the nucleus requires the core macroautophagy genes. Mol. Biol. Cell. 2008;19:4492–4505. [PMC free article] [PubMed]
  • Kvam E., Gable K., Dunn T. M., Goldfarb D. S. Targeting of Tsc13p to nucleus-vacuole junctions: a role for very-long-chain fatty acids in the biogenesis of microautophagic vesicles. Mol. Biol. Cell. 2005;16:3987–3998. [PMC free article] [PubMed]
  • Kvam E., Goldfarb D. S. Nvj1p is the outer-nuclear-membrane receptor for oxysterol-binding protein homolog Osh1p in Saccharomyces cerevisiae. J. Cell Sci. 2004;117:4959–4968. [PubMed]
  • Kvam E., Goldfarb D. S. Nucleus-vacuole junctions in yeast: anatomy of a membrane contact site. Biochem. Soc. Trans. 2006a;34:340–342. [PubMed]
  • Kvam E., Goldfarb D. S. Structure and function of nucleus-vacuole junctions: outer-nuclear-membrane targeting of Nvj1p and a role in tryptophan uptake. J. Cell Sci. 2006b;119:3622–3633. [PubMed]
  • Kvam E., Goldfarb D. S. Nucleus-vacuole junctions and piecemeal microautophagy of the nucleus in. S. cerevisiae. Autophagy. 2007;3:85–92. [PubMed]
  • Lee M. C., Orci L., Hamamoto S., Futai E., Ravazzola M., Schekman R. Sar1p N-terminal helix initiates membrane curvature and completes the fission of a COPII vesicle. Cell. 2005;122:605–617. [PubMed]
  • Levine B., Klionsky D. J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell. 2004;6:463–477. [PubMed]
  • Levine T. Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions. Trends Cell Biol. 2004;14:483–490. [PubMed]
  • Levine T. P., Munro S. Dual targeting of Osh1p, a yeast homologue of oxysterol-binding protein, to both the Golgi and the nucleus-vacuole junction. Mol. Biol. Cell. 2001;12:1633–1644. [PMC free article] [PubMed]
  • Liegeois S., Benedetto A., Garnier J. M., Schwab Y., Labouesse M. The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans. J. Cell Biol. 2006;173:949–961. [PMC free article] [PubMed]
  • Liu J., Brown C. R., Chiang H. L. Degradation of the gluconeogenic enzyme fructose-1,6-bisphosphatase is dependent on the vacuolar ATPase. Autophagy. 2005;1:146–156. [PubMed]
  • Malinska K., Malinsky J., Opekarova M., Tanner W. Visualization of protein compartmentation within the plasma membrane of living yeast cells. Mol. Biol. Cell. 2003;14:4427–4436. [PMC free article] [PubMed]
  • Manolson M. F., Wu B., Proteau D., Taillon B. E., Roberts B. T., Hoyt M. A., Jones E. W. STV1 gene encodes functional homologue of 95-kDa yeast vacuolar H(+)-ATPase subunit Vph1p. J. Biol. Chem. 1994;269:14064–14074. [PubMed]
  • Mao C., Xu R., Bielawska A., Obeid L. M. Cloning of an alkaline ceramidase from Saccharomyces cerevisiae. An enzyme with reverse (CoA-independent) ceramide synthase activity. J. Biol. Chem. 2000;275:6876–6884. [PubMed]
  • Miller J. P., Lo R. S., Ben-Hur A., Desmarais C., Stagljar I., Noble W. S., Fields S. Large-scale identification of yeast integral membrane protein interactions. Proc. Natl. Acad. Sci. USA. 2005;102:12123–12128. [PubMed]
  • Mitchell A. G., Martin C. E. Fah1p, a Saccharomyces cerevisiae cytochrome b5 fusion protein, and its Arabidopsis thaliana homolog that lacks the cytochrome b5 domain both function in the alpha-hydroxylation of sphingolipid-associated very long chain fatty acids. J. Biol. Chem. 1997;272:28281–28288. [PubMed]
  • Mukaiyama H., Baba M., Osumi M., Aoyagi S., Kato N., Ohsumi Y., Sakai Y. Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure. Mol. Biol. Cell. 2004;15:58–70. [PMC free article] [PubMed]
  • Muller O., Neumann H., Bayer M. J., Mayer A. Role of the Vtc proteins in V-ATPase stability and membrane trafficking. J. Cell Sci. 2003;116:1107–1115. [PubMed]
  • Munn A. L., Heese-Peck A., Stevenson B. J., Pichler H., Riezman H. Specific sterols required for the internalization step of endocytosis in yeast. Mol. Biol. Cell. 1999;10:3943–3957. [PMC free article] [PubMed]
  • Nishi T., Forgac M. The vacuolar (H+)-ATPases–nature's most versatile proton pumps. Nat. Rev. Mol. Cell Biol. 2002;3:94–103. [PubMed]
  • Oh C. S., Toke D. A., Mandala S., Martin C. E. ELO2 and ELO3, homologues of the Saccharomyces cerevisiae ELO1 gene, function in fatty acid elongation and are required for sphingolipid formation. J. Biol. Chem. 1997;272:17376–17384. [PubMed]
