QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

Vol. 19, Issue 12, 5131-5142, December 2008

This Article Abstract Full Text (PDF) All Versions of this Article: E08-06-0629v1 19/12/5131    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ryan, M. Articles by Stevens, T. H. Search for Related Content PubMed PubMed Citation Articles by Ryan, M. Articles by Stevens, T. H.

Voa1p Functions in V-ATPase Assembly in the Yeast Endoplasmic Reticulum

Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

Submitted June 20, 2008; Revised August 20, 2008; Accepted September 8, 2008
Monitoring Editor: Sandra Lemmon

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V₁ sector catalyzes ATP hydrolysis and the V₀ sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V₀ assembly. We have discovered a fifth V₀ assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V₀ assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V₀ assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V₀–Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V₀ subunits c, c', and c''. On assembly of the remaining three V₀ subunits (a, d, and e) into the V₀ complex, Voa1p dissociates from the now fully assembled V₀–Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V₀ assembly in the ER, but then it dissociates before exit of the V₀–Vma21p complex from the ER for transport to the Golgi compartment.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The V-type proton-translocating ATPase (V-ATPase) is a ubiquitous multisubunit protein complex present in all eukaryotic cells from yeast to humans (Beyenbach and Wieczorek, 2006). In eukaryotes, the V-ATPase is localized to the membranes of intracellular organelles such as Golgi, endosomes, vesicles, and vacuole or lysosome (Forgac, 2007). The function of the V-ATPase complex is to translocate protons across a lipid bilayer by an ATP-driven rotary mechanism (Hirata et al., 2003; Imamura et al., 2003; Yokoyama et al., 2003). The ATP-driven acidification of the lumen of these organelles by the resident V-ATPase plays a role in regulating a diverse range of cellular processes such as receptor–ligand interactions, accumulation of neurotransmitter in synaptic vesicles, and intracellular trafficking (Forgac, 2007). In addition to the localization throughout the Golgi/endosome/lysosomal network, the V-ATPase also can be found on the plasma membrane in specialized cells such as osteoclasts, kidney cells, and neurons in which it functions in bone resorption, acid–base balance, and neurotransmitter accumulation (Forgac, 2007).

The best-characterized member of the V-ATPase family is present in the yeast Saccharomyces cerevisiae (Graham et al., 2003; Kane, 2006), because the V-ATPase is essential for cell viability in all eukaryotes except fungi. In yeast, the V-ATPase acidifies the lumen of the vacuole, Golgi, and endosomes by establishing an ATP-driven proton gradient across the membrane. In turn, various membrane-bound ion transporters/proton exchangers use this gradient to sequester metal ions to the vacuole and other cellular organelles. The combined action of the V-ATPase and membrane transporters plays a key role in maintaining cellular homoeostasis in yeast (Kane, 2006).

The V-ATPase complex can be functionally separated into two subcomplexes: the V₁ domain involved directly in ATP hydrolysis and the V₀ domain responsible for the translocation of protons across a membrane bilayer. The V-ATPase complex in yeast is composed of 14 integral and peripheral membrane subunits. The V₀ domain is assembled in the endoplasmic reticulum (ER) and is composed of subunits a, c, c', c'', d, and e encoded by the yeast genes VPH1 or STV1, VMA3, VMA11, VMA16, VMA6 and VMA9, respectively. The V₁ domain contains subunits A, B, C, D, E, F, G, and H encoded by the yeast genes VMA1, VMA2, VMA5, VMA8, VMA4, VMA7, VMA10, and VMA13. Subunits A, B, E, G, and c are present in the V-ATPase at more than one copy per complex (Powell et al., 2000; Ohira et al., 2006; Kitagawa et al., 2008).

In yeast, there are two populations of V-ATPase complexes that are identical except for the subunit a isoform present in each complex (Manolson et al., 1992, 1994). The presence of the Vph1p subunit a isoform directs localization of the V-ATPase complex to the membrane of the vacuole, and inclusion of the Stv1p subunit a isoform results in the retention of the V-ATPase complex on Golgi and endosomal membranes (Manolson et al., 1994). The targeting information determining the cellular localization of the Vph1p and Stv1p containing V-ATPase complexes resides in the N-terminal half of each subunit a isoform (Kawasaki-Nishi et al., 2001).

Yeast cells lacking a functional V-ATPase display a characteristic set of growth phenotypes, collectively referred to as the Vma^– phenotype; they are able to grow on rich medium buffered to pH 5.0 but are unable to grow on more alkaline media buffered to pH 7.5 or in the presence of elevated levels of calcium (Kane, 2006). V-ATPase–deficient cells are unable to acidify their vacuoles, a phenomenon that can be observed at the cellular level by failure to accumulate the fluorescent dye quinacrine in the lumen of the vacuoles (Weisman et al., 1987). Failure to assemble the V₁ subcomplex due to loss of any one of the V₁ subunits does not affect the assembly and targeting of the V₀ subcomplex in yeast (Graham et al., 2003). Cells lacking both Vph1p and Stv1p or any one of the other V₀ subunits, fail to assemble a complete V₀ subcomplex, and this partially assembled complex does not exit the ER. Yeast cells lacking any of the other V₀ subunits (except Stv1p) exhibit reduced cellular levels of Vph1p due to increased turnover of this subunit when it is unable to assemble properly into the V₀ subcomplex and exit the ER.

In addition to proteins that comprise the fully functional V-ATPase complex, proteins have been identified and characterized that play a role in the assembly of the V-ATPase. Although the V₁ and V₀ subcomplexes are capable of assembling independently in cells, there is evidence that V₁ subunits associate very early with V₀ subunits in the ER (Kane et al., 1999; Kane, 2006). One interpretation of these data are that although V₁ subunit association is not required for V₀ assembly in the ER, the normal pathway for V-ATPase assembly could involve association of certain V₁ subunits with components of the V₀ subcomplex during its assembly in the ER. Intriguingly, the regulator of the ATPase of vacuolar and endosomal membranes (RAVE) complex (Seol et al., 2001) is required for the stable association of the V₁ domain with the V₀ domain during biosynthesis (Smardon et al., 2002) and for the glucose-regulated reassembly of the V₁ and V₀ domains at the vacuole membrane after glucose depletion (Smardon et al., 2002; Smardon and Kane, 2007).

Assembly of the V₀ subcomplex occurs in the ER and requires four distinct ER-localized assembly factors (Forgac, 2007). The peripheral membrane protein Vma22p, and the integral membrane proteins Vma12p and Vma21p are dedicated to the assembly of the V₀ domain in the ER (Hirata et al., 1993; Hill and Stevens, 1994; Hill and Stevens, 1995; Jackson and Stevens, 1997). In the absence of any one of these proteins, the V₀ subunits fail to assemble properly in the ER, and the V₀ subcomplex does not exit the ER (Graham et al., 2003). In addition, Vma21p has been shown to escort the V-ATPase complex out of the ER, separating from the complex in a post-ER organelle and then retrieved back to the ER by cellular machinery recognizing the C-terminal dilysine motif on Vma21p (Malkus et al., 2004). A fourth V-ATPase assembly factor encoded by the PKR1 gene has been found to play a role in how efficiently the V₀ subcomplex assembles in the ER. In the absence of Pkr1p, yeast cells possess a very low level (5–10%) of properly assembled and fully functional V-ATPase complexes that exit the ER and are found on the membrane of the vacuole (Davis-Kaplan et al., 2006).

