Vam2/Vps41p and Vam6/Vps39p Are Components of a Protein Complex on the Vacuolar Membranes and Involved in the Vacuolar Assembly in the Yeast Saccharomyces cerevisiae*

(Received for publication, August 8, 1996, and in revised form, February 10, 1997)

Norihiro Nakamura Dagger , Aiko Hirata §, Yoshinori Ohsumi Dagger and Yoh Wada Dagger par

From the Dagger  Department of Biology, Graduate School of Arts and Sciences, University of Tokyo, Komaba 3-8-1, Meguro-ku, Tokyo 153, Japan and the § Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The VAM2/VPS41 and VAM6/VPS39 were shown to encode hydrophilic proteins of 113 and 123 kDa, respectively. Deletion of the VAM2 and VAM6 functions resulted in accumulation of numerous vacuole-related structures of 200-400 nm in diameter that were much smaller than the normal vacuoles. Loss of functions of Vam2p and Vam6p resulted in inefficient processings of a set of vacuolar proteins, including proteinase A, proteinase B, and carboxypeptidase Y (CPY), and in severely defective maturation of another vacuolar protein, alkaline phosphatase. A part of newly synthesized CPY was missorted to the cell surface in the mutants. Epitope-tagged versions of Vam2p and Vam6p retained their functions, and they were found mostly in sedimentable fractions. The epitope-tagged Vam2p and Vam6p remained in the sedimentable fractions in the presence of Triton X-100, but they were extracted by urea or NaCl. Vam2p and Vam6p were cross-linked by the treatment of a chemical cross-linker. These observations indicated that Vam2p and Vam6p physically interact with each other and exist as components of a large protein complex. Vam6p fused with a green fluorescent protein were highly accumulated in a few specific regions of the vacuolar membranes. Large portions of Vam2p and Vam6p were fractionated into a vacuolar enriched fraction, indicating that they were localized mainly in the vacuolar membranes. These results showed that Vam2p and Vam6p execute their function in the vacuolar assembly as the components of a protein complex reside on the vacuolar membranes.


INTRODUCTION

The vacuole is the most prominent organelle with significant morphology. In the yeast Saccharomyces cerevisiae, it occupies a large space of about one-quarter of the total cell volume. The assembly of this large compartment requires proper functions of the VAM gene products (1). The vam mutants in one class (class I vam mutants) contain few, if any, small vacuolar compartments and show severe defects in maturation of soluble vacuolar proteins (1, 2). Mutants in the other class (class II vam mutants) accumulate small vesicular structures that are stained with vacuolar marker dyes like ade fluorochrome and lucifer yellow CH (1). From this characteristic phenotype of the class II vam mutants, we suggested that the class II VAM genes (VAM2, VAM3, VAM4, VAM6, and VAM7) are required for the last step of the vacuolar assembly, i.e. the fusion of small vacuolar precursors into the large vacuolar compartment and/or for the maintenance of the assembled large vacuoles (3).

The VAM2 and VAM6 genes belong to the class II VAM. They have been identified several times by different genetic approaches. We identified the vam2 and vam6 mutations by their phenotypes of vacuolar morphology (1). Genetic analyses showed that the vam2 and vam6 mutations were allelic to the vps41 and vps39 mutations, respectively (4). These vps mutations were identified by missorting of the vacuolar soluble proteins to the cell surface (4, 5). Recently, cvt8 and cvt4 mutations (for cytoplasm-vacuole transport) were shown to be allelic to vps41 and vps39 mutations, respectively (6). In this report, we present characterization of phenotypes of the null alleles and functional analyses on their products Vam2/Vps41p and Vam6/Vps39p.


EXPERIMENTAL PROCEDURES

Strains, Media, and Genetic Procedures

Yeast strains used in this study were derived from X2180-1A and -1B, YPH499, YPH500, YPH501 (7), and their hybrids (Table I). Yeast cell culture, standard genetic manipulations, and bacterial methods were carried out as described previously (8).

Table I.

Strains used in this study


Strain Genotype Source or Ref.

