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Originally published In Press as doi:10.1074/jbc.M606451200 on August 22, 2006

J. Biol. Chem., Vol. 281, Issue 42, 32025-32035, October 20, 2006
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PKR1 Encodes an Assembly Factor for the Yeast V-Type ATPase*

Sandra R. Davis-Kaplan{ddagger}1, Mark A. Compton§, Andrew R. Flannery§, Diane M. Ward{ddagger}, Jerry Kaplan{ddagger}2, Tom H. Stevens§3, and Laurie A. Graham§1

From the {ddagger}Division of Immunology and Cell Biology, Department of Pathology, School of Medicine, University of Utah, Salt Lake City, Utah 84132-2501 and the §Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229

Received for publication, July 6, 2006 , and in revised form, August 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells. Reduced levels of the V-ATPase lead to defective copper loading of Fet3p, a component of the high affinity iron transport system. Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V0 subunit or an endoplasmic reticulum (ER) assembly factor. However, unlike yeast mutants lacking a V0 subunit or a V-ATPase assembly factor, low levels of Vph1p present in cells lacking Pkr1p are assembled into a V-ATPase complex, which exits the ER and is present on the vacuolar membrane. The V-ATPase assembled in the absence of Pkr1p is fully functional because the mutant cells are able to weakly acidify their vacuoles. Finally, overexpression of the V-ATPase assembly factor Vma21p suppresses the growth and acidification defects of pkr1{Delta} cells. Our data indicate that Pkr1p functions together with the other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The V-type ATPase (V-ATPase)4 is a ubiquitous multisubunit complex essential for cell viability in all eukaryotes except yeast (1). In the yeast Saccharomyces cerevisiae, the V-ATPase is localized to the membranes of the Golgi, endosomes, and vacuole where it plays a key role in cellular ion homeostasis (2). The acidification of organelles by the V-ATPase, due to proton translocation driven by ATP hydrolysis, is required for intracellular transporters to maintain cytosolic ion balance.

The V-ATPase can be divided into two functionally distinct domains, the V0 proton translocating domain and the V1 ATP-hydrolytic domain. The yeast V-ATPase complex is composed of 14 subunits and requires several additional gene products to assemble a fully functional complex (1, 3-5). One of the subunits is present as two isoforms and the isoform assembled into the complex dictates the localization of the V-ATPase in the cell (6, 7). V-ATPase complexes assembled with the Vph1p isoform are localized to the vacuole and the Stv1p isoform-containing complex is found on the membranes of the Golgi and endosome. Yeast cells lacking the Stv1p Golgi/endosome-localized isoform complex display no obvious growth phenotype suggesting that the Stv1p containing V-ATPase complexes are the minor form present in the cells. Yeast cells lacking the vacuolar-localized V-ATPase isoform display an intermediate growth phenotype due to the decrease of the majority of the V-ATPase complexes present in the cell; they are able to grow in the presence of elevated calcium (100 mM) but are unable to grow in the presence of elevated zinc (4 mM).

In addition to the genes encoding subunits of the V-ATPase complex, there are five previously characterized gene products that are not part of the complex but are required for its function. Three proteins (Vma12p, Vma21p, and Vma22p) are localized to the membrane of the endoplasmic reticulum (8-12). These proteins play a role very early in the assembly of the V0 subcomplex of the V-ATPase following protein synthesis and insertion of the subunit proteins into the membrane of the ER. Vma21p may also function to escort the V-ATPase complex out of the ER (8). Two additional non-subunit proteins, localized to the Golgi (Vma45p also known as Kex2p) and to the cytosol (Vma41p also called Cys4p), presumably function post-assembly (4, 5).

Cells lacking a functional V-ATPase display a distinct set of growth phenotypes related to their inability to maintain cellular homeostasis including the inability to grow at neutral pH, or in the presence of elevated concentrations of calcium or zinc (6) or to grow on low iron medium (13). The ability of yeast to grow on low iron media requires the activity of the high affinity iron transport system. This system is comprised of two cell surface proteins, a multicopper oxidase Fet3p and a transmembrane permease Ftr1p (14). The multicopper oxidase Fet3p is a Type I membrane protein that obtains its copper in a post-Golgi compartment. Copper loading of apoFet3p requires an acidic environment (15). The low pH of the vesicular apparatus is maintained by the V-ATPase and a voltage-regulated chloride channel (Gef1p). In the absence of these elements, apoFet3p fails to be copper loaded, and is localized to the cell surface in an inactive form.

Using a genome-wide screen to identify genes required for growth on low iron we identified CWH36/VMA9 as a subunit of the V-ATPase (13). In the absence of Vma9p the V-ATPase is not assembled, resulting in defective copper loading of apoFet3p. That screen also identified PKR1, a gene encoding a protein that when overexpressed confers Pichia farnosia killer toxin resistance (Saccharomyces Genome Database annotation), as being required for growth on low iron medium.

In this work we show that Pkr1p is an integral membrane protein localized to the ER, similar to the V-ATPase assembly factors Vma12p, Vma21p, and Vma22p. Yeast cells lacking Pkr1p have greatly reduced levels of Vph1p, a subunit of the V-ATPase and component of the V0 subcomplex. The poor growth of pkr1{Delta} cells on low iron results from inefficient copper loading of apoFet3p. Unlike the other V-ATPase assembly factor mutants, cells lacking Pkr1p assemble some functional V-ATPase complex, consistent with the fact that pkr1{Delta} cells are able to grow on media containing 100 mM CaCl2 and these cells have weakly acidified vacuoles. Genetic interactions between PKR1 and VMA21 lead us to conclude that Pkr1p is required for efficient V0 assembly in the ER, but that a low level of V-ATPase assembly occurs in the absence of Pkr1p.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains, Plasmids, and Culture Conditions—Strains and plasmids used in the study are listed in Tables 1 and 2. Yeast were cultured in YEPD either unbuffered or buffered to pH 5.0 using 50 mM succinate/phosphate, or yeast nitrogen base synthetic complete minimal medium with supplements as needed using standard techniques. Low iron growth medium was made by adding 40-90 µM bathophenanthroline disulfonate (BPS), an iron chelator, to complete minimal medium or YEPD and then adding back varying amounts of FeSO4. Media and procedures for growing and analyzing the phenotypes of strains on low iron media have been described (13). Exponentially growing cell cultures were diluted to 1.0 A600 and then serially diluted 10-fold to test the growth phenotype of various yeast strains. 5 µl of the starting culture and each dilution were spotted onto a YEPD, pH 5.0, agar, YEPD + 100 mM CaCl2 agar, or YEPD + 90 µM BPS + FeSO4 and incubated 48 h at 30 °C.