The V-ATPase assembly factors have not yet been as extensively studied as the component proteins of the V-ATPase itself. Factors that function in a subtle manner are difficult to discover because in their absence cells may not exhibit a full Vma^– phenotype and are therefore not recognized by conventional phenotypic screening of mutant yeast strains. We chose instead to search for proteins that physically interact with the V₀ complex, and then examine these for their effect on assembly. Here, we report the identification and characterization of an ER-localized protein, Voa1p, that functions with Vma21p in assembly of the V-ATPase. Voa1p was identified by mass spectrometry as a component of the partially assembled V₀–Vma21p complex in the ER. Voa1p localizes to the ER due to a C-terminal dilysine motif, and it associates with Vma21p when the V₀ subcomplex is partially assembled, but not when this complex is fully assembled. Cells lacking Voa1p have slightly reduced V-ATPase activity but do not show a Vma^– phenotype. An early V-ATPase assembly function for Voa1p is revealed in yeast cells expressing an ER retrieval-deficient form of the assembly factor Vma21p. In these cells, the loss of Voa1p results in a dramatic growth phenotype due to the lack of properly assembled V-ATPase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Plasmids and Yeast Strains
Standard protocols for molecular biology were followed (Sambrook and Russel, 2001). Plasmids used in this study are listed in Table 2. VOA1 plus flanking sequence was amplified by polymerase chain reaction (PCR) from BY4741 (Invitrogen, Carlsbad, CA) genomic DNA. The PCR product was digested with HindIII and MscI to produce a fragment containing VOA1 plus 821 base pairs and 245 base pairs of 5'- and 3'-flanking sequence, respectively. This fragment was inserted into HindIII and SmaI sites of pRS316 to give pMR063. A single hemagglutinin (HA) epitope tag was inserted after Asp25 of VOA1 by using pMR063 as template by overlap extension PCR (Sambrook and Russel, 2001) with two primers having overlapping HA coding sequence at their 5' ends, and two pRS316-specific flanking primers. The PCR product, digested with HindIII and XbaI of the pRS316 multiple cloning site, was ligated into the same sites of the same vector to give pMR072. The same technique was used to place an HA tag after the Met1 start codon of VOA1. A modified QuikChange protocol (Zheng et al., 2004) was used to introduce K262Q, K263Q mutations in pMR072 to give pMR0712. A single c-myc tag was placed after Asp25 in VOA1 by inverse PCR (Ansaldi et al., 1996) by using pMR063 as template and two primers with 5'-overlapping c-myc coding sequence containing a silent BglII site. BglII-digested PCR product was ligated to produce pMR0713. All plasmids inserts prepared were confirmed by DNA sequencing. pLG271 was generated by isolating the VMA21::HA insert from pLG233 (pRS316 VMA21::HA) by digestion with SalI and Not1 and ligating into pRS315 digested with the same enzymes. pGF06 was generated by gap-repair of VPH1::GFP::HIS5 from the genome of the appropriate strain from the Yeast GFP Clone Collection (Invitrogen) by transformation with EcoRI-digested pDJ53 (pRS316 VPH1) and selection on SD minus uracil and histidine.

Affinity Purification for Mass Spectrometry
vph1 (KHY31) yeast cells expressing Vma21p-HA from plasmid pKH28 and control strain vph1 without plasmid were grown in SD medium, diluted to 0.4 OD₆₀₀/ml in YEPD pH 5.0, and grown to OD₆₀₀ = 1. Cells (500 OD) were collected by centrifugation and suspended on ice in phosphate-buffered saline (PBS) (126 mM NaCl, 2.5 mM KCl, and 10 mM Na₂HPO₄/KH₂PO₄, pH 7.1) plus protease inhibitors (EDTA-free 1x Complete; Roche Diagnostics, Indianapolis, IN), 2 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride). Glass bead lysates were obtained using a vortex mixer (Catley, 1988), debris and unlysed cells were removed by centrifugation (500 x g; 10 min; 4°C), and the pellet was washed once. The combined supernatants were centrifuged (13,000 x g; 20 min; 4°C), and the pellet, constituting the membrane fraction, was washed once. Membranes were solubilized by suspension in 4 ml of P13 buffer (1% C₁₂E₉; Sigma-Aldrich, St. Louis, MO) in PBS plus protease inhibitors) and incubated on ice for 1 h with occasional agitation. The solubilized membrane fraction was cleared by centrifugation (16,000 x g; 15 min; 4°C), and the supernatant was transferred to a disposable column containing 40 µl of washed anti-HA agarose (80 µl of 50% slurry, Sigma-Aldrich or Covance Research Products, Berkeley, CA). Proteins were batch adsorbed for 2 h at 4°C with gentle agitation. The affinity resin was washed at 4°C with 4 x 1 ml of P13 buffer and 0.75 ml of Tris-buffered saline (TBS) (150 mM NaCl and 50 mM Tris-HCl, pH 7.5) followed by light centrifugation (1000 x g; 10 s). Columns were moved to room temperature and a two-step elution (60 µl, 10 min followed by 75 µl, 20 min) was used to recover bound proteins. The elution buffer used was either 0.4 mg/ml HA peptide (Sigma-Aldrich), 0.05% C₁₂E₉ in TBS, or 8 M urea, 0.05% C₁₂E₉ in TBS. Affinity purification from vph1 (KHY31) cells with and without HA-Voa1p expressed from plasmid (pMR072) was done in the same manner, except 140 OD of cells were used, and the protocol was scaled accordingly. The elution buffer used was 0.1 M glycine-HCl, pH 2.8. Affinity extracts were separated on analytical 10–20% SDS-polyacrylamide gel electrophoresis (PAGE) minigels (Bio-Rad, Hercules, CA) and silver stained (SilverQuest; Invitrogen) for inspection, and separated on preparative gels stained with silver or Coomassie (SimplyBlue SafeStain; Invitrogen) for mass spectrometry.

Identification of Proteins by Mass Spectrometry
Sections were excised from SDS-PAGE gels, washed with water, and "in gel" protein digestion with trypsin was carried out using published protocols (Shevchenko et al., 1996), both with and without reduction/alkylation before the tryptic digest. Peptide samples were concentrated and desalted using microC₁₈ ZipTips (Millipore, Billerica, MA) and dried. Mass spectrometry data were collected and evaluated at the Proteomics Shared Resource (Fred Hutchinson Cancer Research Center, Seattle, WA) and at the University of Oregon Proteomics Facility (Eugene, OR). The samples were resuspended and analyzed by liquid chromatography (LC)/electrospray ionization tandem mass spectrometry (MS/MS), with a two-dimensional nano-high-performance liquid chromatograph (Eksigent, Dublin, CA) coupled to a LTQ-FT (Thermo Scientific, Waltham, MA) mass spectrometer using an instrument configuration as described (Yi et al., 2003). In-line de-salting (5-µm, 200 Å Magic C₁₈AQ resin, Michrom Bioresources, Auburn, CA) was followed by peptide separations on a 75 µm x 250 mm Picofrit (New Objective, Woburn, MA) reversed-phase column packed with Magic 5-µm 100 Å C₁₈AQ resin (Michrom Bioresources) directly mounted on the electrospray ion source. A 400 nl/min gradient from 2 to 40% acetonitrile in 0.1% formic acid followed by 80% acetonitrile, 0.1% formic acid was used for chromatographic separations. The linear ion trap Fourier transform (FT) instrument was operated in the data-dependent mode, switching automatically between MS survey scans in the FT analyzer and MS/MS spectra acquisition in the linear ion trap. The five most intense ions from the FT full scan were selected for fragmentation. Selected ions were dynamically excluded for 45 s. Collected mass spectrometry data were subjected to the protein database search algorithm X!Tandem (Craig and Beavis, 2004) configured with the COMET score module plug-in (MacLean et al., 2006), and identifications were validated using PeptideProphet (Keller et al., 2002). The protein database used for peptide identification was the translated open reading frame FASTA file from the Saccharomyces Genome Database (www.yeastgenome.org) downloaded 1 November 2006. Peptide identification results were filtered and sorted in Computational Proteomics Analysis System (Rauch et al., 2006) by using a Peptide Prophet minimum probability cut-off of 0.9 and an ion% cut-off of 0.15.

