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Originally published In Press as doi:10.1074/jbc.M703627200 on July 10, 2007

J. Biol. Chem., Vol. 282, Issue 36, 26185-26194, September 7, 2007
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RAVE Is Essential for the Efficient Assembly of the C Subunit with the Vacuolar H+-ATPase*

Anne M. Smardon and Patricia M. Kane1

From the Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, New York 13210

Received for publication, May 2, 2007 , and in revised form, June 21, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The RAVE complex is required for stable assembly of the yeast vacuolar proton-translocating ATPase (V-ATPase) during both biosynthesis of the enzyme and regulated reassembly of disassembled V1 and V0 sectors. It is not yet known how RAVE effects V-ATPase assembly. Previous work has shown that V1 peripheral or stator stalk subunits E and G are critical for binding of RAVE to cytosolic V1 complexes, suggesting that RAVE may play a role in docking of the V1 peripheral stalk to the V0 complex at the membrane. Here we provide evidence for an interaction between the RAVE complex and V1 subunit C, another subunit that has been assigned to the peripheral stalk. The C subunit is unique in that it is released from both V1 and V0 sectors during disassembly, suggesting that subunit C may control the regulated assembly of the V-ATPase. Mutants lacking subunit C have assembly phenotypes resembling that of RAVE mutants. Both are able to assemble V1/V0 complexes in vivo, but these complexes are highly unstable in vitro, and V-ATPase activity is extremely low. We show that in the absence of the RAVE complex, subunit C is not able to stably assemble with the vacuolar ATPase. Our data support a model where RAVE, through its interaction with subunit C, is facilitating V1 peripheral stalk subunit interactions with V0 during V-ATPase assembly.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Vacuolar proton-translocating ATPases (V-ATPases)2 are conserved in all eukaryotic cells where they function to acidify internal organelles such as the lysosome/vacuole, Golgi apparatus, secretory vesicles, and endosomes. V-ATPases couple hydrolysis of cytoplasmic ATP to transport of protons from the cytosol into intracellular compartments. Organelle acidification is essential for a wide range of cellular processes including protein sorting in the biosynthetic and endocytic pathways; protein processing, activation, and degradation; cellular ion homeostasis; and coupled transport of small molecules (1–4). V-ATPases also have been identified in the plasma membrane of certain specialized cells where they pump protons from the cytosol out of the cell (1, 5). The structure and subunit composition of V-ATPases is very similar in all organisms from yeast to humans. They are multisubunit complexes composed of two domains. The V1 domain is a peripheral cytoplasmic complex composed of eight different subunits (subunits A–H), and it contains the sites of ATP hydrolysis. The V0 domain is an integral membrane complex that is composed of six different subunits (subunits a, d, e, c, c', and c''). It comprises the proton pore. The V1 and V0 domains must be structurally and functionally coupled for ATP-driven proton translocation to occur. V1 complexes that are not attached to V0 at the membrane cannot hydrolyze MgATP, and V0 complexes in the membrane that are not attached to V1 are not able to transport protons (6, 7).

The biosynthetic assembly pathway for V-ATPases is not completely understood. Free V1 and V0 complexes exist in vivo in both yeast and mammalian cells (8–11). Independent assembly of preassembled V1 and V0 subcomplexes, however, is probably not the predominant pathway for biosynthetic assembly. There is evidence that supports an integrated assembly of V1 and V0 subunits with initial association of individual V1 and V0 subunits followed by the addition of subunits from both domains (12, 13). In addition to the initial biosynthetic assembly, an important mechanism for regulation of V-ATPase activity is reversible disassembly of assembled complexes into free V1 and V0 domains in response to extracellular glucose concentrations (14). In yeast cells, glucose deprivation for as little as 5 min triggers dissociation of ~70% of V-ATPase complexes. The addition of glucose reverses this process, reassembling the V1 and V0 complexes to original levels (14). Dissociation of the V-ATPase is not unique to yeast. In Manduca sexta midgut epithelial cells, cessation of feeding during molting or starvation results in disassembly of fully assembled, active V-ATPase complexes into inactive cytosolic V1 and membrane-bound V0 complexes (6, 10). There is also recent evidence from renal epithelial cells and maturing dendritic cells that V-ATPase activity is regulated at the level of assembly in mammalian cells as well (15, 16). Reversible dissociation appears to be a widely used mechanism of V-ATPase regulation.

A novel regulator of V-ATPase assembly, the heterotrimeric protein RAVE (regulator of the H+-ATPase of vacuolar and endosomal membranes), mediates both the biosynthetic assembly and the glucose-induced reassembly of the V-ATPase in yeast (17, 18). RAVE was first identified as a Skp1-containing complex that binds to free cytosolic V1 complexes (18). RAVE is a stable complex composed of three subunits: Rav1p, Rav2p, and Skp1p. rav1{Delta} and rav2{Delta} mutants display a very similar partial Vma– phenotype characterized by growth defects at pH 7.5 and 60 mM CaCl2 at elevated temperatures (33–37 °C) (18). Consistent with this phenotype, rav1{Delta} mutants have a loss of quinacrine staining of the vacuole at elevated temperatures (37 °C), indicative of a loss of vacuolar acidification. Even at the permissive temperature of 30 °C, the rav1{Delta} mutant shows a kinetic delay in reassembly of V-ATPase complexes after the readdition of glucose to glucose-deprived cells (18). Vacuoles isolated from cells lacking RAVE subunits have very low levels of V1 subunits and V-ATPase activity even when cells are grown in glucose at the permissive temperature (30 °C) (17). RAVE was shown to bind to V1 released from the vacuolar membrane by glucose deprivation and to release V1 upon glucose readdition (17). The RAVE complex appears to be aiding cytosolic V1 complexes to assemble with V0 at the membrane.

The interaction between RAVE and V1 is lost in mutants lacking two V1 subunits, E and G (17). These two subunits, present in at least two copies/V1 complex, are believed to form one or more peripheral stalks connecting the catalytic head group and the V0 sector. Subunit C is another V1 peripheral stalk component that is associated with subunits E and G of the V1 complex and subunit a of the V0 complex in the assembled V-ATPase (19). Studies in yeast have produced genetic, biochemical, and structural data supporting a role for subunit C in both the stability of the V1/V0 holoenzyme and in regulation of reversible assembly/disassembly of the complex (20, 21).