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  		TABLE 1
S. cerevisiae strains and plasmids used in this study

 


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  		TABLE 2
Plasmids used in this study

 
Cloning and Construction of Disruption Strains—A pkr1{Delta}::Kanr fragment was amplified by PCR from genomic DNA of the pkr1{Delta} yeast genomic deletion strain (Open Biosystems), including ~300 base pairs of the 5' and 3' flanking sequence in addition to the drug resistance cassette. The PCR fragment was transformed into either SF838-1D{alpha} or DY1640 using a standard technique (16), replacing the genomic copy of PKR1 and generating LGY116 (pkr1{Delta}::Kanr) and SDK13-6D or SDK13-5D, haploids obtained following sporulation of drug-resistant diploid transformant. SDK13-5C and SDK13-6A were wild-type haploid strains obtained following transformation and sporulation. A PCR fragment containing the Natr sequence using pAG25 as the template also possessing sequence homologous to 40 bp 5' and 3' of the PKR1 open reading frame was generated and used to replace the genomic deletion of pkr1{Delta}::Kanr generating the pkr1{Delta}::Natr deletion strain (LGY117). A pkr1{Delta}::Natr fragment was generated using genomic DNA prepared from LGY117 by PCR that included ~300 base pairs of 5' and 3' flanking sequence in addition to the open reading frame. The PCR fragment was transformed into yeast strains (KEBY4, KHY31, and LGY146) replacing the genomic copy of PKR1 generating LGY118, LGY119, and LGY147. A similar strategy was used to generate LGY120 and LGY146 using PCR fragments with an ~300-bp flanking sequence amplified from genomic DNA prepared from the appropriate genomic deletion collection strain. A PCR fragment was generated containing the Natr sequence using pAG25 as a template along with primers that also possessed sequences homologous to the 40-bp 5' and 3' of the VPH1 open reading frame. The vph1{Delta}::Natr PCR fragment was transformed into KEBY4 to generate LGY136.

A PCR fragment containing the PKR1 open reading frame and 300-bp flanking DNA was generated from genomic DNA prepared from wild-type yeast strain SF838-1D{alpha}. The fragment was subcloned into pCR4Blunt vector (Invitrogen) generating pLG174, which was then digested with EcoRI to generate a PKR1 fragment that was subcloned into pRS416 also digested with EcoRI. A BglII site was introduced in the PKR1 open reading frame immediately prior to the stop codon by PCR. A BglII fragment from pSMY92 containing a triple HA epitope tag was subcloned into PKR1 to generate pLG221. The construct PKR1::FLAG was generated by PCR amplification from genomic DNA using primers that added a diglycine hinge and a single FLAG epitope at the C terminus prior to the stop codon. The purified PCR product was digested with BamHI and SacI and subcloned into pRS315 generating pRS315PKR1-FLAG.

Immunoblot Analyses of Whole Cell Extracts and Purified Vacuoles—Whole cell protein extracts from various yeast strains were prepared as previously described (17). Briefly, yeast cells were resuspended in Thorner buffer minus beta-mercaptoethanol (8 M urea + 10% SDS + 40 mM Tris, pH 6.8) and solubilized by mixing aggressively in the presence of glass beads (0.5 mm diameter). Cell debris and glass beads were collected by centrifugation at 16,000 x g for 10 min and the supernatant was transferred to a fresh tube. Protein concentrations were determined using the method of Markwell (18). Vacuolar isolations were performed as previously described (19). Briefly, cells were converted to spheroplasts, resuspended in 15% Ficoll + 0.2 M sorbitol, lysed by the addition of 100 µg/ml DEAE-dextran, transferred to a centrifuge tube, and overlaid with 8, 4, and 0% Ficoll in sorbitol, centrifuged for 90 min at 110,000 x g, and vacuoles were collected from the 0/4% interface.

SDS-PAGE analysis was performed on the various protein preparations. Proteins were transferred to 0.2-µm nitrocellulose or polyvinylidene difluoride membrane and probed with antibodies recognizing Vph1p (Molecular Probes 10D7A), Vma1p (Molecular Probes 8B1), Vma2p (Molecular Probes 13D11), Dpm1p (Molecular Probes 5C5), carboxypeptidase Y (Molecular Probes 10A5), or Ccc1p. Bands were visualized by incubation with anti-mouse or anti-rabbit horseradish peroxidase (Jackson ImmunoResearch Laboratories, Inc.) conjugated secondary antibodies and chemiluminescence reagents.

Pulse-Chase Immunoprecipitation of Vph1p—Denaturing immunoprecipitations were performed as previously described (10). Prior to metabolic labeling with [35S]methionine, yeast cells were cultured in synthetic defined medium containing all amino acids except methionine. Briefly, cells (0.5 A600) for each time point were labeled for 10 min with 100 µCi of 35S-Express label (PerkinElmer Life Sciences), unlabeled cysteine/methionine mixture was added and samples were removed at the time points indicated. Cell pellets were denatured by SDS solubilization of spheroplasted cells, diluted in IP buffer (10 mM Tris, pH 8.0, 0.1% SDS, 0.1% Triton X-100 final concentration), precleared, and proteins immunoprecipited using rabbit anti-Vph1p serum (10) and fixed Staphylococcus aureus cells (IgG Sorb, The Enzyme Center). Immunocomplexes were denatured and solubilized in SDS-PAGE-Thorner buffer (8 M urea + 10% SDS + 40 mM Tris, pH 6.8, + 5% beta-mercapthoethanol + 5% glycerol + 0.001% bromphenol blue). Samples were separated by SDS-PAGE, gels were fixed and then dried onto chromatography paper, and exposed to a PhosphorScreen. Data were collected using a STORM PhosphorImager (GE Healthcare), and analyzed using Quality One software (Bio-Rad).