Protein Extraction from Membranes
A glass-bead lysate was prepared from voa1::Hyg^r cells expressing HA-Voa1p (pMR072) as described under Affinity Purification. After removal of large particles by centrifugation, the sample was divided into thirds (22 OD equivalents each). Membrane pellets were obtained by centrifugation (13,000 x g; 20 min; 4°C) and washed once. Membrane protein solubilization was done essentially as published previously (Kaiser et al., 2002). Briefly, the pellets were suspended in 0.2 ml of either PBS or 0.1 M NaCO₃, pH 11.5, or 1% Triton X-100 in PBS with protease inhibitors included in all three treatments. The suspensions were incubated on ice for 45 min and then centrifuged (100,000 x g; 30 min; 4°C). The supernatant fractions were removed and saved separately; the pellet fractions were reconstituted in their respective treatment conditions to the same volume as the supernatant fraction. All fractions were adjusted to contain 0.8x sample buffer (1x sample buffer: 8 M urea, 5% SDS, 5% β-meraptoethanol, 0.01% bromphenol blue, 40 mM Tris-HCl, pH 6.8) and resolved by SDS-PAGE. Western blots were prepared using the following primary antibodies: monoclonal anti-Pho8p (1D3; Invitrogen), rabbit anti-Vma6p (Bauerle et al., 1993), or monoclonal anti-HA (Sigma-Aldrich). Goat anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase were used (Jackson ImmunoResearch Laboratories, West Grove, PA) and detected by enhanced chemiluminescence (ECL).

Endoglycosidase H (Endo H) Assay
Solubilized membranes were prepared from voa1::Kan^r cells expressing HA-Voa1p (pMR072) as described under Affinity Purification, except glass bead lysis was in 10 mM Na₂HPO₄/KH₂PO₄, pH 7.1, and membranes were solubilized with 1% C₁₂E₉ in the same buffer. Both treatments were done in the presence of protease inhibitors. A portion of the cleared, solubilized membrane fraction was withdrawn to serve as untreated control. Of the remainder, 2 OD₆₀₀ equivalents were denatured in 0.5% SDS, 1% C₁₂E₉, 40 mM dithiothreitol, 3.5 mM phosphate buffer, pH 7.1, in a final volume of 40 µl and incubated at 55°C for 15 min. Then, 32 µl of 1.25% C₁₂E₉, 125 mM sodium citrate, pH 5.5, 2.5x protease inhibitors were added, mixed, and 36 µl was dispensed into tubes containing 4 µl of Endo H_f (New England Biolabs, Ipswich, MA) or 4 µl of Endo H_f-storage buffer (50 mM NaCl, 2 mM EDTA, and 50 mM Tris-HCl, pH 7.5). The resulting 40-µl reaction and control were incubated at 37°C for 3 h. Samples were adjusted to contain 0.8x sample buffer and analyzed by SDS-PAGE followed by immunoblotting by using monoclonal anti-HA primary antibody (Sigma-Aldrich) and horseradish conjugated anti-mouse secondary antibody (Jackson ImmunoResearch Laboratories). Blots were developed using ECL.

Whole Cell Extract Preparation and Immunoblotting
Yeast cultures were raised in SD medium, diluted to 0.4 OD₆₀₀/ml in YEPD, pH 5.0, and grown to OD₆₀₀ = 1. Ten OD₆₀₀ of cells were harvested, chilled on ice and suspended in 0.1 ml of 1x sample buffer. Ice-cold glass beads (0.1 ml) were added and the suspension vortexed 4x for 1 min with 1-min cooling intervals on ice. Lysates were removed by pipetting and cleared by centrifugation. The supernatants, constituting the whole cell extracts, were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies. Monoclonal primary antibodies used were anti-HA (Covance Research Products), anti-Vph1p (10D7; Invitrogen), anti-Vma1p (8B1; Invitrogen), and anti-Dpm1p (5C5; Invitrogen), with secondary horseradish-conjugated anti-mouse antibody (Jackson ImmunoResearch Laboratories), followed by ECL detection.

Native Immunoprecipitation
Analysis of c-myc-Voa1p coimmunoprecipitation with Vma21p-HA was done following the Affinity Purification for Mass Spectrometry protocol with the following modifications: 100 OD₆₀₀ of cells were used, and the protocol was scaled proportionally. A portion of the cleared, solubilized membrane fraction was adjusted to 0.8x sample buffer to serve as measure of protein input, the remainder was adsorbed to 10 µl of anti-HA agarose (Sigma-Aldrich). The resin was washed, and bound proteins were recovered by elution with 2x 30 µl of 1x sample buffer but omitting β-meraptoethanol and bromphenol blue. Input and immunoprecipitates were separated on SDS-PAGE gels and analyzed by Western blotting by using monoclonal anti-Vph1 (10D7) and polyclonal anti-c-myc (Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies. Equal amounts of Vma21p-HA were present in input fractions well as immunoprecipitate fractions from all strains tested as ascertained by probing blots with anti-HA antibodies.

Fluorescence Microscopy
The acidification of vacuoles in various yeast strains was visualized using the lysosomotropic fluorescent dye quinacrine. Quinacrine staining of live yeast cells was conducted as described previously (Weisman et al., 1987; Flannery et al., 2004). Quinacrine was used at a final concentration of 200 µM prepared in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 7.7, to stain acidified vacuoles, whereas concanavalin A tetramethylrhodamine (Invitrogen) was added at a final concentration of 50 µg/ml to allow for fluorescent visualization of the cell surface.

Localization of green fluorescent protein (GFP)-tagged proteins and stained DNA were determined by fluorescence microscopy. One milliliter of exponentially growing cells was harvested by centrifugation for 30 s and resuspended in 100 mM MES, pH 7.7, 2% glucose, and 5 mg/ml Hoechst 33342 DNA dye. Cells were fixed by the addition of formaldehyde solution (final concentration, 3.7%) and incubated at room temperature for 5 min. Cells were collected by centrifugation and washed twice with buffered glucose before visualization.

Samples for indirect immunofluorescence were prepared as described previously and probed with anti-HA antibody (Covance Research Products; Conibear and Stevens, 2002). Images were acquired on an Axioplan 2 fluorescence microscope (Carl Zeiss,) using 100x objective and manipulated using AxioVision software (Carl Zeiss, Thornwood, NY).

V-ATPase Activity Assay
Yeast vacuoles were isolated from wild-type (KHY5) and voa1::Hyg^r (MRY9) strains using the method described by Uchida et al. (1985). Freshly prepared vacuoles were assayed for concanamycin A-sensitive ATPase activity by using a coupled spectrophotometric assay as described previously (Conibear and Stevens, 2002). Vacuole membranes prepared from wild-type and voa1::Hyg^r cells and were assayed 11 and 12 times, respectively, with the error expressed as SD.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Voa1p Interacts Directly with the V₀–Vma21p Complex
Previous work has shown that the V-ATPase V₀ subunits assemble in the ER and bind directly to the assembly factor Vma21p (Malkus et al., 2004). It was noted that in addition to the known V₀ subunits, there are other proteins present in this assembly complex that have not yet been identified (Malkus et al., 2004). We chose mass spectrometry as a tool to examine the V₀–Vma21p complex for uncharacterized members in the early stage of assembly in the ER membrane. A vph1 strain background was chosen because partially assembled V₀–Vma21p complex forms in the ER, but it is unable to exit the ER. HA-epitope–tagged Vma21p (Vma21p-HA) was expressed in vph1 cells and purified from solubilized membranes by affinity chromatography using gentle detergent conditions to preserve protein–protein interactions. Affinity extracts were separated by analytical SDS-PAGE and visualized by silver staining (Figure 1A). In addition to known V₀ proteins a prominent band migrated at 35 kDa, just ahead of Vma6p. Gel sections were cut from identical preparative-scale gels and subjected to digestion with trypsin. The peptides were extracted from the gel and analyzed by LC MS/MS. The mass spectrometric results confirmed the presence of V₀ subunits but also identified the prominent unknown protein as the uncharacterized ORF YGR106C, which is predicted to encode a 265-amino acid protein (Figure 1B).