In this work we provide evidence that RAVE interacts with subunit C in the cytosol and that this interaction is independent of RAVE/V1 binding. In the absence of RAVE function, subunit C is not able to stably assemble with the V1 and V0 subcomplexes of the V-ATPase at the vacuole. Our results support a model in which RAVE influences V-ATPase peripheral stalk subunit interactions via a chaperone type assembly function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Growth Media—Oligonucleotides were synthesized by MWG Biotech. TaKaRa LA-Taq was purchased from Fisher, and Pfu I DNA polymerase was from Stratagene. Zymolyase 100T was from MP Biomedicals, and concanamycin A was from Wako Biochemicals. Restriction enzymes and other molecular biology reagents were from New England Biolabs. Monoclonal antibody against the Myc epitope (9E10) was obtained from Roche Applied Science, and HA.11 (16B12) against the HA epitope was from Covance Research Products. Alexa Fluor 488 goat anti-mouse IgG used for immunofluorescence was purchased from Invitrogen. Alkaline phosphatase-conjugated secondary antibody and pGEM-T Easy TA cloning vector were from Promega. Horseradish peroxidase-conjugated c-Myc (9E10):sc-40 was from Santa Cruz Biotechnology. Yeast and Escherichia coli media were from Fisher. All other reagents were purchased from Sigma. Yeast cells were grown in yeast extract –peptone –2% dextrose (YEPD) medium or fully supplemented minimal medium (SD) lacking individual nutrients as described previously (22). For growth and testing of vma mutant strains, YEPD was buffered to pH 5.0 or 7.5 with 50 mM potassium phosphate, 50 mM potassium succinate.

Strain Construction—All of the strains were derived from SF838-5A{alpha} (23) or from the two-hybrid strains PJ69-4A and PJ69-4{alpha} (24). Genotypes of strains used in this study are listed in Table 1, and oligonucleotides used to construct the strains are listed in Table 2. To delete the VMA5 gene we used oligonucleotides VMA5-600/VMA5+350 to amplify vma5{Delta}:: kanMX6 from the BY4741 vma5{Delta}::kanMX6 deletion strain (25). The vma5{Delta}::kanMX6 PCR product was introduced into the SF838-5A strain using a modified lithium acetate transformation protocol (26), and transformants were selected on 200 µg/ml G418 and tested for integration by PCR and by testing for the Vma– phenotype (lack of growth at pH 7.5 and 60 mM CaCl2). A single HA tag (YPYDVPDYA) was fused to the N terminus of VMA5 to generate the integrated HA-VMA5 strain using a fusion PCR protocol in which two individual PCR products were first produced using oligonucleotide pairs VMA5-600/VMA5-HT2 and VMA5-HT/VMA5+350 with genomic DNA as template. This was followed by a second PCR using oligonucleotides VMA5-600/VMA5+350 and the two PCR products previously produced and gel-purified as templates. The resulting fusion PCR product was transformed into the SF838-5A vma5{Delta} strain, and colonies able to grow on YEPD pH 7.5 plates were selected. The integrated HA-VMA5 was confirmed by DNA sequencing. To delete VMA4 in the RAV1-Myc13 and RAV1-Myc13 HA-VMA5 strains, the VMA4-600/VMA4+200 oligonucleotide pair was used to PCR amplify vma4{Delta}::URA3 from the SF838-5A vma4{Delta}::URA3 (9) strain. The vma4{Delta}::URA3 fragment was used to transform the RAV1-Myc13, and the RAV1-Myc13 HA-VMA5 strains as described above and transformants were selected on supplemented minimal plates lacking uracil (SD –ura), followed by testing for integration by PCR and by testing for lack of growth on pH 7.5 and 60 mM CaCl2. We used a previously constructed SF838-5A VMA2-GFP (27) strain to introduce the GFP tag at the C terminus of VMA2 in the SF838-5A rav1{Delta}, SF838-5A vma5{Delta} and SF838-5A vma10{Delta} strains as described (28). The VMA4 open reading frame was deleted in the PJ69-4A and PJ69-4{alpha} strains by integrating a vma4{Delta}::URA3 PCR fragment as described above in the RAV1-Myc13 strains. The VMA10 open reading frame was deleted in a similar manner using the oligonucleotide pair VMA10-2/VMA10-200 to amplify the vma10{Delta}::URA3 PCR fragment from the SF838-5A vma10{Delta}::URA3 strain (12). Transformants were selected on supplemented minimal plates lacking uracil (SD –ura), and disruption of the VMA4 or VMA10 genes was confirmed by PCR and phenotype testing as described above.


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  		TABLE 1
Genotypes of strains used in this study

 


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  		TABLE 2
Oligonucleotides used in this study

 
Two-hybrid Analysis—To introduce RAV1 and RAV2 into the pAS2 two-hybrid plasmid (29), RAV1 and RAV2 were amplified from wild type genomic DNA with primer pairs RAV1NBamHI/YJR9 and RAV2NBamHI/YDR9, respectively. BamHI sites were introduced just upstream of the ATG start codon. The resulting PCR fragments were cloned into the pGEM-T Easy TA cloning vector and sequenced for accuracy. RAV1 and RAV2 inserts were excised using BamHI and SalI and cloned into the BamH1/SalI cleaved pAS2 vector. A similar method was used to clone RAV1, RAV2, VMA4, and VMA10 into the pACT2 (29) two-hybrid vector with the BamHI sites in frame with the activation domain of the pACT2 plasmid and using primer pairs RAV1NBamHI/YJR9, RAV2NBamHI/YDR9, VMA4BamHI/VMA4+200, and VMA10NBamHI/VMA10-2, respectively. PCR inserts were cloned into the pGEM-T Easy vector and sequenced, and inserts were excised with BamHI and SalI and cloned into the BamHI/XhoI cleaved pACT2 vector. To clone VMA5 into the pACT2 vector, the oligonucleotide pair VMA5-5'2hyb/VMA5-3'2hyb was used to amplify VMA5 from wild type genomic DNA and processed as above except that BamHI and SacI were used to excise the VMA5 insert from the pGEM-T Easy vector and cloned into the BamHI/SacI cleaved pACT2 vector. pAS2 plasmids were introduced into the two-hybrid reporter strains PJ69-4A (MATa), PJ69-4A vma4{Delta}, and PJ69-4A vma10{Delta}, and pACT2 plasmids were introduced into PJ69-4{alpha} (MAT{alpha}), PJ69-4{alpha} vma4{Delta}, and PJ69-4{alpha} vma10{Delta} reporter strains as described above, and transformants were selected on SD plates lacking tryptophan or leucine for selection of the pAS2 and pACT2 plasmids, respectively. To test for two-hybrid interactions, a MATa strain containing a pAS2 plasmid construct was crossed to a MAT{alpha} strain containing a pACT2 plasmid and diploids were selected on SD –trp, –leu. The resulting diploid strains were tested for a two-hybrid interaction by plating onto SD plates lacking tryptophan, leucine, adenine and histidine (SD –trp –leu –ade –his) (24).