Microscopy—The acidification of vacuoles in various yeast strains was visualized using the lysosomotropic fluorescent dye quinacrine or LysoSensor Green DND-189 (Molecular Probes). Quinacrine staining of live yeast cells was conducted as previously described (20, 21). Quinacrine was used at a final concentration of 200 µM to stain acidified vacuoles, whereas concanavalin A TRITC (Molecular Probes) was added at a final concentration of 50 µg/ml to allow for fluorescent visualization of the cell surface. Samples for indirect immunofluorescence were prepared as previously described and probed with anti-HA antibody (Covance Research Products) (22). These images were acquired on a Zeiss Axioplan 2 fluorescence microscope using x100 objective and manipulated using AxioVision software (Zeiss).

Staining with LysoSensor Green DND-189 was performed as described (23). Cells where incubated in YEPD + 100 mM HEPES, pH 7.6, + 4 µM LysoSensor Green for 5 min at 30 °C, washed once with buffer minus dye, resuspended in YEPD, and visualized. Immunofluorescence for Fet3p or colocalization of Pkr1p-FLAG and Dpm1p was performed as described previously (19) using either rabbit anti-Fet3p (24) with Alexa 488-conjugated goat anti-rabbit IgG for Fet3p immunofluorescence or rabbit anti-FLAG antibody (1:500) (Sigma) and mouse anti-Dpm1p (1:200) (Molecular Probes) followed by Alexa 594-conjugated goat anti-rabbit IgG and Alexa 488-conjugated goat anti-mouse IgG for colocalization immunofluorescence. Cells were imaged using an Olympus epifluorescence microscope with MagnaFire software.

Localization of Proteins by Subcellular Fractionation—Yeast cells were spheroplasted with zymolyase and lysed in phosphate-buffered saline + 200 mM sorbitol as previously described (17). Unbroken cells were removed by brief centrifugation at 500 x g and supernatant (S5) was re-centrifuged for 15 min at 13,000 rpm (16,000 x g) generating a pellet (P13) and supernatant fraction (S13). Samples were solubilized in SDS-PAGE-Thorner buffer, separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies recognizing Dpm1, HA, and 3-phosphoglycerate kinase (Molecular Probes 22C5). Samples were visualized as described above.

Extraction of Peripheral Membrane Proteins with Carbonate—P13 membranes were prepared and treated with TE (10 mM Tris, pH 7.4, 1 mM EDTA), 100 mM sodium carbonate, or 1% Triton X-100 as previously described (9). Samples were analyzed using antibodies against HA, Vph1p, or Vma2p.

Iron Transport Activity—Measurement of iron transport activity was performed as previously described (13). Briefly, cells where grown in YEPD + 90 µM BPS + 7.5 µM FeSO4, cells were collected by centrifugation, and incubated in assay buffer either with or without 2.5 µM CuSO4 + 1.0 mM ascorbate for 30 min on ice. Cells were collected, washed, resuspended to 4-6 x 107 cells, then incubated with 0.50 µM 59Fe for 10 min at 30 °C, placed at 0 °C and washed, onto filter paper, and iron uptake was measured by counting using a Packard 5000 series {gamma}-counter.


Figure 1
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  		FIGURE 1.
Deletion of PKR1 results in a low iron growth deficit. Cells with a deletion in PKR1 homozygous diploid BY4743 (S288c derivative) (A) or haploid SDK13-6D (W303) (B) were transformed with a low copy control plasmid (pRS315) or a plasmid containing PKR1 (pRS315PKR1-FLAG). Serial dilutions of wild-type (BY4743 or SDK13-6A) and pkr1{Delta} cells were spotted and grown on either YEPD or YEPD medium made limiting for iron by the addition of the iron chelator BPS (90 µM) plus 2.5 µM FeSO4 or YEPD + BPS (90 µM) + 100 µM FeSO4.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Deletion of PKR1 Leads to a Low Iron Growth Phenotype Due to Defective Copper Loading of apoFet3p—A homozygous diploid strain BY4743 (S288c derivative) with a deletion in PKR1 showed a growth defect on iron-limited medium (BPS + 2.5 µM Fe) that could be overcome with additional iron (BPS + 100 µM Fe; Fig. 1A). Transformation of pkr1{Delta} cells with either a low or high copy plasmid containing PKR1 under the control of its endogenous promoter complemented the low iron growth deficit (data not shown). Addition of a carboxyl-terminal FLAG epitope to Pkr1p did not affect the ability of the plasmid to complement the phenotype of the deletion strain (Fig. 1A). The low iron growth defect of pkr1{Delta} was not specific to the S288c homozygous diploid strain. A haploid W303 derivative strain with a deletion in PKR1 (SDK13-6D) also showed a low iron growth defect (Fig. 1B). The low iron growth deficit could be complemented by transformation of SDK13-6D yeast cells with a low copy PKR1 containing plasmid.

An inability to grow on low iron medium may reflect a defect in iron transport activity or an inability to utilize iron once transported. Growth of cells in low iron medium results in an induction of the high affinity iron transport system (25). Wild-type yeast cells grown in iron-limited medium showed high levels of 59Fe transport activity (Fig. 2A, open bars). Measurement of 59Fe transport in pkr1{Delta} cells grown in iron-limited medium showed much lower rates of 59Fe transport than wild-type cells. Cells with deletions in genes that encode a structural subunit of the V-ATPase (vma2{Delta}) or a protein required for the assembly of the V-ATPase (vma21{Delta}) possess even lower iron transport activities compared with pkr1{Delta} cells.