View larger version (27K): [in this window] [in a new window]   Figure 1. Voa1p associates with the V0–Vma21p complex. (A) Voa1p copurifies with the Vma21p–HA bait protein. Detergent-solubilized membranes were prepared from vph1 (KHY31) in the absence (–) or in the presence (+) of Vma21p-HA expressed from pKH28. Vma21p-HA and copurifying proteins were captured using anti-HA agarose and then eluted and analyzed by SDS-PAGE and silver staining. Protein identifications were made by LC MS/MS using gel sections cut from identical gels but at a preparative scale. V-ATPase subunit bands were assigned based on previously observed apparent molecular mass and identification by mass spectrometry in the appropriate gel section. An unidentified protein band is marked with an asterisk. (B) Amino acid sequence of Voa1p. The position of the subsequently introduced HA epitope tag (YPYDVPDYA) is indicated. Tryptic peptides identified by mass spectrometry for the untagged protein are underlined. Gray bars indicate predicted transmembrane segments (SOSUI; Hirokawa et al., 1998). The predicted ER retrieval motif (KKXX) at the C terminus is shown in bold. (C) Two peptides generated by digestion of HA-tagged Voa1p with trypsin allow the detection of N-terminal signal sequence cleavage. Placement of the HA-tag (black background) adjacent to the predicted signal cleavage site between Ala-24 and Asp-25 (SignalP; Bendtsen et al., 2004) results in the two possible tryptic peptides shown, depending on whether signal cleavage occurs. (D) Topology model of Voa1p. The model shows cleavage of the 24-amino acid signal sequence (scissors), leaving Asp25 at the newly formed N terminus. The bulk of Voa1p is exposed to the lumenal side of the ER and consists of a predicted globular domain anchored to the ER membrane by a transmembrane segment near the C terminus of the protein. The three possible sites for N-glycosylation are indicated (N69, N104, and N172). The C terminus contains an ER retention and retrieval signal (KKNN) facing the cytosol.

Mass spectrometric analysis of Voa1p having an HA-tag after Asp25 confirmed cleavage of the signal sequence. HA-Voa1p was expressed in vph1 cells, affinity purified, and resolved by SDS-PAGE. A section containing the appropriate band was cut from the gel, treated with trypsin, and the resulting peptides were recovered. Two signature peptides were detected by LC MS/MS; the first is as expected from the amino acid sequence of the full-length protein, but the second is formed only when Voa1p has Asp25 at its N terminus as a result of signal peptide cleavage (Figure 1C). Consequently, signal sequence cleavage produces a processed protein where the bulk of Voa1p is located on the lumenal side of the ER and the C-terminal transmembrane segment serves as an anchor (Figure 1D). This topology results in the cytosolic exposure of the C-terminal, dilysine ER retention motif, KKNN, and would make available three asparagine residues in the lumenal domain as sites of possible N-linked glycosylation (Figure 1D).

Voa1p Is an Integral Membrane Glycoprotein
To confirm that Voa1p is an integral membrane protein, membrane fractions were prepared from yeast cells expressing HA-Voa1p and treated with either PBS, Na₂CO₃, pH 11.5, or 1% Triton X-100. Alkali treatment with Na₂CO₃ extracts peripherally associated membrane proteins from isolated membranes but not integral membrane proteins, which can only be extracted by solubilization with detergent (Fujiki et al., 1982). The integral membrane protein alkaline phosphatase (Pho8p) and the V-ATPase peripheral membrane protein Vma6p served as controls. As shown in Figure 2A, the integral membrane protein control Pho8p remained in the membrane pellet fraction (P) after treatment with either buffer or Na₂CO₃. Treatment with the detergent Triton X-100 was required to produce soluble Pho8p that can be extracted into the supernatant fraction (S). The peripheral membrane protein control Vma6p was extracted from the membrane pellet fraction into the supernatant fraction by treatment with either Na₂CO₃ or detergent. Like Pho8p, Voa1p remained with the membrane pellet fraction when treated with buffer or Na₂CO₃ and was only extracted from the membranes by treatment with detergent, indicating that Voa1p is an integral membrane protein.

View larger version (64K): [in this window] [in a new window]   Figure 2. Voa1p is a glycosylated, integral membrane protein. (A) Membrane proteins prepared from voa1::Hygr (MRY14) cells expressing HA-Voa1p (pMR072) were divided into equal aliquots and treated with buffer only (PBS), 100 mM Na2CO3, pH 11.5, or with 1% Triton X-100. After centrifugation at 100,000 x g for 30 min, the pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE followed by Western blotting using antibodies against Pho8p (integral membrane protein), Vma6p (peripheral membrane protein), and HA to detect HA-Voa1p. Gels contained equal amounts of protein per lane. (B) Membrane proteins prepared from voa1::Kanr cells (MRY1) expressing HA-Voa1p (pMR072) were solubilized [left lane] and then incubated for 3 h with (+) or without (–) Endo H. Samples were analyzed by SDS-PAGE and Western blotting using anti-HA antibodies to detect HA-Voa1p. The molecular mass (kilodaltons) of the nearest marker protein is given on the left.

Voa1p Is Required for V₀ Assembly in vma21QQ Cells
Voa1p and Vma21p are encoded by neighboring ORFs on chromosome VII, and the two proteins are encoded on opposite strands, separated by only 156 base pairs of 3'-noncoding sequence (Figure 3A). Western blots of extracts from a voa1::Kan^r strain with an integrated HA-tagged VMA21 allele (VMA21::HA) revealed that Vma21p levels were reduced two- to threefold in the voa1::Kan^r strain relative to wild-type cells (Figure 3B), with Dpm1p (an unrelated ER membrane protein) as a loading control. Furthermore, introducing plasmid-expressed Voa1p into voa1::Kan^r cells did not return Vma21p levels to those found in wild-type cells. Thus, replacing the VOA1 ORF with Kan^r interfered with the expression level of Vma21p. A second voa1 null mutant was prepared where the VOA1 coding sequence was disrupted by replacing the first two nucleotides of the start codon with Hyg^r. This leaves intact 952 nucleotides of VMA21 3'-flanking sequence (Figure 3A), and in this strain Vma21p levels were indistinguishable from wild-type cells (Figure 3B). All subsequent experiments characterizing the loss of Voa1p were carried out using the insertional disruption of VOA1 by Hyg^r (voa1::Hyg^r).

View larger version (23K): [in this window] [in a new window]   Figure 3. Disruption of Voa1p by insertional mutagenesis does not affect protein levels of Vma21p. (A) Schematic of the genomic location of VOA1 and VMA21. The two ORFs are separated by 156 base pairs. Deletion of the VOA1 ORF by replacement with the kanMX4 cassette (Kanr) generating voa1::Kanr (MRY1) is outlined, in addition to the construction of the voa1::Hygr (MRY14) allele, wherein the VOA1 ORF is disrupted by insertion of the hphMX4 cassette (Hygr) with concomitant deletion of the first two nucleotides of the VOA1 start codon. The voa1::Hygr allele maintains 952 nucleotides of native VMA21 3' sequence. (B) Western blot analysis of whole cell extracts from yeast strains each of which has an integrated VMA21::HA allele; wild-type VOA1 (WT; KHY5), voa1::Hygr (voa1::H; MRY9), and voa1::Kanr (voa1K; MRY11). Blots were probed with anti-HA antibodies to detect Vma21p-HA and antibodies against Dpm1p as a loading control. Protein samples in lanes 2 and 3 and 4 and 5 are prepared from two independent isolates. The molecular mass (kilodaltons) of the nearest marker protein is shown on the left.