Immunoprecipitations and Immunoblots—For coimmunoprecipitations of Rav1pMyc with HA-Vma5p and Vma2p, cytosolic fractions were obtained from the Rav1p-Myc HA-Vma5p strains as described (30). Briefly, the cells (100 A600 of each strain) were lysed by agitation with glass beads, and cytosol was obtained by centrifugation for 30 min at 100,000 x g in a Beckman TLA-100 ultracentrifuge. Protein concentrations of the cytosolic fractions from the various strains were measured by Lowry assay (31); 0.4 mg of protein was directly precipitated with trichloroacetic acid, and 4.0 mg was combined with 100 µl of anti-Vma2p monoclonal antibody (13D11) or 6 µl(~30 µg) anti-HA (16B12) monoclonal antibody followed by the addition of 60 µl of a 50% suspension of protein A-Sepharose CL4B. The trichloroacetic acid-precipitated and immunoprecipitated proteins were solubilized at 75 °C in cracking buffer (50 mM Tris-HCl, pH 6.8, 8 M urea, 5% SDS, 5% beta-mercaptoethanol) for analysis on SDS-PAGE and immunoblotted as described below.

Other Biochemical Techniques—Vacuolar vesicles were isolated as described (32). ATP hydrolysis activity was determined at 37 °C using a coupled enzyme assay (33) in the presence and absence of 100 nM concanamycin A. For immunoblotting, vacuolar vesicles were solubilized in cracking buffer. For all immunoblots, the samples were separated by SDS-PAGE and then transferred to either nitrocellulose or polyvinylidene difluoride membranes. The blots were probed with monoclonal antibodies 13D11, 7A2, and 10D7 against V1 subunits B and C and V0 subunit a, respectively (34). Primary antibodies were bound by alkaline phosphatase-conjugated goat anti-mouse secondary antibody and detected by colorimetric assay as described (35). Western blot signals were quantitated using Image J (National Institutes of Health). Rav1p-Myc was detected with mouse monoclonal antibody 9E10 or horseradish peroxidase-conjugated 9E10 antibody. The antibodies were detected on blots either as described above or by using ECL detection reagents from Amersham Biosciences to detect the primary horseradish peroxidase-conjugated 9E10 antibody.

Microscopy and Immunofluorescence—Strains carrying the VMA2-GFP construct were grown in YEPD at 30 °C to 1.0 A600/ ml, and GFP fluorescence was observed on a Zeiss Axioskop II microscope under fluorescein fluorescence optics. The images were captured using a Hamamatsu CCD camera. Indirect immunofluorescence microscopy was performed as described (32). Anti-Vma2p (13D11) and anti-HA monoclonal antibodies were used at dilutions of 1:10 and 1:100, respectively. Secondary Alexa Fluor 488 goat anti-mouse IgG (2 µg/µl) was used at a 1:200 dilution. The cells were visualized by fluorescence microscopy as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
RAVE Binds to Subunit C—The binding of RAVE to V1 is almost completely lost in the vma4{Delta} and vma10{Delta} mutants, which lack subunits E and G, respectively (17). Based on these results we hypothesized that RAVE binds to the stalk region of the cytosolic V1 complex, specifically subunits E and/or G. To examine further the nature of RAVE/V1 binding, we looked at pairwise interactions between RAVE subunits and V1 subunits in two-hybrid assays (see "Experimental Procedures"). We found that Rav1p interacts with Rav2p and Skp1p of the RAVE complex and Vma4p (subunit E), Vma10p (subunit G), and Vma5p (subunit C) of the V1 complex. Rav2p interacts with Rav1p and Vma5p, but not with Skp1p, Vma4p, or Vma10p (Fig. 1A). The remaining V1 subunits (Vma1p, Vma2p, Vma7p, Vma8p, and Vma13p) did not interact in two-hybrid tests with Rav1p or Rav2p (data not shown).

The disruption of the RAVE/V1 interaction in subunit E and G deletion strains (17) predicted positive interactions in the two-hybrid assay between RAVE and subunits E and/or G, but the RAVE interaction with subunit C was surprising. As noted above, subunit C is released from both V1 and V0 domains during disassembly. Subunit C is isolated only at very low levels with cytosolic V1 subcomplexes from glucose-deprived cells or yeast cells containing only V1 subcomplexes because of a deletion of a V0 subunit (36). An interaction between RAVE and subunit C raises the question as to whether this interaction is independent of the RAVE/V1 interaction. It is possible that the two-hybrid interactions between the RAVE subunits and Vma5p are not due to direct binary interactions but instead result from other V1 subunits bridging the interaction. To address this question we tested these interactions in two-hybrid strains that lacked either subunit E or subunit G; loss of these subunits disrupts the RAVE/V1 interaction. Rav2p is able to interact with subunit C in these deletion strains (Fig. 1B). Similar tests with Rav1p were not possible because we were unable to make the appropriate diploid in a vma{Delta} background. Overexpression of either Rav1p or Vma5p is somewhat toxic (37, 38), and this toxicity may be exacerbated in the vma{Delta} strains. Nevertheless, the results suggest that the interaction between Rav2p and subunit C does not occur via a bridging interaction with V1 subunits and that the RAVE/V1 and RAVE/subunit C interactions can occur independently.


Figure 1
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  		FIGURE 1.
Two-hybrid interactions between RAVE and V1 subunits. A, wild type RAV1 and RAV2 were cloned into the pAS2 two-hybrid vector and RAV2, SKP1, VMA4 (subunit E), VMA5 (subunit C), and VMA10 (subunit G) were cloned into the pACT2 two-hybrid vector as described under "Experimental Procedures." pAS2-RAV1 and pAS2-RAV2 plasmids were transformed independently into the MATa two-hybrid strain PJ69-4A and pACT2-RAV2, pACT2-SKP1, pACT2-VMA4, pACT2-VMA5, and pACT2-VMA10 were transformed into the MAT{alpha} two-hybrid strain PJ69-4{alpha}. To test for interactions, the PJ69-4A strains containing pAS2-RAV1 and pAS2-RAV2 were crossed separately to the PJ69-4{alpha} strains containing the pACT2 plasmid constructs containing the RAVE and V1 subunits described above and to the empty pACT2 vector as a control. The diploid strains resulting from these crosses were plated on supplemented minimal medium lacking tryptophan and leucine (SD –trp –leu) to select for the pAS2 and pACT2 plasmids, respectively. To test for interactions equal numbers of cells were applied (from left to right; undiluted and 1:10, 1:100, and 1:1000 dilutions) on control (SD –trp –leu) and on selective medium that also lacked adenine and histidine (SD –trp –leu –ade –his). Growth on –ade, –his plates indicates an interaction. B, the pAS-RAV2 plasmid was transformed separately into strains PJ69-4A vma4{Delta} and PJ69-4A vma10{Delta}, and the pACT2-VMA5 and pACT plasmids were transformed separately into PJ69-4{alpha} vma4{Delta} and PJ69-4{alpha} vma10{Delta}. Two-hybrid tests were performed as in A.