Figure 2
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  		FIGURE 2.
Deletion of PKR1 affects the high affinity iron transport system. A, BY4743 wild-type (WT), pkr1{Delta}, vma21{Delta},or vma2{Delta} cells were grown in YEPD made iron limited by the addition of 90 µM BPS plus 7.5 µM FeSO4 overnight at 30 °C. Cells were washed and preloaded with (gray bars) or without (open bars) Cu+ for 30 min at 0 °C. Cells were washed and then assayed for 59Fe uptake in the presence of ascorbate. B, BY4743 wild-type, pkr1{Delta}, or fet3{Delta} cells grown in iron-limited medium, YEPD + 90 µM BPS + 5 µM FeSO4, or iron-replete medium (YEPD) were processed for immunofluorescence as described under "Experimental Procedures." Cells were stained for Fet3p using a rabbit anti-Fet3p antibody.

 
In the absence of copper loading, an apoFet3p can still localize to the cell surface (26). The presence of apoFet3p at the cell surface can be determined by measuring iron transport activity in cells that had been incubated with reduced copper, low pH, and chloride at 0 °C (15). Wild-type or pkr1{Delta} cells were copper-loaded at 0 °C and then assayed for 59Fe transport activity. Wild-type cells treated this way did not show increased transport activity, showing that cell surface Fet3p is fully copper loaded (Fig. 2A, gray bars). In contrast, copper loading of pkr1{Delta} cells resulted in increased 59Fe transport activity. Copper loading of apoFet3p in vma2{Delta} and vma21{Delta} cells also increased the 59Fe transport activity of these mutant cells. The decrease in induction of the iron transport system is a reflection of the low levels of cell surface apoFet3 in these deletion strains relative to pkr1{Delta} cells (see below).


Figure 3
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  		FIGURE 3.
Compromised growth of pkr1{Delta} yeast cells compared with wild-type cells in the presence of elevated levels of calcium. A, exponentially growing cultures of wild-type (WT; SF838-1D{alpha}), pkr1{Delta} (LGY117), and the assembly factor mutant vma21{Delta} (TASY006) yeast were serially diluted and spotted onto YEPD buffered to pH 5.0 with 50 mM succinate/phosphate or YEPD + 100 mM CaCl2. B, exponentially growing cultures of wild-type (SF838-1D{alpha}), pkr1{Delta} (LGY117), pkr1{Delta} stv1{Delta} (LGY118), pkr1{Delta} vph1{Delta} (LGY119), vph1{Delta} (LGY120), stv1{Delta} (KEBY4), and vph1{Delta} stv1{Delta} (LGY136) were serially diluted and spotted onto YEPD buffered to pH 5.0 with 50 mM succinate/phosphate yeast or YEPD + 100 mM CaCl2.

 
To confirm that pkr1{Delta} cells express Fet3p on their cell surface, cells grown in iron-limited (BPS + 5 µM iron) and iron-replete media (YEPD) were fixed and stained with an antibody to Fet3p. Both wild-type and pkr1{Delta} cells grown in iron-limited medium showed high levels of Fet3p on the cell surface (Fig. 2B, top panels). In contrast, when grown in iron-replete medium only pkr1{Delta} cells showed cell surface Fet3p (Fig. 2B, middle panels). These results demonstrate that the defect in copper loading of Fet3p in pkr1{Delta} cells leads to the presence of enzymatically inactive apoFet3p on the cell surface.

Cells Lacking Pkr1p Demonstrate Compromised Growth Suggesting Reduced V-ATPase Function—Yeast cells lacking a functional V-ATPase are unable to grow on media containing 100 mM CaCl2, yet are able to grow on media buffered to an acidic pH 5.0. The growth phenotype of cells lacking Pkr1p was compared with wild-type yeast cells and to cells lacking a required V-ATPase assembly factor Vma21p (8, 10). As shown in Fig. 3A, wild-type cells containing a fully functional V-ATPase complex are able to grow on buffered acidic media (YEPD, pH 5.0) as well as in the presence of 100 mM CaCl2. Yeast cells lacking Vma21p, thus lacking any functional V-ATPase, show some compromised growth on YEPD, pH 5.0, compared with wild-type cells and are unable to grow in the presence of elevated CaCl2. Interestingly, yeast cells lacking Pkr1p show wild-type growth on YEPD media buffered to pH 5.0, yet pkr1{Delta} cells grow poorly in the presence of 100 mM CaCl2. One explanation is that cells lacking Pkr1p might have a reduced level of functional V-ATPase compared with wild-type cells and thus exhibit compromised growth in the presence of elevated CaCl2. Alternatively, the V-ATPase complex could be present at wild-type levels in cells lacking Pkr1p but the function of the complex could be impaired. Because cells lacking Pkr1p are able to grow in the presence of elevated CaCl2, unlike cells lacking any Vma protein such as Vma21p, pkr1{Delta} cells exhibit a novel partial Vma- growth phenotype.

V-ATPase complexes assembled with the Vph1p isoform are localized to the vacuolar membrane, whereas Stv1p-containing V-ATPase complexes are less abundant and localize to Golgi and endosomal membranes (6, 7, 27). To examine if the loss of Pkr1p affects both the Vph1p- and Stv1p-containing V-ATPase complexes, the growth of single and double deletion yeast strains were compared in the presence of elevated CaCl2. Cells lacking Vph1p exhibit only a slight growth defect in the presence of 100 mM CaCl2 (Fig. 3B, fifth row). Consistent with Stv1p being assembled into a minor non-vacuolar population of V-ATPase complexes, cells lacking the Stv1p isoform grow the same as wild-type in the presence of elevated calcium (Fig. 3B, sixth row). Cells lacking both Vph1p and Stv1p have no functional V-ATPase and thus are unable to grow in the presence of CaCl2 (Fig. 3B, seventh row). By comparison, cells lacking Pkr1p grew slower in the presence of elevated calcium than cells lacking Vph1p (Fig. 3B, second row). Double deletion mutants lacking both Pkr1p and Stv1p exhibit only slightly more compromised growth than pkr1{Delta} single mutant cells (Fig. 3B, third row). Only the loss of both Pkr1p and Vph1p compromised yeast cell growth on elevated calcium to a level approaching that of a vma mutant (Fig. 3B, fourth row). Because pkr1{Delta} vph1{Delta} cells are more growth defective than vph1{Delta} cells, it is likely that the loss of Pkr1p also affects the levels of the Stv1p-containing V-ATPase complexes still present in these cells. Similarly, pkr1{Delta} stv1{Delta} cells grew less robustly than stv1{Delta} cells, suggesting that the loss of Pkr1p affects the levels of Vph1p-containing V-ATPase complexes present in these cells. These growth data suggest that the loss of Pkr1p affects the assembly of both Vph1p- and Stv1p-containing V-ATPase complexes.