  • Pan X., Roberts P., Chen Y., Kvam E., Shulga N., Huang K., Lemmon S., Goldfarb D. S. Nucleus-vacuole junctions in Saccharomyces cerevisiae are formed through the direct interaction of Vac8p with Nvj1p. Mol. Biol. Cell. 2000;11:2445–2457. [PMC free article] [PubMed]
  • Peri F., Nusslein-Volhard C. Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell. 2008;133:916–927. [PubMed]
  • Peters C., Bayer M. J., Buhler S., Andersen J. S., Mann M., Mayer A. Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature. 2001;409:581–588. [PubMed]
  • Pichler H., Riezman H. Where sterols are required for endocytosis. Biochim. Biophys. Acta. 2004;1666:51–61. [PubMed]
  • Proszynski T. J., Klemm R. W., Gravert M., Hsu P. P., Gloor Y., Wagner J., Kozak K., Grabner H., Walzer K., Bagnat M., Simons K., Walch-Solimena C. A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast. Proc. Natl. Acad. Sci. USA. 2005;102:17981–17986. [PubMed]
  • Qie L., Nagiec M. M., Baltisberger J. A., Lester R. L., Dickson R. C. Identification of a Saccharomyces gene, LCB3, necessary for incorporation of exogenous long chain bases into sphingolipids. J. Biol. Chem. 1997;272:16110–16117. [PubMed]
  • Ramos C., Rafikova E. R., Melikov K., Chernomordik L. V. Transmembrane proteins are not required for early stages of nuclear envelope assembly. Biochem. J. 2006;400:393–400. [PubMed]
  • Roberts P., Moshitch-Moshkovitz S., Kvam E., O'Toole E., Winey M., Goldfarb D. S. Piecemeal microautophagy of nucleus in Saccharomyces cerevisiae. Mol. Biol. Cell. 2003;14:129–141. [PMC free article] [PubMed]
  • Ryan M., Graham L. A., Stevens T. H. Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum. Mol. Biol. Cell. 2008;19:5131–5142. [PMC free article] [PubMed]
  • Sackmann E., Feder T. Budding, fission and domain formation in mixed lipid vesicles induced by lateral phase separation and macromolecular condensation. Mol. Membr. Biol. 1995;12:21–28. [PubMed]
  • Sakai Y., Oku M., van der Klei I. J., Kiel J. A. Pexophagy: autophagic degradation of peroxisomes. Biochim. Biophys. Acta. 2006;1763:1767–1775. [PubMed]
  • Schaffer E., Thiele U. Dynamic domain formation in membranes: thickness-modulation-induced phase separation. Eur. Phys. J. E Soft Matter. 2004;14:169–175. [PubMed]
  • Schmid S., Fuchs R., Kielian M., Helenius A., Mellman I. Acidification of endosome subpopulations in wild-type Chinese hamster ovary cells and temperature-sensitive acidification-defective mutants. J. Cell Biol. 1989;108:1291–1300. [PMC free article] [PubMed]
  • Severs N. J., Jordan E. G., Williamson D. H. Nuclear pore absence from areas of close association between nucleus and vacuole in synchronous yeast cultures. J. Ultrastruct. Res. 1976;54:374–387. [PubMed]
  • Simons K., Ehehalt R. Cholesterol, lipid rafts, and disease. J. Clin. Invest. 2002;110:597–603. [PMC free article] [PubMed]
  • Sun-Wada G. H., Toyomura T., Murata Y., Yamamoto A., Futai M., Wada Y. The a3 isoform of V-ATPase regulates insulin secretion from pancreatic beta-cells. J. Cell. Sci. 2006;119:4531–4540. [PubMed]
  • Uttenweiler A., Schwarz H., Neumann H., Mayer A. The vacuolar transporter chaperone (VTC) complex is required for microautophagy. Mol. Biol. Cell. 2007;18:166–175. [PMC free article] [PubMed]
  • Watkins P. A., Lu J. F., Steinberg S. J., Gould S. J., Smith K. D., Braiterman L. T. Disruption of the Saccharomyces cerevisiae FAT1 gene decreases very long-chain fatty acyl-CoA synthetase activity and elevates intracellular very long-chain fatty acid concentrations. J. Biol. Chem. 1998;273:18210–18219. [PubMed]
  • Wenk M. R., De Camilli P. Protein-lipid interactions and phosphoinositide metabolism in membrane traffic: insights from vesicle recycling in nerve terminals. Proc. Natl. Acad. Sci. USA. 2004;101:8262–8269. [PubMed]
  • Yamamoto A., Tagawa Y., Yoshimori T., Moriyama Y., Masaki R., Tashiro Y. Bafilomycin A1 prevents maturation of autophagic vacuoles by inhibiting fusion between autophagosomes and lysosomes in rat hepatoma cell line, H-4-II-E cells. Cell Struct. Funct. 1998;23:33–42. [PubMed]
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