 


Figure 2
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  		FIGURE 2.
Interaction of cytosolic RAVE with V1 and subunit C in wild type, vma4{Delta}, and vma5{Delta} Cells. A, cytosolic fractions were prepared from a strain carrying a C-terminal Myc-tagged RAV1 and a vma5{Delta} mutation (Rav1p-Myc vma5{Delta}) or an N-terminal HA-tagged VMA5 (Rav1p-Myc HA-Vma5p). Cytosolic proteins (4 mg) from both strains were immunoprecipitated separately with an antibody against the HA epitope (16B12) or a monoclonal antibody against subunit B (13D11). Immunoprecipitates were solubilized in cracking buffer, separated by SDS-PAGE, and immunoblotted with an antibody against the Myc epitope (9E10). B, cytosolic fractions were prepared from a Rav1p-Myc vma4{Delta} strain and from a Rav1p-Myc HA-Vma5p vma4{Delta} strain. Protein was directly trichloroacetic acid-precipitated from 0.4 mg of cytosol (input) from each strain, and 4 mg of cytosol from each strain was immunoprecipitated separately with an antibody against the HA epitope (16B12) or a monoclonal antibody against subunit B (13D11). Myc-tagged Rav1p was detected as in A.

 
RAVE Binds Subunit C Independent of V1 in Vivo—Based on the two-hybrid data presented in Fig. 1A above and on previous immunoprecipitation data in strains deleted for individual RAVE and V1 subunits, Rav1p appears to connect Skp1p, Rav2p, and V1 (17, 18, 39). To confirm a RAVE/subunit C interaction in vivo, we tested the ability of Rav1p to coprecipitate subunit C. A strain that expressed Vma5p containing a single HA epitope tag at the N terminus was constructed (see "Experimental Procedures"). HA-VMA5 was integrated into the yeast genome of a previously constructed Rav1p-Myc strain (17) creating a strain in which the sole copies of both VMA5 and RAV1 are epitope-tagged. This strain has a wild type growth phenotype and a V-ATPase complex with wild type assembly and activity properties in isolated vacuolar vesicles (data not shown). A cytosolic protein extract from the Rav1p-Myc HA-Vma5p strain was immunoprecipitated with anti-HA under native conditions, blotted, and probed with anti-Myc. Fig. 2A (left panel, second lane) shows that HA-Vma5p coimmunoprecipitates Rav1p-Myc, confirming an interaction between Rav1p and subunit C in vivo. Immunoprecipitation with anti-Vma2p (V1) in a vma5{Delta} strain is able to pull down Rav1p-Myc, demonstrating that RAVE can interact with V1 in the absence of its interaction with subunit C (Fig. 2A, right panel, first lane).

The two-hybrid data above suggested that the Rav1p/subunit C interaction occurs independent of the RAVE/V1 interaction. To verify this in vivo, we deleted the VMA4 gene in the Rav1p-Myc HA-Vma5p strain so that we could repeat the immunoprecipitation in a background where RAVE is not able to interact with V1. Fig. 2B (right panel, center lane) shows that HA-Vma5p coimmunoprecipitates Rav1p-Myc in a vma4{Delta} strain, confirming that RAVE can interact with subunit C in the cytosol independent of its interaction with V1. Loss of coimmunoprecipitation of this same strain with anti-Vma2p (subunit B) verifies that Rav1p does not interact with V1 when VMA4 is deleted (Fig. 2B, right panel, last lane).

As noted above, stalk subunit C is required for activity and proper assembly of the yeast vacuolar ATPase, but after in vivo dissociation of the V-ATPase induced by glucose deprivation, very little subunit C is bound to either V1 or V0 (14, 40). Yeast strains lacking subunit C (vma5{Delta}) have a strong Vma– phenotype characterized by failure to grow at pH 7.5 and high Ca2+ concentrations. vma5{Delta} mutants are distinct from most other V1 subunit deletion strains, however, in that both V1 and V0 subcomplexes assemble (9). Additionally, isolated vacuoles from vma5{Delta} strains, although they have no V-ATPase activity, do have low levels of V1 subunits at the vacuole (40). An interaction between RAVE and subunit C suggests that RAVE may be involved in establishing a functional interaction between subunit C and the V-ATPase.

rav1{Delta} Assembly Phenotypes Resemble vma5{Delta} Assembly Phenotypes—To examine the assembly defects of rav1{Delta} and vma5{Delta} mutants in vivo, we employed strains expressing an integrated GFP-tagged V1 subunit B (Vma2p-GFP) (27) that would allow us to visualize the localization of V1 in these mutants. As shown in the top row of Fig. 3, the Vma2p-GFP subunit localizes specifically to the vacuolar membrane in wild type cells. In most V-ATPase subunit deletion mutants, V1 subunits are found in the cytosol and are visualized by Vma2p-GFP as cytosolic fluorescent staining. The bottom row in Fig. 3 shows an example of this diffuse cytosolic staining in a vma10{Delta} mutant. In the vma5{Delta} mutant (Fig. 3, third row from the top), Vma2p-GFP is clearly visible at the vacuolar membrane and at lower levels in the cytosol. This localization of Vma2p-GFP in a vma5{Delta} mutant confirms that the vma5{Delta} mutant allows some assembly of V1 subunits with the vacuolar membrane in vivo. In a rav1{Delta} mutant Vma2p-GFP shows a localization that is very similar to that of the vma5{Delta} mutant (Fig. 3, second row from top). It has been reported previously that rav{Delta} mutants appear to support some functional V-ATPase assembly and activity in vivo, but they, like vma5{Delta} mutants, are highly unstable in vitro (17, 40).