Western Analysis Confirms Reduced Levels of the Vacuole-associated V-ATPase in Cells Lacking Pkr1p—To determine whether cells lacking Pkr1p have reduced levels of V-ATPase complexes we analyzed whole cell extracts from various strain backgrounds by Western blot analysis for the V0 subunit protein Vph1p and the V1 subunit Vma2p. The levels of Vma2p are similar in wild-type and pkr1{Delta} cells (Fig. 4A). The levels of Vph1p, however, are lower in pkr1{Delta} cells but similar to extracts prepared from cells lacking either a V0 subunit (vma3{Delta}) or a V-ATPase assembly factor (vma21{Delta}). To control for the amount of protein loaded the extracts were also probed for Dpm1p, a protein localized to the endoplasmic reticulum. Interestingly, the five other V0 subunits (Vma3p, Vma6p, Vma9p, Vma11p, and Vma16p) were present at normal levels in pkr1{Delta} cells (data not shown).

Decreased levels of both V0 (Vph1p) and V1 subunits (Vma1p and Vma2p) were observed in vacuoles isolated from pkr1{Delta} cells (Fig. 4B). Whereas there was a decrease in V-ATPase subunits, other vacuolar components, such as Ccc1p, the vacuolar Fe2+/Mn2+ transporter, and carboxypeptidase Y, a soluble vacuolar hydrolase, were present at levels comparable with that seen in wild-type vacuoles. These data suggest that the compromised growth phenotype of cells lacking Pkr1p is due to a decrease in the level of V-ATPase complexes present in vacuoles.


Figure 4
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  		FIGURE 4.
Decreased levels of V-ATPase subunits in vacuoles isolated from pkr1{Delta} cells. A, immunoblot analysis of whole cell extracts isolated from wild-type (WT) (SF838-1Da), pkr1{Delta} (LGY117), vma1{Delta} (LGY146), vma3{Delta} (LGY113), vma21{Delta} (TASY006), and vph1{Delta} (LGY120) yeast cells. 12 µg of protein were loaded into each lane of an 8-20% SDS-PAGE, resolved by electrophoresis, transferred to nitrocellulose, and probed with antibodies recognizing Vph1p (10D7), Vma2p (13D11), and Dpm1p (5C5). B, purified vacuoles from wild-type (SKD13-5C) and pkr1{Delta} (SDK13-5D) cells were examined by Western blot analysis using antibodies directed against Vph1p, Vma1p (8B1), Vma2p, Ccc1p, and carboxypeptidase Y (CPY).

 
Cells Lacking Pkr1p Exhibit a Low Level of V-ATPase-dependent Vacuole Acidification—The above data shows that pkr1{Delta} cells have reduced levels of assembled V-ATPase. Deletion of PKR1 results in a reduced ability to acidify vacuoles, as shown by use of the pH-sensitive membrane dye LysoSensor Green DND-189 (Fig. 5A). In wild-type cells stained with Lyso-Sensor Green DND-189 the vacuolar membrane was clearly delineated. In pkr1{Delta} cells the amount of staining is reduced and no clear vacuolar outlines could be detected.

Evidence of low levels of vacuolar acidification could be seen in cells containing a deletion of PKR1 (LGY117) using quinacrine, a fluorescent dye that accumulates within acidified cellular compartments (20). When visualized under the microscope following quinacrine uptake, wild-type cells have highly fluorescent vacuoles, shown as bright green disks within the yeast cell, whereas Vma- (vma1{Delta}) cells do not contain fluorescent/acidified vacuoles (Fig. 5B). The outline of the yeast cell, depicted in red, was accomplished by the addition of concanavalin A-TRITC, which binds to the yeast cell wall. The pkr1{Delta} cells exhibit an intermediate fluorescent staining indicating that the vacuoles are acidified, but to a lesser extent. To ascertain if vacuole acidification observed in pkr1{Delta} cells was due to the V-ATPase, quinacrine uptake was examined in vma1{Delta} pkr1{Delta} double mutant cells. The intermediate quinacrine staining present in the pkr1{Delta} cells was completely absent in the vma1{Delta} pkr1{Delta} cells due to the complete loss of an active V-ATPase complex. These results indicate that cells lacking Pkr1p still have a small fraction of active V-ATPase complexes on the vacuole resulting in a weakly acidified vacuole.


Figure 5
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  		FIGURE 5.
Deletion of PKR1 affects vacuolar pH. A, BY4743 wild-type or pkr1{Delta} cells transformed with pRS315 or pRS315PKR1-FLAG were incubated with LysoSensor Green DND-189 and examined by fluorescence microscopy. B, wild-type (WT) (SF838-1D{alpha}), pkr1{Delta} (LGY117), vma1{Delta} (LGY146), and pkr1{Delta} vma1{Delta} (LGY147) cells were stained with quinacrine and concanavalin A-TRITC as described under "Experimental Procedures." The panels on the left show cell morphology as viewed by differential interference contrast (DIC). The panels on the right show quinacrine depicted in green and concanavalin A-TRITC in red. C, immunocomplexes from C12E9-solubilized membranes were isolated using anti-Vma2p serum and protein A-Sepharose under native conditions. Samples were denatured, separated by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies recognizing Vph1p and Vma1p. IP, immunoprecipitation.

 


Figure 6
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  		FIGURE 6.
Pkr1p is a predicted transmembrane protein with homologs in other fungi. A, sequence comparison of S. cerevisiae (Sc) Pkr1p with homologs from C. albicans (Ca) and S. pombe (Sp). Multisequence alignment was generated using the program Clustal 1.8 (31). * indicates exact match, colon indicates strong similarity, and dot indicates weak similarity. B, model of Pkr1p, boxed amino acids represent two putative transmembrane domains as predicted by the SOUSI program (32). The position of the COOH-terminal HA epitope tag is also shown. Epitope tagging of Pkr1p with the FLAG tag was in the identical COOH-terminal location as the HA tag.