View larger version (36K): [in this window] [in a new window]   Figure 4. Loss of Voa1p in cells expressing Vma21pQQ results in calcium sensitivity and lack of vacuolar acidification. (A) Exponentially growing cultures of wild-type (WT; SF838–1D), voa1::Hygr (voa1::H; MRY14), vma21QQ (21QQ; LGY183), voa1::Hygr vma21QQ (voa1::H 21QQ; MRY5), and vma21 (21; TASY006) were serially diluted and spotted onto rich media buffered to pH 5.0 or rich media buffered to pH 7.5 plus 50 mM CaCl2. (B) The same strains were stained with quinacrine (green) and concanavalin A-tetramethylrhodamine (red) and viewed by either fluorescent or DIC microscopy.

Because the function of the V-ATPase is to bring about acidification of the vacuole in yeast, we tested for a defect in vacuolar acidification in voa1::Hyg^r vma21QQ yeast cells. Quinacrine is a fluorescent dye that accumulates in acidic cellular compartments (Uchida et al., 1985; Conibear and Stevens, 2002). After quinacrine uptake, wild-type cells exhibited highly fluorescent vacuoles that showed up as bright green disks when visualized by microscopy (Figure 4B). The binding of concanavalin A-tetramethylrhodamine isothiocyanate to the yeast cell wall showed the outline of the yeast cells in red. Yeast cells expressing Vma21pQQ also accumulate quinacrine, demonstrating vacuolar acidification even though these cells have reduced levels of V-ATPase on the vacuolar membrane. Similarly, voa1::Hyg^r cells accumulate quinacrine in their vacuoles. Cells lacking Voa1p and expressing Vma21pQQ (voa1::Hyg^r vma21QQ) failed to accumulate quinacrine, similar to cells completely lacking V-ATPase function (vma21 cells). This result indicates that voa1::Hyg^r vma21QQ yeast cells lack a functional V-ATPase on their vacuolar membranes and are unable to acidify their vacuoles.

Loss of Voa1p Enhances the V-ATPase Assembly Defect of vma21QQ Cells
The demonstrated loss of V-ATPase function in cells lacking Voa1p and expressing Vma21pQQ could be a result of either a nonfunctional V-ATPase on the vacuole or reduced levels of V-ATPase on the vacuole due to an assembly defect. The V₀ subunit Vph1p is normally a very stable protein if upon synthesis it is assembled into the V₀ subcomplex and transported to the vacuole membrane (Graham et al., 2003). However, the levels of Vph1p are dramatically reduced in cells that are defective in the assembly of a complete V₀ subcomplex, which is the case when individual V₀ subunits or assembly factors (Vma21p, Vma12p, Vma22p, or Pkr1p) are absent (Graham et al., 2003; Compton et al., 2006; Davis-Kaplan et al., 2006). To test for defective V₀ assembly, cell extracts were prepared and analyzed by Western blotting for the presence of Vph1p, the V₁ protein Vma1p, and Dpm1p as a loading control. As expected, we found that cellular levels of Vma1p present at steady-state are similar in all strains tested; wild-type, the single mutants vma21QQ and voa1::Hyg^r, the double mutant voa1::Hyg^r vma21QQ, and vma21 (Figure 5). Because the V₁ subcomplex can assemble independently of the V₀ subcomplex (Doherty and Kane, 1993; Graham et al., 2003), V₁ protein levels are generally unaffected by any defect in V₀. Consistent with near wild-type growth and quinacrine accumulation for the single mutants vma21QQ and voa1::Hyg^r, Vph1p levels in these strains were similar to that of wild-type cells (Figure 5). Likewise, a Western blot of more dilute extracts from wild-type and voa1::Hyg^r cells failed to reveal differences in Vph1p levels between these cells types (unpublished data). In contrast, the levels of Vph1p were clearly reduced in voa1::Hyg^r vma21QQ yeast cells compared with wild-type cells, and they were more similar to Vph1p levels found in vma21 cells, which have no detectable functional V-ATPase on the vacuole.

View larger version (65K): [in this window] [in a new window]   Figure 5. Loss of Voa1p in cells expressing Vma21pQQ results in reduced levels of V0 subunit Vph1p. Whole cell extracts were prepared from wild type (WT) (KHY5), voa1::Hygr (voa1::H; MRY9), vma21QQ (21QQ; LGY183), voa1::Hygr vma21QQ (voa1::H 21QQ; MRY5), and vma21 (21; TASY006). Proteins were separated by SDS-PAGE and probed with anti-Vph1p and anti-Vma1p antibodies. Anti-Dpm1p antibody was included as a loading control. The molecular mass (kilodaltons) of the nearest marker protein is shown on the left.

View larger version (37K): [in this window] [in a new window]   Figure 6. Vph1p fails to exit the ER in voa1::Hygr vma21QQ cells. Wild-type (WT; SF838–1D), vma21 (TASY006), and voa1::Hygr (voa1::H) vma21QQ (MRY5) cells expressing Vph1p-GFP (pGF06) were treated with the DNA binding dye Hoechst 33342 (shown as magenta) and visualized by fluorescent or DIC microscopy. Merged images show localization of Vph1p-GFP (green) to either the ER perinuclear and cortical membranes (vma21 and voa1::H vma21QQ) or the vacuole (WT).

Voa1p Is a V-ATPase Assembly Factor Localized to the ER with Vma21p
The subcellular localization of Voa1p was determined by indirect immunofluorescence by using the HA epitope-tagged version of the protein (HA-Voa1p). Immunolocalization of HA-Voa1p revealed a perinuclear (the nucleus visualized by 4',6-diamidino-2-phenylindole [DAPI]) and cortical ER membrane staining pattern, indicating that Voa1p localizes to the ER membrane (Figure 7). Voa1p, like Vma21p, possesses a C-terminal dilysine motif that may function in the retention of Voa1p in the ER. To test this, we examined by indirect immunofluorescence the localization of HA-Voa1p and Voa1p in which the dilysines have been changed to diglutamines (HA-Voa1pQQ). As shown in Figure 7, modification of the two amino acids in Voa1p changed the localization pattern from ER to vacuole membrane. These results indicate that Voa1p is an ER localized V-ATPase assembly factor possessing a functional dilysine ER-retrieval motif.

View larger version (35K): [in this window] [in a new window]   Figure 7. Voa1p requires its C-terminal dilysine motif for localization to the ER. Indirect immunofluorescence was performed on voa1::Hygr (MRY14) cells expressing either HA-Voa1p (pMR072) or HA-Voa1pQQ (pMR0712). Voa1p and Voa1pQQ were visualized using anti-HA antibodies, followed by goat anti-mouse biotin conjugated antibody treatment, and a streptavidin-fluorescein isothiocyanate treatment. DNA was visualized by labeling with DAPI and shown as magenta. Vacuoles were visualized by DIC microscopy. Merged images show localization of Voa1p or Voa1pQQ (green) to either the ER perinuclear and cortical membranes (HA-Voa1p) or the vacuole (HA-Voa1pQQ).

View larger version (42K): [in this window] [in a new window]   Figure 8. Retrieval deficient Voa1pQQ does not function as a V-ATPase assembly factor. Exponentially growing cultures of vma21QQ (LGY183), voa1::Hygr (voa1::H) vma21QQ (MRY5), and voa1::Hygr vma21QQ expressing either Voa1p (pVOA1; pMR072) or Voa1pQQ (pvoa1QQ; pMR0712) were serially diluted and spotted onto rich media buffered to pH 5.0 or rich media plus 100 mM CaCl2.

Immunoprecipitates obtained from wild-type, vph1, and vma1 cells were probed for the presence of Voa1p and the V₀ subunit Vph1p. As shown in Figure 9A, very little Voa1p was found associated with Vma21p in wild-type cells. At the same time significant amounts of Vph1p copurified with Vma21p from these cells (Figure 9A, top), indicating that V₀–Vma21p subcomplexes were present in wild-type cells but that they did not contain Voa1p. A similar result was observed for V₀–Vma21p subcomplexes immunoprecipitated from vma1 cells. Cells lacking the V₁ protein Vma1p assemble a complete V₀–Vma21p subcomplex that exits the ER normally, even though these cells do not form a V₁ complex and therefore have no functional V-ATPase (Graham et al., 2003).