To further characterize and compare assembly defects of V-ATPase complexes in rav1{Delta} and vma5{Delta} mutants, we isolated vacuolar vesicles from these strains and also from a vma3{Delta} strain that is not able to assemble V0 or V1/V0 complexes at the vacuolar membrane (34). Immunoblots of the isolated vesicles shown in Fig. 4A indicate that although both rav1{Delta} and vma5{Delta} vesicles have levels of V0 subunit a approximately equal to wild type vesicles, they have much lower levels of V1 subunits A and B. The rav1{Delta} mutant vesicles, however, have significantly higher levels of subunits A and B than the vma5{Delta} vesicles, and also a small amount of subunit C can be detected in rav1{Delta} vesicles. By quantitating bands on immunoblots containing different dilutions of wild type and mutant vesicles (Fig. 4B), we were able to estimate that rav1{Delta} vesicles have ~9.3% of the V1 B subunit when compared with wild type and less than 2% of the C subunit. vma5{Delta} vesicles have less than 2% of subunit B compared with wild type. These results correlate well with previous observations in rav1{Delta} and vma5{Delta} mutant vesicles (17, 40). Although the Vma2p-GFP data described above indicate that V1/V0 is able to assemble in rav1{Delta} and vma5{Delta} mutants in vivo, these complexes are highly unstable in vitro because very small percentages of V1 subunits are found in isolated vacuolar vesicles from these strains, and V-ATPase activity is extremely low in isolated vesicles from these mutants. rav1{Delta} V-ATPase activity is 5.3%, and vma5{Delta} is 2.0% of wild type activity. Although the assembly defects in the rav1{Delta} and vma5{Delta} strains have striking similarities, rav1{Delta} mutants have more V1 subunits in isolated vesicles, including a small amount of subunit C, when compared with vma5{Delta} vesicles, and rav1{Delta} mutants are able to produce a population of V-ATPase complexes in vivo that provide some function at the permissive temperature of 30 °C. A similarity in V-ATPase assembly phenotypes in RAVE mutants and subunit C mutants could be explained by a loss of functional association of subunit C with the V-ATPase in the absence of RAVE.


Figure 3
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  		FIGURE 3.
Localization of Vma2p-GFP in Wild Type, rav1{Delta}, and Vma– Mutant Strains. Vma2p-GFP was introduced into wild type, rav1{Delta}, vma5{Delta}, and vma10{Delta} cells and visualized by fluorescence microscopy (right panels) as described under "Experimental Procedures." The same cells visualized under Nomarski optics are shown in the left panels.

 


Figure 4
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  		FIGURE 4.
Levels of V-ATPase subunits and V-ATPase activity in vacuolar vesicles isolated from wild type, rav1{Delta}, vma5{Delta}, and vma3{Delta} strains. A, vacuolar vesicles were prepared from wild type cells (SF838-5A) and the congenic rav1{Delta}, vma5{Delta}, and vma3{Delta} mutants. The vesicles were solubilized in cracking buffer, separated by SDS-PAGE, transferred to nitrocellulose, and probed with monoclonal antibodies against V0 subunit a, and V1 subunits A, B, and C or alkaline phosphatase, a vacuolar membrane protease. 9 µg of vacuolar protein was loaded for visualization of the A and B subunits, 25 µg of protein was loaded for visualization of the a and C subunits, and 10 µg of protein was loaded for visualization of alkaline phosphatase. (The higher molecular mass band in the alkaline phosphatase blot from vma3{Delta} and vma5{Delta} strains represents unprocessed alkalinephosphatase.) ATPase activity sensitive to 100 nM concanamycin A was measured for each vacuole preparation and compared with the activity of the wild type strain. V-ATPase activity in the wild type vesicles was 1.34 µmol of ATP consumed/min/mg of protein. The percentage of wild type activity in each strain is shown at the bottom. B, protein from the vacuolar vesicle preparations for wild type, rav1{Delta} and vma5{Delta} strains described in A above were loaded in the amounts indicated at the top of each lane, and immunoblots were prepared and processed as in A above. Levels of subunit B and C were quantitated using Image J, and relative assembly of subunits B and C in rav1{Delta} and vma5{Delta} were compared with wild type. A similar relative assembly pattern was observed on three independent experiments.

 
RAVE Is Required for Subunit C Binding at the Vacuole—The localization of Vma2p-GFP in a rav1{Delta} strain shown in Fig. 3 indicates that a fairly significant amount of V1 subunits assemble at the vacuole in vivo. However, isolated vacuoles from the rav1{Delta} mutant strain shown in Fig. 4B have only 9.3% of subunit B compared with wild type vacuoles. Presumably the unstable V-ATPase complexes in the rav1{Delta} mutant do not remain intact through the vacuole vesicle isolation process, and significantly, the fraction of subunit C in rav1{Delta} vesicles is less than 20% of the fraction of subunit B found in rav1{Delta} compared with wild type vesicles (Fig. 4B). This result suggests that subunit C is depleted from V-ATPase complexes in rav1{Delta} mutants.

To compare the localization of subunit B and subunit C in the rav1{Delta} mutant in vivo, we examined these subunits using indirect immunofluorescence microscopy. Subunit B can be detected by the monoclonal antibody 13D11 in immunofluorescence, and Vma5p was visualized via an HA epitope (14). As shown in the top row of Fig. 5A V1 subunit B localizes predominantly at the vacuolar membrane in wild type cells. The bottom row in Fig. 5A shows significant staining of subunit B at the vacuolar membrane in rav1{Delta} cells with some cytosolic staining as well. This agrees with VMA2p-GFP results shown in Fig. 3. In wild type cells subunit C (HA-Vma5p) displays essentially the same staining pattern at the vacuolar membrane as subunit B. Conversely, subunit C in rav1{Delta} cells shows a diffuse cytosolic staining pattern and little or no vacuolar localization (Fig. 5B, bottom row). This result suggests that RAVE is required for subunit C to assemble efficiently with the vacuolar ATPase. This experiment also suggests that the depletion of subunit C from the vacuole in rav1{Delta} mutants (Fig. 4B) is not caused by loss of subunit C from the cell. To confirm that subunit C was not destabilized in the rav1{Delta} mutant, we compared the total cell levels of HA-tagged subunit C in wild type and rav1{Delta} mutant cells by immunoprecipitation with the anti-HA antibody and immunoblotting. Very similar levels of HA-tagged subunit C are present in the two cell types (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
RAVE and Subunit C Stabilize V-ATPase Stator Stalk Assembly—The prior identification of subunits E and G as mediators of the RAVE-V1 interaction suggested an involvement of the RAVE complex in assembling the V1 peripheral stalk to the membrane-bound V0 complex. The results reported here provide additional support for the notion that RAVE, through its interaction with subunit C, may be facilitating V1/V0 peripheral stalk subunit interactions during V-ATPase assembly.