 
To further test if the vacuolar acidification observed in pkr1{Delta} cells is due to a functional V-ATPase complex, native immunoprecipitations were performed to detect the presence of fully assembled V-ATPase complexes. Membrane fractions were solubilized under non-denaturing conditions using a detergent (C12E9) that maintains interactions between V-ATPase subunits (8), then incubated with an antibody against a V1 subunit Vma2p. The immunocomplexes formed were isolated by centrifugation following the addition of Protein A-Sepharose and were examined for the presence of both V1 (Vma1p) and V0 (Vph1p) subunits. Fig. 5C reveals that both Vma1p and Vph1p were co-immunoprecipitated with Vma2p from wild-type and pkr1{Delta} cells. Consistent with the lack of assembled V-ATPase complexes in vma21{Delta} cells (8, 10), neither Vph1p nor Vma1p co-immunoprecipitated with Vma2p from vma21{Delta} membrane fractions. Therefore, the association of V1 subunits with the membrane fraction in pkr1{Delta} cells is not a nonspecific association, but rather is due to the presence of low levels of fully assembled V-ATPase complexes.

Pkr1p Is an Integral Membrane Protein Localized to the ER—Pkr1p homologues have been identified in many species of fungi. An alignment of Pkr1p from S. cerevisiae with homologues from Candida albicans and Schizosaccharomyces pombe is shown in Fig. 6A. Interestingly, comparing the alignment of the three proteins reveals that the majority of the amino acid identity occurs in the NH2-terminal portion of the protein sequences. All three proteins also possess a COOH-terminal tail rich in acidic residues, which does not align by the sequence comparison. The S. cerevisiae gene PKR1 encodes a 14-kDa protein that is predicted to form two transmembrane domains, thus orienting the NH2 and COOH termini on the same side of the membrane (Fig. 6B). We introduced a 3x HA epitope tag or a FLAG tag on the extreme COOH terminus immediately before the stop codon to allow detection of the protein using antibodies directed against the epitope. The introduction of either epitope tag did not affect the function of the protein, because cells expressing only this tagged version of the protein exhibit wild-type growth in the presence of elevated calcium, wild-type levels of the V-ATPase subunit Vph1p (data not shown), high levels of LysoSensor fluoresecence similar to wild-type cells (Fig. 5A), and normal growth on low iron medium (Fig. 1).

The subcellular localization of Pkr1p was determined by indirect immunofluorescence utilizing the HA epitope-tagged protein that is fully functional. As shown in Fig. 7A, the localization pattern of Pkr1p revealed bright perinuclear staining (the nucleus is defined by 4',6-diamidino-2-phenylindole staining), consistent with Pkr1p being localized to the ER membrane. Second, the immunofluorescent pattern of the Pkr1p-FLAG protein, expressed by a CEN plasmid was shown to co-localize with the endoplasmic reticulum enzyme dolichol-phosphate mannose synthase (Dpm1p) (Fig. 7B), although the Dpm1p antibody shows a small amount of additional punctate (and possibly non-ER) staining.


Figure 7
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  		FIGURE 7.
Pkr1p is localized to the membrane of the ER. A, indirect immunofluorescence was performed on wild-type (SF838-1D{alpha}) or pkr1{Delta} (LGY117) cells transformed with plasmid carrying PKR1::HA (pLG221). Pkr1p-HA was visualized using a monoclonal anti-HA antibody, followed by goat anti-mouse biotin-conjugated antibody treatment, and a streptavidin-fluorescein isothiocyanate treatment. DNA was visualized by labeling with 4',6-diamidino-2-phenylindole (DAPI). To observe vacuoles, differential interference contrast (DIC) images were collected. Merged images show the simultaneous localization of the nucleus (blue) and Pkr1p-HA (green). B, indirect immunofluorescence was performed on pkr1{Delta} (SDK13-6D) transformed with a plasmid containing PKR1::FLAG (pRS315PKR1::FLAG). Pkr1p-FLAG was visualized using a rabbit polyclonal anti-FLAG antibody followed by an Alexa 488-conjugated goat anti-rabbit IgG. The endoplasmic reticulum protein Dpm1p was visualized using a monoclonal antibody followed by an Alexa 594-conjugated goat anti-mouse IgG.

 
To determine whether Pkr1p fractionates as a membrane protein, whole cell lysates (S5) were prepared and differential centrifugation was used to separate membranes (P13) from soluble cellular proteins (S13). Fractions were analyzed by Western blot analysis by probing for Pkr1p (HA), the ER membrane protein Dpm1p, and the soluble cytosolic protein 3-phosphoglycerate kinase. As expected, the membrane protein Dpm1p fractionated exclusively to the P13 membrane fraction and the soluble protein 3-phosphoglycerate kinase was present in the S13 fraction (Fig. 8A). Pkr1p-HA fractionated entirely to the P13 membrane fraction, consistent with it being a membrane protein as predicted.

To determine the nature of the association of Pkr1p with membranes, P13 membranes were treated with alkaline carbonate. Alkaline carbonate is able to extract peripherally associated membrane proteins, but not those integrally associated with the membrane (28). Treated membranes were separated from soluble proteins by centrifugation and the protein content of the samples subjected to Western blot analysis probing for Pkr1p (HA), and the vacuolar membrane V-ATPase subunits Vph1p and Vma2p. In membrane samples treated with buffer only (TE) all three proteins remained associated with the membrane fraction (P13) (Fig. 8B). Treatment with alkaline carbonate extracted the well characterized peripheral membrane protein Vma2p from the membrane, but the integral membrane protein Vph1p remained with the membrane fraction. Pkr1p also remained with the membrane fraction upon alkaline carbonate treatment, consistent with it being an integral membrane protein. Only after detergent solubilization of the membranes did the integral membrane proteins Vph1p and Pkr1p partition to the supernatant fraction (S13). These data show that Pkr1p is an integral membrane protein localized to the ER and not a component of the V-ATPase.