View larger version (15K): [in this window] [in a new window]   Figure 9. Voa1p associates with the assembling V0 complex in the ER. Solubilized membranes were prepared from various vma strains expressing both Vma21p-HA (pLG271) and c-myc-Voa1p (pMR0713), followed by immunoprecipitation of Vma21p-HA with anti-HA antibody. Proteins of the solubilized membrane input (I) and immunoprecipitate (IP) were separated by SDS-PAGE and used to generate Western blots. (A) Strains used were wild-type (WT; SF838-1D), vph1 (LGY120) and vma1 (LGY146), and Western blots were probed with antibodies recognizing either Vph1p or c-myc-Voa1p. (B) Strains used were vma3 (LGY113), vma11 (LGY114), vma16 (LGY115), vma6 (LGY184) and vma9 (GFY96), and Western blots were probed with antibodies recognizing c-myc-Voa1p. The molecular mass (kilodaltons) of the nearest marker protein is shown on the left.

We also examined how the loss of any of the remaining five V₀ subunits affects the interaction between Voa1p and Vma21p. In the absence of either the peripheral membrane subunit Vma6p or the integral membrane subunit Vma9p, Voa1p is found associated with the V₀–Vma21p complex (Figure 9B). As we have reported previously, vma6 or vma9 strains contain Vma3p, Vma11p, and Vma16p in incomplete V₀ subcomplexes associated with Vma21p (Malkus et al., 2004; Compton et al., 2006). These incomplete V₀ complexes remain in the ER. In addition, a small amount of Voa1p can associate with the incomplete V₀–Vma21p complex lacking Vma16p.

Vma3p, Vma11p, and Vma16p are modeled to form a "proteolipid" ring that bridges the interaction between Vma21p and the remaining V₀ subunits (Malkus et al., 2004; Compton et al., 2006; Wang et al., 2007). Not surprisingly, very little Voa1p immunoprecipitated with Vma21p in vma3 or vma11 cells. Vma21p and all three of the proteolipid subunits (Vma3p, Vma11p, and Vma16p) are required to form a stable proteolipid ring, and Voa1p preferentially associates with this complex. Together, our results reveal that Voa1p associates with an incompletely assembled V₀–Vma21p complex in the ER; specifically, Voa1p binds to V₀–Vma21p complexes lacking Vph1p, Vma6p, or Vma9p but containing a partially formed or preferably, a completely formed proteolipid ring.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Voa1p Functions Early in V₀ Domain Assembly
With 14 different integral and peripheral membrane subunits, some of them present in multiple copies, the V-ATPase is a complex enzyme, requiring additional protein factors for its assembly and function. Factors have been identified for the stable assembly and glucose-regulated reassembly of the V₁ and V₀ domains (Kane, 2006; Forgac, 2007). Previous work in yeast has identified four ER-localized proteins (Vma12p, Vma21p, Vma22p, and Pkr1p) that are required for the assembly of the V₀ domain of the V-ATPase (Forgac, 2007), and one of these proteins (Vma21p) escorts the V₀ domain out of the ER (Malkus et al., 2004). In this report, we identify a fifth V₀ assembly factor, Voa1p.

Voa1p is an ER-localized integral membrane glycoprotein, which when deleted reduces V-ATPase activity to 75% wild type but results in no apparent Vma^– phenotype, i.e., no calcium-sensitive growth and normal acidification of the vacuole. Because it takes at least a three- to fivefold reduction in the level of V-ATPase to observe increased sensitivity to Ca²⁺ in yeast cells (Liu et al., 1996; Curtis and Kane, 2002), the effect on the level of the V-ATPase due to loss of Voa1p is not sufficiently strong to confer a Vma^– growth phenotype. This might explain why a clear role for Voa1p in V₀ assembly was revealed in cells expressing an ER retrieval-deficient form of Vma21p (Vma21pQQ). vma21QQ yeast cells have reduced V-ATPase levels (Hill and Stevens, 1994) and a very mild growth defect in the presence of elevated Ca²⁺ (Figure 4). vma21QQ yeast cells lacking Voa1p (voa1::Hyg^r vma21QQ) exhibit a severe Ca²⁺ growth defect and fail to acidify their vacuoles, comparable with cells completely lacking V-ATPase. This is consistent with an absence of functional V-ATPase on the vacuole membrane. V₀ domain assembly was severely compromised in voa1::Hyg^r vma21QQ cells, as evidenced by the decreased cellular levels of the V₀ subunit Vph1p and the ER localization of the remaining Vph1p, due to its inability to assemble into a complete V₀ domain and exit the ER. In addition, ER localization of Voa1p was found to be critical for its function in V₀ domain assembly, because a retrieval-deficient form of Voa1p (Voa1pQQ) could not support V₀ assembly in voa1::Hyg^r vma21QQ cells. The strong enhancement of the Ca²⁺-sensitive phenotype in vma21QQ cells by deletion of Voa1p supports the model that Voa1p functions together with Vma21p in V₀ assembly in the ER.

Characterization of the association of Voa1p with the V₀–Vma21p complex revealed that Voa1p is associated with the partially assembled V₀–Vma21p complex but did not associate significantly with the fully assembled V₀–Vma21p complex found in wild-type cells. Voa1p did not appreciably associate with Vma21p in the absence of the c or c' components of the core proteolipid ring (composed of subunits c, c', and c''; Wang et al., 2007). This suggests that partial formation of this V₀ core is the minimum requirement for Voa1p association with the Vma21p complex, although the presence of all three components of the proteolipid ring led to the highest level of association. This is corroborated by finding Voa1p most strongly associated with the assembling V₀–Vma21p complex in cells lacking subunit a (vph1), subunit d (vma6), or subunit e (vma9), wherein the full proteolipid ring is associated with Vma21p. Together, these results indicate that Voa1p associates early with the assembling V₀–Vma21p complex in the ER, that it dissociates from the fully assembled V₀-Vma21p complex, and that it does not exit the ER in close association with the V₀–Vma21p complex. In support of these results, a recent proteomics publication reported proximity between Voa1p (YGR106C) and several V-ATPase subunits by using a split DHFR reporter system (Tarassov et al., 2008).

V₀ Sector Assembly Pathway
With the identification of Voa1p, the assembly of the V₀ domain in the ER has been shown to be dependent on at least five proteins: Vma12p, Vma21p, Vma22p, Pkr1p, and now Voa1p. Although a specific role for Pkr1p in V₀ assembly has remained elusive (Davis-Kaplan et al., 2006), the model in Figure 10 outlines specific roles for the four other proteins in the V₀ assembly pathway. Vma21p associates with subunits c, c', and c'' to form the core Vma21p–proteolipid ring complex, and loss of neither Vma12p nor Vma22p interferes with the formation of this complex (Malkus et al., 2004). Voa1p associates stably with this core Vma21p–proteolipid complex (Figure 10, complex 1). A complex of Vma12p and Vma22p interacts with newly synthesized subunit a (Vph1p; Graham et al., 1998) to form complex 2 in the assembly pathway depicted in Figure 10. The addition of subunits a, d, and e to the core Vma21p–proteolipid complex results in the dissociation of the Vma12–22p complex and Voa1p, and the formation of a stable and fully assembled V₀–Vma21p ER-localized complex (Figure 10, complex 3; Malkus et al., 2004; Compton et al., 2006; this work). The V₀–Vma21p complex is then loaded onto ER-derived coat protein (COP) II vesicles for transport to the cis-Golgi. After dissociation of Vma21p from the V₀ domain in the Golgi compartment, COPI-dependent retrieval of Vma21p to the ER (due to Vma21p's C-terminal dilysine motif) allows this assembly/escort factor to function in multiple rounds of V₀ assembly and ER-to-Golgi transport of this essential portion of the V-ATPase complex (Malkus et al., 2004).