There is significant homology between the V-ATPase and the bacterial and mitochondrial F1F0-ATP synthase (F-ATPase) in both structure and mechanism of action (1). Both the V-ATPase and the F-ATPase utilize a rotational catalytic mechanism (41). In the V-ATPase, ATP hydrolysis on the V1 A subunits drives rotation of the central stalk that is composed of the V1 subunits D and F and the V0 subunits d and the proteolipid ring (41, 42). Proton translocation occurs at the interface between the rotating proteolipid ring and the fixed V0 a subunit. For proper coupling of ATP hydrolysis at the catalytic sites and proton transport across the membrane, a stator structure is required that prevents rotation of the static domain (43). Numerous studies have shown that the stator of the E. coli F-ATPase is composed of a dimer of the b subunit of F0 (44). V-ATPase subunit G shows some homology to subunit b of the F-ATPase, and subunit G has been shown to interact strongly with subunit E (45–47). The V-ATPase E-G complex could be functioning in a manner similar to the b dimer of the F-ATPase, but it is notable that unlike the b subunit, subunit G has no membrane domain. Unlike the F-ATPase, the activity of the V-ATPase is down-regulated by reversible dissociation of the V1 and V0 domains of the enzyme in response to low extracellular glucose, and also unlike the F-ATPase, the dissociated V1 and V0 complexes are enzymatically inactive (6, 7). The role of the stator stalk (or stalks) of the V-ATPase, therefore, is 2-fold. It not only must act to stabilize the interaction between the V1 and V0 sectors but also must allow for the regulated disassembly/reassembly of the enzyme. Most of the V-ATPase subunits have functional homologues (although not necessarily sequence homologues) in the F-ATPase. The exceptions are V-ATPase peripheral stalk V1 subunits C and H and V0 subunit d and the cytoplasmic domain of subunit a.


Figure 5
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  		FIGURE 5.
Immunolocalization of V1 subunits B and C in wild type and rav1{Delta} cells. Wild type strain SF838-5A and congenic strains SF838-5A rav1{Delta}, SF838-5A HA-VMA5, and SF838-5A HA-VMA5 rav1{Delta} were grown in YEPD pH 5.0 medium at 30 °C to an 1.0 A600/ml. The strains were fixed, spheroplasted, permeabilized, and mounted onto slides as described under "Experimental Procedures." A, Vma2p was visualized using the monoclonal antibody 13D11 followed by a secondary Alexa Fluor 488 goat anti-mouse IgG. The cells were visualized and recorded by fluorescence microscopy as described under "Experimental Procedures." B, HA-Vma5p was visualized using the anti-HA monoclonal antibody HA.11 (16B12) followed by the secondary Alexa Fluor 488 goat anti-mouse IgG and visualized and recorded as in A above. All of the images were obtained using the same exposure time. In some cases separate images were combined to create the frame. DIC, differential interference contrast microscopy.

 


Figure 6
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  		FIGURE 6.
RAVE delivers the C subunit to V1/V0 in both biosynthesis and reassembly. RAVE subunits are shaded in gray. V1 subunits are white, and V0 subunits are black.

 
Recent studies have implicated V1 subunit C as part of the stator holding the V1 and V0 domains together and as the primary regulator of reversible dissociation of the complex. Based on cysteine-mediated cross-linking, subunit C binds to key subunits of the peripheral stalk including E and G of the V1 complex and to subunit a of the V0 complex (19). This localization of subunit C has been confirmed by immunoelectron microscopy that shows subunit C bound to the stalk region at the V1/V0 interface (21). Interestingly, subunit C binds on the opposite side of V1V0 from subunit H, the other V1 subunit implicated in bridging subunits E and G of V1 and V0 subunit a (21, 48). This arrangement suggests that the two copies of subunits E and G (45) might be present as two distinct EG stalks, one bridged to V0 by subunit H and the other by subunit C. In yeast, subunit C deletion (vma5{Delta}) mutants display a typical V-ATPase null phenotype, but a closer examination of the vma5{Delta} mutant reveals characteristics that distinguish it from other V1 subunits. In most V1 deletion mutants the V1 subcomplex is not able to assemble in the cytosol, whereas vma5{Delta} mutants can assemble both V1 and V0 subcomplexes (12). In this study we show that in a vma5{Delta} mutant a significant portion of a GFP-tagged V1 subunit is able to bind V0 at the vacuolar membrane in vivo. These V1V0 complexes, however, are not stable in in vitro isolations and do not support V-ATPase activity. We conclude from these data that a nonfunctional V1V0 complex is able to form in the absence of subunit C. RAVE mutants, rav1{Delta} and rav2{Delta}, show many similarities to vma5{Delta} mutants. A GFP-tagged V1 subunit in a rav1{Delta} mutant shows a localization that is very much like that seen in cells lacking subunit C. RAVE mutants have a partial Vma– phenotype that supports some assembly and some activity of the V-ATPase in vivo in contrast to the strong Vma– phenotype of vma5{Delta} mutants. Interestingly, a number of subunit C mutations exhibit growth phenotypes that are identical to rav mutants; that is they do not have a strong Vma– phenotype in vivo, but the V-ATPase complexes show assembly defects in vitro (49). The similarity in defects in subunit C function and RAVE function suggested to us that RAVE might be involved in the attachment of subunit C with the peripheral stalk of the V-ATPase.

One notable difference between the rav1{Delta} mutant and the vma5{Delta} mutant is the temperature conditional nature of the Vma– phenotype in the rav1{Delta} mutant. We cannot explain this difference at present. It may be that there is a more stringent requirement for V-ATPase function at high temperature, so that the rav1{Delta} mutant, which retains a low level of V-ATPase activity (Fig. 4A), can support sufficient V-ATPase function at 30 °C, but not at 37 °C. It could also be that there is an alternate, inefficient route of C subunit assembly in the rav1{Delta} mutant that is functional at 30 °C but not at 37 °C or that the C subunit assumes a conformation at 37 °C that makes it less readily assembled in the absence of RAVE. Further experiments will be necessary to distinguish these possibilities.