Figure 8
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  		FIGURE 8.
Pkr1p is an integral membrane protein. A, subcellular fractionation of Pkr1p-HA expressed in pkr1{Delta} (LGY117) cells. Exponentially growing cells were converted to spheroplasts, osmotically lysed, and fractionated by differential centrifugation generating whole cell lysate (S5), membrane pellet (P13), and supernatant (S13) fractions. Proteins from equal portions of each fraction were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies recognizing the HA epitope (Pkr1p-HA), the cytosolic protein 3-phosphoglycerate kinase (PGK), and the ER membrane protein Dpm1p. B, membrane fractions (P13) were prepared from pkr1{Delta} (LGY117) cells expressing Pkr1p-HA. Washed membrane fractions were treated with TE, 100 mM sodium carbonate, pH 11.5, or TE + 1% Triton X-100 for 30 min on ice. Samples were fractionated by centrifugation to generate pellet (P13) or supernatant (S13) fractions. Proteins from equal portions of each fraction were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with antibodies recognizing the HA epitope (Pkr1p-HA), the integral membrane protein Vph1p, or the peripheral membrane protein Vma2p.

 
A Population of Newly Synthesized Vph1p Is Stable in Cells Lacking Pkr1p—The above data shows that Pkr1p may be involved in the assembly or trafficking of the V-ATPase. The low levels of Vph1p observed in pkr1{Delta} cell extracts may be due to increased turnover of this V-ATPase subunit, reflecting its failure to assemble into the V0 subcomplex in the ER resulting in overall lower levels of Vph1p present in the cell (29). Kinetic analysis of newly synthesized Vph1p in cells lacking Pkr1p revealed a unique turnover profile as compared with wild-type cells or those lacking a V-ATPase assembly factor, Vma21p. In wild-type cells newly synthesized radiolabeled Vph1p is very stable over the course of two cell divisions (180 min) and is consistent with an estimated half-life in excess of 400 min (Fig. 9A, top panel) (11). In the absence of the assembly factor Vma21p, newly synthesized Vph1p is unstable with a half-life of ~30 min and almost completely degraded after 120 min (Fig. 9A, bottom panel). In cells lacking Pkr1p, newly synthesized Vph1p exhibited a high turnover rate for the first 90 min and was then stable throughout the remainder of the experiment (Fig. 9A, middle panel). A graphic representation of the turnover rates from a similar second independent data set is shown in Fig. 9B. The Vph1p remaining in pkr1{Delta} cells after a 3-h chase may represent the population of the newly synthesized Vph1p that is assembled into V0 subcomplexes that is then localized to vacuoles. These V0 subcomplexes formed in the absence of Pkr1p would be able to exit the ER and assemble with V1 subunits to form a functional vacuolar membrane-localized V-ATPase. These data suggest that Pkr1p is an ER protein required for the efficient assembly of the V-ATPase.


Figure 9
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  		FIGURE 9.
Increased turnover of newly synthesized Vph1p in pkr1{Delta} cells. A, wild-type (WT) (SF838-1D{alpha}), pkr1{Delta} (LGY117), and vma21{Delta} (TASY006) cells were labeled with [35S]methionine for 10 min and chased in the presence of 50 mM unlabeled methionine. Samples were removed at 0, 30, 60, 120, and 180 min following chase. Denaturing cell extracts were prepared and Vph1p was immunoprecipitated using polyclonal sera. Immunoprecipitated proteins were resuspended in SDS-PAGE-Thorner buffer, resolved by SDS-PAGE, and gels were dried and exposed to a phosphorscreen. Images were collected using a phosphorimager system. B, graphical representation of a second independent pulse-chase experiment data set different from that shown above.

 
Overexpression of Vma21p Suppresses the Growth and Acidification Defects of pkr1{Delta} Cells—We have shown that Pkr1p is a small integral membrane protein, localized to the ER, that plays a role in the assembly of the vacuolar-localized V-ATPase. We have previously reported on three ER-localized V-ATPase assembly factors Vma12p, Vma21p, and Vma22p (1, 9-11, 29). Vma21p has been shown to interact directly with the V-ATPase V0 subcomplex during assembly in the ER (8). As with pkr1{Delta} cells, cells lacking any one of the three V-ATPase assembly factors result in the rapid degradation of Vph1p due to its inability to assemble into and form a stable V0 subcomplex (10, 11). To assess the relationship between Pkr1p and the other V-ATPase assembly factors we tested for possible interactions between Pkr1p and the other assembly factors. Transformation of pkr1{Delta} cells with a plasmid overexpressing Vma21p, as determined by immunoblot analysis (data not shown), was able to suppress the growth defects on both iron-limiting media (Fig. 10A) and in the presence of elevated calcium (Fig. 10B). The growth phenotypes of pkr1{Delta} cells were not affected by the elevated expression of either Vma12p or Vma22p (data not shown). Overexpression of Vma21p did not suppress the growth defect of cells lacking VPH1 and STV1 (Vma-).

The growth of pkr1{Delta} cells overexpressing Vma21p was near that of wild-type cells, suggesting that more V-ATPase complexes were correctly assembled in these cells. To determine whether the improved growth of pkr1{Delta} cells overexpressing Vma21p reflected an increase in vacuole acidification, we tested for the accumulation of quinacrine by these cells. As shown in Fig. 10C, pkr1{Delta} cells overexpressing Vma21p have highly acidified vacuoles compared with pkr1{Delta} cells, consistent with increased levels of functional and correctly localized V-ATPase. These data suggest that Pkr1p may function at or near the Vma21p-related step and may aid or increase the efficiency of Vma21p in its role as a V-ATPase assembly factor.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The de novo formation of the multisubunit V-ATPase requires the coordinated assembly of 14 different subunits, some of which are present in the complex in multiple copies such as Vma1p, Vma2p, and Vma3p. Previous research has identified five proteins that are not components of the final V-ATPase complex but are required for its proper assembly or function (4, 5, 10-12). In this work we have characterized an additional protein, Pkr1p, which plays a role in the assembly of the V-ATPase in yeast.