YPH499 MATa ade2-101 his-3Delta 200 leu2-Delta 1 lys2-801 trp1-Delta 63 ura3-52 (7)
YPH500 MATalpha ade2-101 his3-Delta 200 leu2-Delta 1 lys2-801 trp1-Delta 63 ura3-52 (7)
YPH501 MATa/alpha ade2-101/ade2-101 his3-Delta 200/his3-Delta 200 leu2-Delta 1/leu2-  Delta 1 lys2-801/lys2-801 trp1-Delta 63/trp1-Delta 63 ura3-52/ura3-52 (7)
X2180-1A MATa SUC2 mal mel gal2 CUP1 Y.G.S.C.a
X2180-1B MATalpha SUC2 mal mel gal2 CUP1 Y.G.S.C.
NNY33 YPH499; Delta vam2::HIS3 Delta vam6::LEU2 This study
NNY60L YPH499; Delta vam6::LEU2 This study
STY1 MATa Delta pep4::LEU2 leu2-Delta 1 trp1-Delta 63 ura3-52 This study
YN6-1D MATalpha vam6-1 trp1-Delta 63 ura3-52 This study
YN6-47D MATa vam6-1 ade1 leu2-Delta 1 ura3-52 This study
YW25-1C YPH499; Delta vam2::HIS3 This study
YWK008-2A MATa vam2-3 ade1 leu2 ura3-52 This study

a Y.G.S.C., Yeast Genetic Stock Center.

Molecular Cloning and Nucleotide Sequence Analysis of VAM2 and VAM6

Yeast genomic libraries constructed on YEp13 (9) or on YCp50 were introduced into mutant strains YWK008-2A (MATa ade1 ura3-52 leu2 vam2-3) and YN6-47D (MATa ade1 ura3-52 leu2-Delta 1 vam6-1) by the lithium acetate method (10, 11). Vam+ colonies were identified by the pigmentation/depigmentation assay of vam ade1 strains and subsequent microscopic analyses (3, 8). Various subregions of the complementing plasmids were introduced into pRS314, pRS315, and pRS316 (7) to map the complementing activities. Minimum essential regions for the complementation of the vam2-3 and vam6-1 mutations were subcloned into pBluescript II KS+ and pBluescript II SK+. Unidirectional deletion series were generated by the treatment with exonuclease III and mung bean nucleases (12). The nucleotide sequences of both strands were determined using Sequenase Version 2 (Amersham Corp.) or Prism AmpliTaq/Dye terminator kit (Perkin-Elmer).

Disruption of the Chromosomal VAM2 and VAM6

A 2.0-kb BamHI-Csp45I fragment of the VAM2 (containing 434 bp encoding the N-terminal region of the open reading frame and the 1.6-kb 5' flanking region) was introduced into the BamHI-ClaI site of an integration type plasmid pRS303 (7). The generated intermediate plasmid was digested with XbaI and ligated to a 1.8-kb XbaI fragment of VAM2 (containing 831 bp of the VAM2 ORF encoding the C-terminal region and the 1-kb 3' untranslated region) to yield plasmid pYVQ213. pYVQ213 was digested with BamHI and transformed into the diploid strain YPH501. Plasmid pVAM6::LEU2-BSSK has a truncated VAM6 in which a 1.8-kb EcoRI-EcoRI region was removed and replaced by a 2.0-kb LEU2 fragment (13). This plasmid was digested with XbaI and introduced into a diploid strain YPH501 or a haploid strain YPH499. Targeting of the constructs to the VAM2 and VAM6 loci were verified by diagnostic PCR amplification and Southern hybridization analyses (data not shown).

Influenza Hemagglutinin and Green Fluorescent Protein Tagging of the VAM2 and VAM6 Gene Products

The triplicate 9-amino acid peptide (YPYDVPDYA) of a part of the influenza virus hemagglutinin protein (HA)1 (14) was introduced at the N terminus of Vam2p by sticky feet mutagenesis as described (15) using a pair of primers, V2HA-S (5'-GAGTATATACCTACTATTAGACATTAATGTACCCATATG-3') and V2HA-A (5'-AATCATTCTGATGATTATCTGTAGTAGCGTAGTCAGGTA-3'; phosphorylated by T4 kinase prior to the PCR). The resultant 3xHA-VAM2 fragment was introduced into pRS316 to obtain pYVQ215.