RAVE Is Required for Efficient Assembly of Subunit C with the V-ATPase—Vacuolar vesicles isolated from a rav1{Delta} mutant strain show lower levels of all V1 subunits, but Vma5p is present at significantly lower levels than the other V1 subunits. This suggests that in the absence of RAVE function, the association of subunit C with V1 is defective. Further evidence for this assumption is provided in the immunofluorescent localization of HA-Vma5p in a rav1{Delta} strain. We show in Fig. 5 that although a significant fraction of V1 subunit B is found at the vacuolar membrane in a strain lacking Rav1p, subunit C localizes to the cytosol and shows no staining at the vacuole. Our data support a model in which RAVE is directly involved in the attachment of subunit C to the peripheral stalk either just prior to or during the assembly of the V1 complex with the membrane-bound V0.

RAVE Delivers Subunit C to V1/V0 in Biosynthesis and Reassembly—Both biosynthetic assembly of the enzyme and reversible disassembly of the assembled complexes establish the overall level of assembly of the V-ATPase. The RAVE complex is required for stable V-ATPase assembly in both processes, but where and when in the assembly process do V1, subunit C, and V0 come together? Does RAVE play a similar role in the assembly of the C subunit in both biosynthetic assembly and regulated reassembly? A clue to the assembly function of RAVE was provided by the independent isolation of a mutation in Rav1p (soi3-1) that suppresses the mislocalization of a Kex2p mutant with a defective trans-Golgi network localization signal (50). These studies identified a membrane-bound fraction of Rav1p in addition to the cytosolic fraction. In sucrose gradient fractionation, the membrane-associated Rav1p cofractionated in a dense fraction characteristic of early endosomes and localization of Rav1p-GFP was also consistent with localization to early endosomes (50). The conclusion from this work was that acidification of early endosomes was required for proper localization of trans-Golgi network proteins and that this acidification is regulated at the level of assembly of the V-ATPase. Therefore, a mutation in Rav1p (soi3-1) resulted in defective V-ATPase assembly at the early endosome. One possibility is that the fraction of Rav1p that is in the early endosome is functioning in the initial biosynthetic assembly of the V-ATPase, and the Rav1p fraction in the cytosol is involved in the regulated reassembly of disassembled V-ATPase complexes. The biochemical function of the RAVE complex could be the same in both places, and based on our current findings, RAVE is likely "chaperoning" or directing subunit C to properly orient the peripheral stalk subunits of the V1 and V0 complexes and assemble a stable active V-ATPase (Fig. 6).

Why Would Subunit C Need a Specialized Chaperone?—If there are two peripheral stalks, as suggested by electron microscopy (21), then forming distinct interfaces with the V0 sector may require RAVE intervention during biosynthesis. In addition, RAVE may serve to keep subunit C away from cytosolic V1 sectors. If subunit C is, as has been proposed, the regulator of reversible dissociation of the V-ATPase, a chaperone may be needed to ensure that V1 and V0 functionally interact only when conditions in the cell require an active V-ATPase.


   FOOTNOTES
 
* This work was supported by a postdoctoral fellowship from the American Heart Association, New York State Affiliate (to A. M. S.) and National Institutes of Health Grant RO1 GM63742 (to P. M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: SUNY Upstate Medical University, 750 East Adams St., Syracuse, NY 13210. Tel.: 315-464-8742; Fax: 315-464-8750; E-mail: kanepm{at}upstate.edu.

2 The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; F-ATPase, F1F0-ATP synthase; HA, influenza hemagglutinin epitope; GFP, green fluorescence protein; YEPD, yeast extract-peptone-2% dextrose media; SD, supplemented minimal medium with 2% dextrose. Back