We have shown that Pkr1p is an integral membrane protein that localizes to the ER. Cells lacking Pkr1p have drastically reduced levels of V-ATPase complexes, as reflected by compromised growth in the presence of elevated calcium or on low iron medium, low levels of the V-ATPase subunit Vph1p, and weak vacuole acidification compared with wild-type cells. In addition, the V-ATPase subunit Vph1p was rapidly degraded in the absence of Pkr1p, indicative of its inability to assemble efficiently into the V0 complex in the ER.

Initially, Pkr1p appeared to function in a manner similar to the other well characterized ER localized V-ATPase assembly factors (Vma12p, Vma21p, and Vma22p) because in its absence the V0 subunit Vph1p undergoes increased turnover (9-11). Yet, unlike the other V-ATPase assembly factors Pkr1p is not absolutely required for V-ATPase assembly. Whereas V-ATPase assembly is completely blocked in cells lacking Vma12p, Vma21p, or Vma22p, cells lacking Pkr1p do possess some low level of fully assembled and functional V-ATPase on the vacuolar membrane.


Figure 10
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  		FIGURE 10.
Elevated expression of VMA21 suppresses growth defects of pkr1{Delta} cells. A, wild-type (WT; BY4743), pkr1{Delta} and pkr1{Delta} transformed with a plasmid containing a HA-tagged VMA21 gene (pKH28) were spotted on either YEPD or YEPD made iron-limited through the addition of the iron chelator BPS (90 µM) plus 2.5 µM FeSO4. B, exponentially growing cultures of wild-type (WT; SF838-1D{alpha}), pkr1{Delta} (LGY117), pkr1{Delta} carrying plasmid containing HA-tagged Vma21p (pKH28), and a Vma- strain (LGY136) also carrying plasmid containing HA-tagged Vma21p (pKH28) yeast were serially diluted and spotted onto YEPD buffered to pH 5.0 with 50 mM succinate/phosphate and YEPD + 100 mM CaCl2. C, wild-type (SF838-1D{alpha}), pkr1{Delta} (LGY117), and pkr1{Delta} cells carrying plasmid containing HA-tagged Vma21p (pKH28) were stained with quinacrine and concanavalin A-TRITC as described under "Experimental Procedures." The panels on the left show cell morphology as viewed by differential interference contrast (DIC). The panels on the right show quinacrine depicted in green and concanavalin A-TRITC in red.

 
The ER localization of Pkr1p strongly suggests that it has a role very early in the assembly of the V-ATPase and in particular the assembly of the V0 subcomplex of the V-ATPase. That another vacuolar membrane protein Ccc1p localizes to the vacuole in the absence of Pkr1p suggests that the loss of Pkr1p does not globally affect membrane trafficking to the vacuole, but is specific to trafficking of the V-ATPase complex. The kinetic analysis of Vph1p degradation in pkr1{Delta} cells suggests a previously unobserved biphasic degradation profile for newly synthesized Vph1p. In cells lacking Pkr1p, a portion of newly synthesized Vph1p is subject to rapid degradation, presumably by the ER quality control pathways, due to the inability of Vph1p to assemble into V-ATPase V0 subcomplexes (30). The initial turnover rate of Vph1p in pkr1{Delta} cells is very similar to the rate observed in cells lacking Vma12p, Vma21p, or Vma22p (9-11). Interestingly, there is a small pool (10%) of Vph1p remaining in pkr1{Delta} cells after the initial rapid degradation. This Vph1p pool is stably maintained in the cell by its successful incorporation into V0 subcomplexes and eventually V-ATPase complexes. V-ATPase complexes formed in the absence of Pkr1p are targeted correctly to the vacuolar membrane and can function to bring about modest acidification of the vacuole in these mutant cells. We propose that Pkr1p plays a role in promoting the efficiency in which newly synthesized V-ATPase V0 subunits are assembled into the V0 subcomplex in the ER, thus protecting the Vph1p subunit from rapid degradation.

Another clue to the function of Pkr1p in V-ATPase assembly is the observation of genetic interactions between PKR1 and VMA21. We found that overexpression of the V-ATPase assembly factor Vma21p suppresses the V-ATPase assembly defects in cells lacking Pkr1p. One possible explanation for this genetic interaction is that Pkr1p is required for the stability of Vma21p; however, Western analysis revealed that pkr1{Delta} cells have normal levels of Vma21p (data not shown). It is thus likely that the genetic interaction between Pkr1p and Vma21p reflects that these two proteins may function at the same assembly step, or that Vma21p functions most efficiently when Pkr1p is present, because only a low level of V-ATPase complexes are assembled in the absence of Pkr1p even when all the required V-ATPase assembly factor proteins are still present in the cell. Unfortunately, numerous attempts to demonstrate a physical interaction between Vma21p and Pkr1p have proven unsuccessful. In addition, efforts to test for an interaction between Pkr1p and V0 subunits have also been unsuccessful. Future work will focus on the precise role of Pkr1p in V-ATPase assembly, and in particular on identifying the nature of the interaction between Pkr1p and the V-ATPase subunits assembling in the ER, as well as the functional relationship between Pkr1p and the other V-ATPase assembly factors.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM38006 (to T. H. S.) and DK30534 (to J. K.), and support for DNA oligonucleotides and sequencing was provided by Cancer Center Support Grant CA43014. 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 Both authors contributed equally to this work. Back

2 To whom correspondence may be addressed. Tel.: 801-581-7427; Fax: 801-581-6001; E-mail: jerry.kaplan{at}path.utah.edu. 3 To whom correspondence may be addressed. Tel.: 541-346-5884; Fax: 541-346-4854; E-mail: stevens{at}molbio.uoregon.edu.

4 The abbreviations used are: V-ATPase, V-type ATPase; BPS, bathophenanthroline disulfonate; ER, endoplasmic reticulum; YEPD, yeast extract-peptone-dextrose; Pkr, Pichia farnosia killer toxin resistance; HA, hemagglutinin; TRITC, tetramethylrhodamine isothiocyanate. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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