An NheI site was introduced after the initiation ATG of VAM6 by PCR-based mutagenesis. The DNA fragment encoding 3xHA sequence with NheI sites at both ends, a gift from Dr. Anraku of University of Tokyo, was introduced into the NheI site created in the VAM6. Sequencing analysis showed that the resultant plasmid, pVAM6-N6HA, contained six repeats of HA epitope sequence. The DNA fragment encoding a GFP was amplified by PCR using a pair of primers, 5'-ATGGCTAGCATGGTGAGCAAGGGCGAGGAG-3' and 5'-TAAGCTAGCCTTGTACAGCTCGTCCATGCCG-3', and plasmid pblue-sGFP(S65T)-nos SK (16) as a template. The PCR product was digested by NheI and then introduced into the NheI site created at the N-terminal of the VAM6 ORF.

Anti-Vam6p Antibodies

A 1.8-kb EcoRI fragment encoding amino acids 315-916 of Vam6p was subcloned into pGEX-5X-1 (Pharmacia Biotech Inc.) to yield pGST-Vam6p. The fusion protein was expressed in E. coli strain NM522, purified by SDS-PAGE, and immunized in a rabbit. Anti-Vam6p antibodies were purified from serum on cyanogen bromide-activated Sepharose 4B (Sigma) coupled with a 6xHis-tagged Vam6p (amino acids 315-465) that was also produced in Escherichia coli and purified on an Ni2+-nitrilotri acetic acid resin column (Qiagen Inc.).

Immunoblotting Analysis and Vacuolar Protein Targeting

Yeast total cell lysates were prepared and subjected to immunoblotting analysis as described previously (2). The HA-epitope was decorated by a monoclonal antibody 12CA5 (2 µg/ml, Boehringer Mannheim) and then detected by a horseradish peroxidase-conjugated rabbit anti-mouse IgG (Jackson Immunoresearch Laboratories) and by ECL detection system (Amersham Corp.).

Cell labeling was carried out as descrived (17) with minor modifications. Spheroplasts were labeled for 10 min at 30 °C with Expre35S35S protein labeling mix (DuPont NEN) at final concentration of 0.925 MBq/107 cells. They were chased for 0, 15, 30, or 45 min at 30 °C in the presence of 0.2% yeast extract, 5 mM methionine, and 1 mM cysteine.

Subcellular Fractionation and Solubilization of Vam2p and Vam6p

Spheroplasts (5 × 108 cells) were resuspended in 125 µl of 0.1 M Tris-HCl (pH 7.6) containing 1.2 M sorbitol, 10 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A. After addition of 1.25 ml of lysis buffer (0.1 M Tris-HCl (pH 7.6), 0.2 M sucrose, 10 mM EDTA, 1 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A), the lysate was spun for 2 min at 500 × g. A 1-ml sample from resulting supernatant (S5) was spun at 13,000 × g for 15 min to generate an S13 (supernatant) and a P13 (pellet) fraction. 800 µl of the S13 fraction was centrifuged at 100,000 × g for 60 min in a TLA100.3 rotor (Beckman Instruments) to obtain P100 (pellet) and S100 (supernatant) fractions.

To analyze the solubilities of Vam2p and Vam6p, a 0.25-ml aliquot of the S5 fraction was adjusted to 312.5 µl with one of the following reagents to give the indicated final concentrations: 1% Triton X-100, 2 M urea, or 1 M NaCl. The lysates were incubated on ice for 10 min and then spun at 100,000 × g for 60 min to obtain the supernatant and pellet fractions.

Sucrose Velocity Gradients

Spheroplasts (4.3 × 109 cells) of YW25-1C harboring pYVQ215 (3xHA-VAM2) were lysed in 3.9 ml of lysis buffer (0.2 M sucrose, 0.1 M Tris-Cl (pH 7.5)) and spun at 500 × g for 2 min to yield a supernatant fraction (S5). Triton X-100 was added to the S5 fraction to give a final concentration of 1%, and then the mixture was spun at 100,000 × g for 60 min to obtain a pellet fraction. The pellet was suspended in 500 µl of 50 mM Tris-Cl (pH 7.5), 0.1% Triton X-100, and proteinase inhibitors (5 µg/ml antipain, 1 µg/ml of 1-chloro-3-tosylamido-7-amino-2-heptanone, leupeptin, aprotinin, and pepstatin A, 0.5 mM PMSF, and 50 µg/ml alpha 2-macroglobulin). 450 µl of the suspension was layered on top of 10-30% (wt/wt) sucrose gradients (10 ml) and spun at 280,000 × g for 5 h in a Hitachi P40ST rotor. Fifteen fractions (0.7 ml each) were obtained by pipetting from the top and subjected to the immunoblotting analyses.