   ACKNOWLEDGMENTS
 
We thank Ester Cobb for the pACT2-VMA5 plasmid and Maureen Tarsio for technical assistance and helpful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nishi, T., and Forgac, M. (2002) Nat. Rev. Mol. Cell. Biol. 3, 94–103[CrossRef][Medline] [Order article via Infotrieve]
  2. Mellman, I., Fuchs, R., and Helenius, A. (1986) Annu. Rev. Biochem. 55, 663–700[CrossRef][Medline] [Order article via Infotrieve]
  3. Stevens, T. H., and Forgac, M. (1997) Annu. Rev. Cell Dev. Biol. 13, 779–808[CrossRef][Medline] [Order article via Infotrieve]
  4. Dietz, K. J., Tavakoli, N., Kluge, C., Mimura, T., Sharma, S. S., Harris, G. C., Chardonnens, A. N., and Golldack, D. (2001) J. Exp. Bot. 52, 1969–1980[Abstract/Free Full Text]
  5. Wieczorek, H., Brown, D., Grinstein, S., Ehrenfeld, J., and Harvey, W. R. (1999) Bioessays 21, 637–648[CrossRef][Medline] [Order article via Infotrieve]
  6. Graf, R., Harvey, W. R., and Wieczorek, H. (1996) J. Biol. Chem. 271, 20908–20913[Abstract/Free Full Text]
  7. Beltran, C., and Nelson, N. (1992) Acta Physiol. Scand. 607, (suppl.) 41–47
  8. Myers, M., and Forgac, M. (1993) J. Cell. Physiol. 156, 35–42[CrossRef][Medline] [Order article via Infotrieve]
  9. Doherty, R. D., and Kane, P. M. (1993) J. Biol. Chem. 268, 16845–16851[Abstract/Free Full Text]
  10. Sumner, J. P., Dow, J. A., Earley, F. G., Klein, U., Jager, D., and Wieczorek, H. (1995) J. Biol. Chem. 270, 5649–5653[Abstract/Free Full Text]
  11. Tomashek, J. J., Sonnenburg, J. L., Artimovich, J. M., and Klionsky, D. J. (1996) J. Biol. Chem. 271, 10397–10404[Abstract/Free Full Text]
  12. Kane, P. M., Tarsio, M., and Liu, J. (1999) J. Biol. Chem. 274, 17275–17283[Abstract/Free Full Text]
  13. Ochotny, N., Van Vliet, A., Chan, N., Yao, Y., Morel, M., Kartner, N., von Schroeder, H. P., Heersche, J. N., and Manolson, M. F. (2006) J. Biol. Chem. 281, 26102–26111[Abstract/Free Full Text]
  14. Kane, P. M. (1995) J. Biol. Chem. 270, 17025–17032[Abstract/Free Full Text]
  15. Sautin, Y. Y., Lu, M., Gaugler, A., Zhang, L., and Gluck, S. L. (2005) Mol. Cell. Biol. 25, 575–589[Abstract/Free Full Text]
  16. Trombetta, E. S., Ebersold, M., Garrett, W., Pypaert, M., and Mellman, I. (2003) Science 299, 1400–1403[Abstract/Free Full Text]
  17. Smardon, A. M., Tarsio, M., and Kane, P. M. (2002) J. Biol. Chem. 277, 13831–13839[Abstract/Free Full Text]
  18. Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384–391[CrossRef][Medline] [Order article via Infotrieve]
  19. Inoue, T., and Forgac, M. (2005) J. Biol. Chem. 280, 27896–27903[Abstract/Free Full Text]
  20. Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1148–1152[CrossRef][Medline] [Order article via Infotrieve]
  21. Zhang, Z., Inoue, T., Forgac, M., and Wilkens, S. (2006) FEBS Lett. 580, 2006–2010[CrossRef][Medline] [Order article via Infotrieve]
  22. Sherman, F., Fink, G. R., and Hicks, J. B. (1982) Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  23. Stevens, T. H., Rothman, J. H., Payne, G. S., and Schekman, R. (1986) J. Cell Biol. 102, 1551–1557[Abstract/Free Full Text]
  24. James, P., Halladay, J., and Craig, E. A. (1996) Genetics 144, 1425–1436[Abstract]
  25. Winzeler, E. A., Shoemaker, D. D., Astromoff, A., Liang, H., Anderson, K., Andre, B., Bangham, R., Benito, R., Boeke, J. D., Bussey, H., Chu, A. M., Connelly, C., Davis, K., Dietrich, F., Dow, S. W., El Bakkoury, M., Foury, F., Friend, S. H., Gentalen, E., Giaever, G., Hegemann, J. H., Jones, T., Laub, M., Liao, H., Liebundguth, N., Lockhart, D. J., Lucau-Danila, A., Lussier, M., M'Rabet, N., Menard, P., Mittmann, M., Pai, C., Rebischung, C., Revuelta, I. L., Riles, L., Roberts, C. J., Ross-MacDonald, P., Scherens, B., Snyder, M., Sookhai-Mahadeo, S., Storms, R. K., Véronneau, S., Voet, M., Volckaert, G., Ward, T. R., Wysocki, R., Yen, G. S., Yu, K., Zimmermann, K., Philippsen, P., Johnston, M., and Davis, R. W. (1999) Science 285, 901–906[Abstract/Free Full Text]
  26. Ito, H., Fukuda, Y., Murata, K., and Kimura, A. (1983) J. Bacteriol. 153, 163–168[Abstract/Free Full Text]
  27. Sambade, M., Alba, M., Smardon, A. M., West, R. W., and Kane, P. M. (2005) Genetics 170, 1539–1551[Abstract/Free Full Text]
  28. Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998) Yeast 14, 953–961[CrossRef][Medline] [Order article via Infotrieve]
  29. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge, S. J. (1993) Cell 75, 805–816[CrossRef][Medline] [Order article via Infotrieve]
  30. Seol, J. H., Feldman, R. M., Zachariae, W., Shevchenko, A., Correll, C. C., Lyapina, S., Chi, Y., Galova, M., Claypool, J., Sandmeyer, S., Nasmyth, K., and Deshaies, R. J. (1999) Genes Dev. 13, 1614–1626[Abstract/Free Full Text]
  31. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265–275[Free Full Text]
  32. Roberts, C. J., Raymond, C. K., Yamashiro, C. T., and Stevens, T. H. (1991) Methods Enzymol. 194, 644–661[Medline] [Order article via Infotrieve]
  33. Lotscher, H. R., deJong, C., and Capaldi, R. A. (1984) Biochemistry 23, 4140–4143[CrossRef][Medline] [Order article via Infotrieve]
  34. Kane, P. M., Kuehn, M. C., Howald-Stevenson, I., and Stevens, T. H. (1992) J. Biol. Chem. 267, 447–454[Abstract/Free Full Text]
  35. Kane, P. M., and Stevens, T. H. (1992) J. Bioenerg. Biomembr. 24, 383–393[CrossRef][Medline] [Order article via Infotrieve]
  36. Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761–21767[Abstract/Free Full Text]
  37. Brace, E. J., Parkinson, L. P., and Fuller, R. S. (2006) Eukaryot. Cell 5, 2104–2113[Abstract/Free Full Text]
  38. Keenan Curtis, K., and Kane, P. M. (2002) J. Biol. Chem. 277, 2716–2724[Abstract/Free Full Text]
  39. Kane, P. M., and Smardon, A. M. (2003) J. Bioenerg. Biomembr. 35, 313–321[CrossRef][Medline] [Order article via Infotrieve]
  40. Ho, M. N., Hill, K. J., Lindorfer, M. A., and Stevens, T. H. (1993) J. Biol. Chem. 268, 221–227[Abstract/Free Full Text]
  41. Hirata, T., Iwamoto-Kihara, A., Sun-Wada, G. H., Okajima, T., Wada, Y., and Futai, M. (2003) J. Biol. Chem. 278, 23714–23719[Abstract/Free Full Text]
  42. Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312–2315[Abstract/Free Full Text]
  43. Dunn, S. D., McLachlin, D. T., and Revington, M. (2000) Biochim. Biophys. Acta 1458, 356–363[Medline] [Order article via Infotrieve]
  44. Grabar, T. B., and Cain, B. D. (2003) J. Biol. Chem. 278, 34751–34756[Abstract/Free Full Text]
  45. Ohira, M., Smardon, A. M., Charsky, C. M., Liu, J., Tarsio, M., and Kane, P. M. (2006) J. Biol. Chem. 281, 22752–22760[Abstract/Free Full Text]
  46. Fethiere, J., Venzke, D., Diepholz, M., Seybert, A., Geerlof, A., Gentzel, M., Wilm, M., and Bottcher, B. (2004) J. Biol. Chem. 279, 40670–40676[Abstract/Free Full Text]
  47. Jones, R. P., Durose, L. J., Findlay, J. B., and Harrison, M. A. (2005) Biochemistry 44, 3933–3941[CrossRef][Medline] [Order article via Infotrieve]
  48. Wilkens, S., Inoue, T., and Forgac, M. (2004) J. Biol. Chem. 279, 41942–41949[Abstract/Free Full Text]
  49. Curtis, K. K., Francis, S. A., Oluwatosin, Y., and Kane, P. M. (2002) J. Biol. Chem. 277, 8979–8988[Abstract/Free Full Text]
  50. Sipos, G., Brickner, J. H., Brace, E. J., Chen, L., Rambourg, A., Kepes, F., and Fuller, R. S. (2004) Mol. Biol. Cell 15, 3196–3209[Abstract/Free Full Text]

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