View larger version (24K): [in this window] [in a new window]   Figure 10. Model of Voa1p function in the assembly of the V-ATPase. Vma21p associates with the V0 subunits c, c', and c'' (Vma3p, Vma11p, and Vma16) that assemble to form the proteolipid ring. Voa1p associates with the partially assembled V0–Vma21p complex in the ER, and this association may be mediated by the proteolipid ring subunits (1). In a parallel assembly pathway, the Vma12p/Vma22p complex interacts with subunit a (Vph1p or Stv1p) (2). The association of Vph1p (subunit a), Vma6p (subunit d), and Vma9p (subunit e) with the proteolipid ring subunits and the dissociation of Voa1p from this complex lead to the formation of the fully assembled V0–Vma21p complex (3). The fully assembled V0–Vma21p complex is then competent to exit the ER.

Genetic and genome-wide screens in yeast have identified the numerous proteins that are required for assembly and regulation of the V-ATPase complex (Kane, 2006). The identification of the new V₀ assembly factor Voa1p by proteomic approaches indicates that there may be many additional factors yet to be discovered to fully understand the coordinated assembly of the V₀ domain, the sorting of the V₀–Vma21p complex into COPII vesicles for ER-to-Golgi transport, the selective sorting of the Vph1p-containing and Stv1p-containing V-ATPase complexes, and the regulated dissociation/reassembly of the V₁V₀ complex. voa1 mutants would not have been identified in any of the previous V-ATPase/vacuole acidification screens, because its function was only revealed in a compromised genetic background (vma21QQ). The identification of proteins such as Voa1p that enhance V₀ assembly (or proteins that have redundant functions) will require enhancer or suppressor screens in sensitized genetic backgrounds (such as vma21QQ) to identify mutations that act synthetically with mutations that compromise known factors or to identify proteins that when overexpressed alleviate or exacerbate partial V-ATPase deficiencies. Such approaches will reveal new components of the V-ATPase assembly pathway and help elucidate the detailed mechanism of assembly and regulation of the V-ATPase enzyme complex.

	ACKNOWLEDGMENTS

 
We thank Dave Anderson (University of Oregon) and Phil Gafken and Lisa Nader (Fred Hutchinson Cancer Research Center) for providing mass spectrometry analysis. We also thank members of the Stevens laboratory for helpful comments related to this manuscript. We thank Greg Finnigan for generation of the vma9 yeast strain and the Vph1p-GFP plasmid used in this work. Additionally we want to thank Mark Compton for time and effort creating the artwork used to generate the final model figure. This work was supported by National Institutes of Health grant GM-38006 (to T.H.S.).

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-06-0629) on September 17, 2008.

* These authors contributed equally to this work.

Address correspondence to: Tom H. Stevens (stevens{at}molbio.uoregon.edu)

Abbreviations used: DAPI, 4',6-diamidino-2-phenylindole; GFP, green fluorescent protein; ER, endoplasmic reticulum; HA, hemagglutinin; V-ATPase, vacuolar-type proton-translocating ATPase; YEPD, yeast extract peptone dextrose.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Ansaldi, M., Lepelletier, M., and MÈjean, V. (1996). Site-specific mutagenesis by using an accurate recombinant polymerase chain reaction method. Anal. Biochem 234, 110–111.[CrossRef][Medline]

Bauerle, C., Ho, M. N., Lindorfer, M. A., and Stevens, T. H. (1993). The Saccharomyces cerevisiae VMA6 gene encodes the 36-kDa subunit of the vacuolar H⁺-ATPase membrane sector. J. Biol. Chem 268, 12749–12757.[Abstract/Free Full Text]

Bendtsen, J. D., Nielsen, H., von Heijne, G., and Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. J. Mol. Biol 340, 783–795.[CrossRef][Medline]

Beyenbach, K. W., and Wieczorek, H. (2006). The V-type H⁺ ATPase: molecular structure and function, physiological roles and regulation. J. Exp. Biol 209, 577–589.[Abstract/Free Full Text]

Catley, B. J. (1988). Isolation and analysis of cell walls. In: Yeast: A Practical Approach, I. Campell and J. H. Duffus, Oxford, England: IRL Press, 163–183.

Compton, M. A., Graham, L. A., and Stevens, T. H. (2006). Vma9p (subunit e) is an integral membrane V₀ subunit of the yeast V-ATPase. J. Biol. Chem 281, 15312–15319.[Abstract/Free Full Text]

Conibear, E., and Stevens, T. H. (2002). Studying yeast vacuoles. Methods Enzymol 351, 408–432.[Medline]

Craig, R., and Beavis, R. C. (2004). TANDEM: matching proteins with tandem mass spectra. Bioinformatics 20, 1466–1467.[Abstract/Free Full Text]

Curtis, K. K., and Kane, P. M. (2002). Novel vacuolar H⁺-ATPase complexes resulting from overproduction of Vma5p and Vma13p. J. Biol. Chem 277, 2716–2724.[Abstract/Free Full Text]

Davis-Kaplan, S. R., Compton, M. A., Flannery, A. R., Ward, D. M., Kaplan, J., Stevens, T. H., and Graham, L. A. (2006). PKR1 encodes an assembly factor for the yeast V-type ATPase. J. Biol. Chem 281, 32025–32035.[Abstract/Free Full Text]

Doherty, R. D., and Kane, P. M. (1993). Partial assembly of the yeast vacuolar H⁺-ATPase in mutants lacking one subunit of the enzyme. J. Biol. Chem 268, 16845–16851.[Abstract/Free Full Text]

Flannery, A. R., Graham, L. A., and Stevens, T. H. (2004). Topological characterization of the c, c', and c'' subunits of the vacuolar ATPase from the yeast Saccharomyces cerevisiae. J. Biol. Chem 279, 39856–39862.[Abstract/Free Full Text]

Forgac, M. (2007). Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat. Rev. Mol. Cell Biol 8, 917–929.[CrossRef][Medline]

Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982). Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J. Cell Biol 93, 97–102.[Abstract/Free Full Text]

Goldstein, A. L., and McCusker, J. H. (1999). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.[CrossRef][Medline]

Graham, L. A., Flannery, A. R., and Stevens, T. H. (2003). Structure and assembly of the yeast V-ATPase. J. Bioenerg. Biomembr 35, 301–312.[CrossRef][Medline]

Graham, L. A., Hill, K. J., and Stevens, T. H. (1998). Assembly of the yeast vacuolar H⁺-ATPase occurs in the endoplasmic reticulum and requires a Vma12p/Vma22p assembly complex. J. Cell Biol 142, 39–49.[Abstract/Free Full Text]

Hill, K. J., and Stevens, T. H. (1994). Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H⁺-ATPase complex. Mol. Biol. Cell 5, 1039–1050.[Abstract]

Hill, K. J., and Stevens, T. H. (1995). Vma22p is a novel endoplasmic reticulum associated protein required for assembly of the yeast vacuolar H⁺-ATPase complex. J. Biol. Chem 270, 22329–22336.[Abstract/Free Full Text]

Hirata, R., Umemoto, N., Ho, M. N., Ohya, Y., Stevens, T. H., and Anraku, Y. (1993). VMA12 is essential for assembly of the vacuolar H⁺-ATPase subunits onto the vacuolar membrane in Saccharomyces cerevisiae. J. Biol. Chem 268, 961–967.[Abstract/Free Full Text]

Hirata, T., Iwamoto-Kihara, A., Sun-Wada, G. H., Okajima, T., Wada, Y., and Futai, M. (2003). Subunit rotation of vacuolar-type proton pumping ATPase: relative rotation of the G as to c subunit. J. Biol. Chem 278, 23714–23719.[Abstract/Free Full Text]

Hirokawa, T., Boon-Chieng, S., and Mitaku, S. (1998). SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics 14, 378–379.[Abstract/Free Full Text]

Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003). Evidence for rotation of V₁-ATPase. Proc. Natl. Acad. Sci. USA 100, 2312–2315.[Abstract/Free Full Text]