Cross-linking of Vam2p and Vam6p

Spheroplast lysates were prepared as described above except that the lysis buffer contained 50 mM potassium phosphate (pH 7.5) instead of 0.1 M Tris-Cl (pH 7.5). The lysates (8 × 108 cells) were spun at 100,000 × g for 60 min to give pellet fractions. The pellets were dissolved in a buffer (50 mM potassium phosphate (pH 7.5), the proteinase inhibitors) by passing through 26-gauge needles 10 times. Then, DTSSP was added to the suspension as powder to yield final concentration of 10 mM, and the mixture was incubated at room temperature for 30 min. The reaction was terminated by adding 50 µl of 1 M Tris-Cl (pH 7.5), 1 mg/ml bovine serum albumin, and the proteinase inhibitors and was further incubated for 15 min. The proteins were recovered by trichloroacetic acid precipitation, resolved in 100 µl of boiling buffer (0.1 M Tris-Cl (pH 7.5), 1 mM EDTA, and 1% SDS) and boiled for 4 min. The samples were diluted with 0.5 ml of Tween 20 IP buffer (50 mM Tris-Cl, 150 mM NaCl, 0.5% Tween 20, 0.1 mM EDTA, pH 7.5), incubated with 25 µl of 50% slurry of protein A-Sepharose coupled with anti-HA MmAb 12CA5 or affinity purified anti-Vam6p antibodies overnight. The beads were sedimented and washed 6 times with Tween 20 IP buffer, and immunoreactive materials were released by suspension in 22 µl of 0.1 M glycine (pH 2.5) for 15 min. After removing the beads, the solution was neutralized by adding 2.5 µl of 1.5 M Tris-Cl (pH 8.8) and 6 µl of 5 × SDS-PAGE sample buffer and was then subjected to SDS-PAGE and immunoblotting analysis.

Microscopy

For detection of GFP-Vam6p, cells were grown in SCD(-Ura) (8) for 40 h at 23 °C, fixed in SCD containing 3.7% formaldehyde and 0.1 M potassium phosphate (pH 7.5) for 30 min, and then mounted on a polylysine-coated slide glass and viewed under a laser-scan confocal microscope (Zeiss LSM410 and Axiovert 135M). The images were processed by AdobeTM Photoshop 3.01J on a MacintoshTM computer.

For electron microscopy, cells were grown on YPD (8) plates overnight at 30 °C, harvested by scraping with copper grids, and then rapidly frozen by plunging into liquid propane cooled with liquid N2 in a Reichert KF80. Frozen cells were transferred to 2% OsO4 in anhydrous acetone, kept at -80 °C for 2 days, and then transferred to -35 °C for 2 h, 4 °C for 2 h, and to room temperature for 2 h. After washing with anhydrous acetone three times, samples were infiltrated with increasing concentrations of Spurr's resin in anhydrous acetone and finally with 100% Spurr's resin. These samples were then polymerized in capsules at 50 °C for 5 h and 70 °C for 30 h. Thin sections were cut on a Reichert Ultracut S, stained with uranyl acetate and lead citrate, and were viewed on a JEOL 1210 electron microscope at 80 kV.


RESULTS

Structures of VAM2, VAM6 and Their Products

Yeast genomic libraries were screened for the complementation of the vam2-3 and vam6-1 mutations. Structural analyses on the cloned genomic DNAs showed that a 6.5-kb BamHI-ClaI region of the chromosome IV was sufficient for complementation of the vam2-3 mutation. By integration and mapping of this region, we confirmed that the cloned segment contains the authentic VAM2. Similarly, we found that a 3.4-kb ApaI-PvuII fragment of the chromosome IV was enough to complement the vam6-1 mutation, and this region corresponded to the VAM6 locus.

The nucleotide sequences showed that the VAM2 (GenBankTM/EBI/DDBJ accession number AB000223) corresponded to the ORF YDR080w and the VAM6 (GenBankTM/EBI/DDBJ accession number D83058[GenBank]) did to the ORF YDL077c. The product of VAM2 (Vam2p) was predicted to be a protein of 992 amino acids with a molecular mass of 113.4 kDa. The entire molecule was highly hydrophilic, and there were no hydrophobic regions capable of forming transmembrane domains. Vam2p contained highly acidic (79DDDDDDDDDDDEDEDDEDE97) and basic (239KKKKKKTRK247) regions. The VAM6 appeared to encode a 1,049 amino acid protein with a molecular mass of 122.9 kDa. The Vam6p was also hydrophilic. A search for related sequences in the GenBankTM/EBI/DDBJ data bases using the BLAST program and FASTA algorithm revealed no proteins with significant similarity to the Vam6p.

vam2 and vam6 Null Mutants Exhibit Severe Defects in the Vacuolar Morphology

The requirement of Vam2p and Vam6p functions was examined by construction of strains carrying the null alleles of VAM2 and VAM6. The null mutants exhibited highly fragmented vacuolar morphologies (Fig. 1). The mutant cells accumulated numerous spherical structures of 0.2-0.4 µm in diameter. These structures were stained with uranylacetate in various levels, suggesting that they may not be homogeneous. The mutant cells did not show a significant accumulation of typical transport vesicles that are usually smaller than 80 nm in diameter. We also observed the null mutants by light microscopy. Under DIC optics, the mutant cells exhibited numerous vesicular structures scattered in the cytoplasm (data not shown). This phenotype was indistinguishable to those shown by the original vam2 and vam6 mutant cells (1). The null mutant cells accumulated a fluorescent endocytic marker dye, lucifer yellow CH, in the fragmented compartments, indicating that the internalization of lucifer yellow CH was not affected in the absence of the Vam2p and Vam6p function and that the morphologically fragmented compartments were related to vacuoles, the destination of endocytic trafficking (18). The fragmented compartments accumulated the ade fluorochrome, an endogeneous marker for the yeast vacuoles (19). Quinacrine, which is known to be accumulated in acidic compartments, was also accumulated in the fragmented compartments, indicating that the inside of the fragmented compartments was acidified (data not shown). These cytological observations indicated that the fragmented compartments shared the characteristics with the vacuoles in the wild-type cells.


Fig. 1. Vacuolar morphology of Delta vam2 and Delta vam6 mutant cells by electron microscopy. YPH499 (A), YW25-1C (Delta vam2) (B), and NNY60L (Delta vam6) (C) cells were fixed and processed for electron microscopy by the freeze-substitution technique. The scale bar indicates 1 µm.
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The Processing of Vacuolar Proteins Is Defective in Delta vam2 and Delta vam6 Cells

The Delta vam2 and Delta vam6 mutant cells appeared to be defective in maturation of vacuolar proteins, including proteinase A (PrA), proteinase B (PrB), and carboxypeptidase Y (CPY) (Fig. 2). PrA and PrB appeared mostly as aberrantly processed forms migrating between the normal Golgi and vacuolar forms. However, minor amounts of PrA and PrB and a considerable amount of CPY existed as their mature forms in the mutants. In contrast to these severe but partially defective processings of PrA, PrB, and CPY, maturation of another vacuolar protein alkaline phosphatase (ALP) was more severely affected. ALP remained as its Golgi-modified form (71 kDa), and no mature form of ALP (69 kDa) was found in the mutant cells.