Jackson, D. D., and Stevens, T. H. (1997). VMA12 encodes a yeast endoplasmic reticulum protein required for vacuolar H⁺-ATPase assembly. J. Biol. Chem 272, 25928–25934.[Abstract/Free Full Text]

Kaiser, C. A., Chen, E. J., and Losko, S. (2002). Subcellular fractionation of secretory organelles. Methods Enzymol 351, 325–338.[Medline]

Kane, P. M. (2006). The where, when, and how of organelle acidification by the yeast vacuolar H⁺-ATPase. Microbiol. Mol. Biol. Rev 70, 177–191.[Abstract/Free Full Text]

Kane, P. M., Tarsio, M., and Liu, J. (1999). Early steps in assembly of the yeast vacuolar H⁺-ATPase. J. Biol. Chem 274, 17275–17283.[Abstract/Free Full Text]

Kawasaki-Nishi, S., Bowers, K., Nishi, T., Forgac, M., and Stevens, T. H. (2001). The amino-terminal domain of the vacuolar proton-translocating ATPase a subunit controls targeting and in vivo dissociation, and the carboxyl-terminal domain affects coupling of proton transport and ATP hydrolysis. J. Biol. Chem 276, 47411–47420.[Abstract/Free Full Text]

Keller, A., Nesvizhskii, A. I., Kolker, E., and Aebersold, R. (2002). Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search. Anal. Chem 74, 5383–5392.[Medline]

Kitagawa, N., Mazon, H., Heck, A. J., and Wilkens, S. (2008). Stoichiometry of the peripheral stalk subunits E and G of yeast V₁-ATPase determined by mass spectrometry. J. Biol. Chem 283, 3329–3337.[Abstract/Free Full Text]

Liu, J. Z., and Kane, P. M. (1996). Mutational analysis of the catalytic subunit of the yeast vacuolar proton-translocating ATPase. Biochemistry 35, 10938–10948.[CrossRef][Medline]

Liu, Q., Kane, P. M., Newman, P. R., and Forgac, M. (1996). Site-directed mutagenesis of the yeast V-ATPase B subunit (Vma2p). J. Biol. Chem 271, 2018–2022.[Abstract/Free Full Text]

MacLean, B., Eng, J. K., Beavis, R. C., and McIntosh, M. (2006). General framework for developing and evaluating database scoring algorithms using the TANDEM search engine. Bioinformatics 22, 2830–2832.[Abstract/Free Full Text]

Malkus, P., Graham, L. A., Stevens, T. H., and Schekman, R. (2004). Role of Vma21p in assembly and transport of the yeast vacuolar ATPase. Mol. Biol. Cell 15, 5075–5091.[Abstract/Free Full Text]

Manolson, M. F., Proteau, D., Preston, R. A., Stenbit, A., Roberts, B. T., Hoyt, M. A., Preuss, D., Mulholland, J., Botstein, D., and Jones, E. W. (1992). The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H⁺-ATPase. J. Biol. Chem 267, 14294–14303.[Abstract/Free Full Text]

Manolson, M. F., Wu, B., Proteau, D., Taillon, B. E., Roberts, B. T., Hoyt, M. A., and Jones, E. W. (1994). STV1 gene encodes functional homologue of the 95-kDa yeast vacuolar H⁺-ATPase subunit Vph1p. J. Biol. Chem 269, 14064–14074.[Abstract/Free Full Text]

Neubert, C., Graham, L. A., Black-Maier, E. W., Coonrod, E. M., Liu, T. Y., Stierhof, Y. D., Seidel, T., Stevens, T. H., and Schumacher, K. (2008). Arabidopsis has two functional orthologs of the yeast V-ATPase assembly factor Vma21p. Traffic 9, 1618–1628.[CrossRef][Medline]

Ohira, M., Smardon, A. M., Charsky, C. M., Liu, J., Tarsio, M., and Kane, P. M. (2006). The E and G subunits of the yeast V-ATPase interact tightly and are both present at more than one copy per V₁ complex. J. Biol. Chem 281, 22752–22760.[Abstract/Free Full Text]

Powell, B., Graham, L. A., and Stevens, T. H. (2000). Molecular characterization of the yeast vacuolar H⁺-ATPase proton pore. J. Biol. Chem 275, 23654–23660.[Abstract/Free Full Text]

Rauch, A. et al. (2006). Computational Proteomics Analysis System (CPAS): an extensible, open-source analytic system for evaluating and publishing proteomic data and high throughput biological experiments. J. Proteome Res 5, 112–121.[CrossRef][Medline]

Rothman, J. H., and Stevens, T. H. (1986). Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell 47, 1041–1051.[CrossRef][Medline]

Sambrook, J., and Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Seol, J. H., Shevchenko, A., Shevchenko, A., and Deshaies, R. J. (2001). Skp1 forms multiple protein complexes, including RAVE, a regulator of V-ATPase assembly. Nat. Cell Biol 3, 384–391.[CrossRef][Medline]

Shevchenko, A., Wilm, M., Vorm, O., and Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem 68, 850–858.[Medline]

Sikorski, R. S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.[Abstract/Free Full Text]

Smardon, A. M., and Kane, P. M. (2007). RAVE is essential for the efficient assembly of the C subunit with the vacuolar H⁺-ATPase. J. Biol. Chem 282, 26185–26194.[Abstract/Free Full Text]

Smardon, A. M., Tarsio, M., and Kane, P. M. (2002). The RAVE complex is essential for stable assembly of the yeast V-ATPase. J. Biol. Chem 277, 13831–13839.[Abstract/Free Full Text]

Tarassov, K., Messier, V., Landry, C. R., Radinovic, S., Molina, M. M., Shames, I., Malitskaya, Y., Vogel, J., Bussey, H., and Michnick, S. W. (2008). An in vivo map of the yeast protein interactome. Science 320, 1465–1470.[Abstract/Free Full Text]

Uchida, E., Ohsumi, Y., and Anraku, Y. (1985). Purification and properties of H⁺-translocating, Mg⁺²-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J. Biol. Chem 260, 1090–1095.[Abstract/Free Full Text]

Veses, V., Casanova, M., Murgui, A., Dominguez, A., Gow, N. A., and Martinez, J. P. (2005). ABG1, a novel and essential Candida albicans gene encoding a vacuolar protein involved in cytokinesis and hyphal branching. Eukaryot. Cell 4, 1088–1101.[Abstract/Free Full Text]

Wang, Y., Cipriano, D. J., and Forgac, M. (2007). Arrangement of subunits in the proteolipid ring of the V-ATPase. J. Biol. Chem 282, 34058–34065.[Abstract/Free Full Text]

Weisman, L. S., Bacallao, R., and Wickner, W. (1987). Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J. Cell Biol 105, 1539–1547.[Abstract/Free Full Text]

Yi, E. C., Lee, H., Aebersold, R., and Goodlett, D. R. (2003). A microcapillary trap cartridge-microcapillary high-performance liquid chromatography electrospray ionization emitter device capable of peptide tandem mass spectrometry at the attomole level on an ion trap mass spectrometer with automated routine operation. Rapid Commun. Mass Spectrom 17, 2093–2098.[CrossRef][Medline]

Yokoyama, K., Nakano, M., Imamura, H., Yoshida, M., and Tamakoshi, M. (2003). Rotation of the proteolipid ring in the V-ATPase. J. Biol. Chem 278, 24255–24258.[Abstract/Free Full Text]

Zheng, L., Baumann, U., and Reymond, J. L. (2004). An efficient one-step site-directed and site-saturation mutagenesis protocol. Nucleic Acids Res 32, e115.[Abstract/Free Full Text]

This article has been cited by other articles:

				B. Ediger, S. D. Melman, D. L. Pappas Jr., M. Finch, J. Applen, and K. J. Parra The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d J. Biol. Chem., July 17, 2009; 284(29): 19522 - 19532. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: E08-06-0629v1 19/12/5131    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Ryan, M. Articles by Stevens, T. H. Search for Related Content PubMed PubMed Citation Articles by Ryan, M. Articles by Stevens, T. H.