Fig. 2. Processing of vacuolar proteins in Delta vam2 and Delta vam6 cells. Whole cell lysates from YPH499 (wild-type), YW25-1C (Delta vam2), NNY60L (Delta vam6), YW25-1C harboring pYVQ215 (Delta vam2+3xHA-VAM2), NNY60L harboring pVAM6-6HA (Delta vam6+6xHA-VAM6), and STY1 (Delta pep4) were resolved by SDS-PAGE and analyzed by immunoblotting using specific antibodies against proteinase A (PrA), proteinase B (PrB), carboxypeptidase Y (CPY), and alkaline phosphatase. The mature, vacuolar forms of PrA, PrB, CPY, and ALP were indicated by mPrA, mPrB, mCPY, and mALP, respectively. The Golgi-modified forms of these vacuolar proteins were shown by proPrA, proPrB, p2CPY, and proALP.
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Genetic analyses showed that loss of the functions of VPS41/VAM2 and VPS39/VAM6 results in the partial missorting of the soluble vacuolar proteins (4, 5). The Delta vam2 and Delta vam6 cells secreted approximately 20% of newly synthesized CPY to the external medium, confirming that vam2/vps41 and vam6/vps39 mutations cause the defective vacuolar protein sorting (Fig. 3). A cytosolic protein alcohol dehydrogenase (ADH) was found only in the internal fractions; thus, the integrity of spheroplasts was not affected during the chase. In the wild-type cells, most of the newly synthesized CPY was processed to its mature form within a 15-min chase period. In contrast, most of CPY remained as the Golgi-modified 69-kDa molecule in Delta vam2 and Delta vam6 cells even after a 45-min chase. Processing of CPY took place significantly slowly in the mutant cells. Only about 40% of CPY was converted to its mature form even after 3 h of chase (data not shown), whereas most CPY existed as its mature form, exclusively, after 30 min in the wild-type cells (Fig. 3). In contrast, ALP remained as its Golgi form in the mutants even after the prolonged chase in the vam2 and vam6 mutant cells (data not shown). These observations were well consistent with the results of immunoblotting analysis that showed accumulation of the mature CPY and pro-ALP at the steady-state levels in the Delta vam2 and Delta vam6 mutant cells.


Fig. 3. Intracellular sorting of carboxypeptidase Y in Delta vam2 and Delta vam6 cells. Spheroplasts of YPH499 (wild-type), YW25-1C (Delta vam2), and NNY60L (Delta vam6) were pulse labeled for 10 min with Expre35S35S labeling mix and chased for the indicated times at 30 °C. The cultures were fractionated into spheroplast (internal, I) and medium (external, E) fractions. Carboxypeptidase Y and alcohol dehydrogenase in these fractions were analyzed by immunoprecipitation, SDS-PAGE, and fluorography. The positions of ER, Golgi, and vacuolar forms of CPY are indicated by p1CPY, p2CPY, and mCPY, respectively.
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Vam2p and Vam6p Are Components of a Large Protein Complex

To facilitate characterization of Vam2p and Vam6p, triplicate repeats of the HA epitope sequence (YPYDVPDYA) were inserted between the first amino acid Met and the second Thr of Vam2p, and six repeats of the HA epitope sequence were introduced at the N terminus of Vam6p. The monoclonal antibody 12CA5 recognized proteins of approximately 120 and 130 kDa for the 3xHA-Vam2p and 6xHA-Vam6p, respectively. The epitope-tagged versions of the Vam2p and Vam6p were functional; the expression of the modified genes in the corresponding mutant cells complemented the defective vacuolar morphologies (data not shown) and maturation of vacuolar proteins (Fig. 2).

The 3xHA-Vam2p and 6xHA-Vam6p showed similar fractionation patterns in differential centrifugation fractionation (Fig. 4). Both proteins were primarily fractionated in the P13 fraction (approximately 60%). In this fraction, most of ALP, a vacuolar membrane marker protein was fractionated. About 20% of the proteins were also found in the P100 fraction where most of the late Golgi marker protein, Kex2p, distributed. The remainders (approximately 20%) were found in the S100 fraction in which the cytosolic marker protein ADH was fractionated. The structures of Vam2p and Vam6p predicted that both proteins were hydrophilic; however, they were found in the sedimentable fractions. The 3xHA-Vam2p and 6xHA-Vam6p were not extracted by treating with a detergent, Triton X-100. In contrast, they were readily solubilized in the presence of M NaCl and partially extracted by 2 M urea from the sedimentable fractions (Fig. 5). These solubilization profiles suggested that Vam2p and Vam6p associated with pelletable materials by an ionic interaction rather than a hydrophobic interaction.


Fig. 4. Subcellular fractionation of epitope-tagged Vam2p and Vam6p. Cells of YW25-1C (Delta vam2) harboring pYVQ215 (left panels) and NNY60L (Delta vam6) harboring pVAM6-6HA (right panels) were converted to spheroplasts and osmotically lysed, and then the lysates were subjected to centrifugation at 13,000 × g to yield pellet (P13) and supernatant (S13) fractions. The S13 fractions were further subjected to centrifugation at 100,000 × g to yield pellet (P100) and supernatant (S100) fractions. Proteins in these fractions were resolved by SDS-PAGE, and then the HA-tagged Vam2p and Vam6p were probed by MmAb 12CA5. Organelle marker proteins Kex2p (late Golgi), ALP (vacuolar membrane), and ADH (cytosol) were also detected by specific antibodies.
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Fig. 5. Differential extraction of Vam2p and Vam6p by various reagents. Spheroplast lysates of NNY60L (Delta vam6) harboring pVAM6-6HA (for 6xHA-Vam6p) and YW25-1C (Delta vam2) harboring pYVQ215 (for 3xHA-Vam2p) were treated with distilled water (DW), 1% Triton X-100, 2 M urea, or 1 M NaCl and then spun at 100,000 × g to yield pellet (Ppt) and supernatant (Sup) fractions. The Vam6p and Vam2p in these fractions were probed by MmAb 12CA5.
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We speculated that Vam2p and Vam6p may form a large sedimentable protein complex by interacting with each other. When the detergent-insoluble materials were resolved through a sucrose velocity gradient, 3xHA-Vam2p and 6xHA-Vam6p showed similar sedimentation patterns (Fig. 6). This underscored a possibility that Vam2p and Vam6p were components of a quite large complex that was sedimented much faster than the 20 S marker molecule thyroglobulin. The physical interaction between Vam2p and Vam6p was shown by co-immunoprecipitation of Vam2p and Vam6p after a treatment of a chemical cross-linker DTSSP (Fig. 7). The 3xHA-Vam2p was immunoprecipitated by anti-Vam6p antibodies from the sedimentable materials (lane 4). This co-immunoprecipitation was not due to nonspecific precipitation of Vam2p because the 3xHA-Vam2p was not precipitated by the anti-Vam6 antibodies from Delta vam6 cells derived materials (lane 6). We concluded from these observations that Vam2p and Vam6p are present in a large sedimentable complex.


Fig. 6. Sucrose velocity gradient analysis of Vam2p and Vam6p. Cell lysate from YW25-1C harboring pYVQ215 was treated with Triton X-100, then spun at 100,000 × g. The resulting pellet was resuspended in a buffer containing 1% Triton X-100, layered on top of 10-30% (wt/wt) continuous sucrose gradient, and spun at 280,000 × g for 5 h. Fractions were collected from the top of the tube, and the 3xHA-Vam2p and Vam6p were analyzed by immunoblotting using anti-HA monoclonal antibody and anti-Vam6p polyclonal antibody. The markers used were ovalbumin (4S), gamma -globulin (7S), and thyroglobulin (20S).
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Fig. 7. Cross-linking of Vam2p and Vam6p. Spheroplast lysates from YW25-1C (Delta vam2) harboring plasmid pYVQ206 (VAM2, lanes 1 and 3) or plasmid pYVQ215 (3xHA-VAM2, lanes 2 and 4), and from NNY33 (Delta vam2 Delta vam6) harboring pYVQ215 (lanes 5 and 6) were subjected to centrifugation at 100,000 × g, and the pellet fractions were incubated with dithiobis(sulfosuccinimidyl propionate). After quenching the cross-linker, the lysate was incubated with anti-HA (lanes 1, 2, and 5) or with anti-Vam6p (lanes 3, 4, and 6) antibodies immobilized on beads. The immunoprecipitates were analyzed by immunoblotting analysis using anti-HA antibody to detect the 3xHA-Vam2p.
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The Subcellular Localization of GFP-Vam6p

-We found that immunofluorescence localization of the 3xHA-Vam2p and 6xHA-Vam6p by MmAb 12CA5 was difficult presumably due to the low abundance of these proteins. We used a green fluorescent protein (GFP) (20) as a tag for detecting the subcellular localization of Vam6p (Fig. 8). The introduction of GFP sequence into the N-terminal of Vam6p (GFP-Vam6p) did not disrupt the Vam6p function; expression of the fusion protein from a low copy plasmid complemented the Vam- phenotype of Delta vam6 cells (Fig. 8A). The GFP fluorescence was localized to the vacuolar membrane. The GFP-Vam6p was highly accumulated in one or two distinct locations of the vacuolar membranes (Fig. 8, A-C), giving a few bright "spots." When the GFP-Vam6p was expressed from a multicopy plasmid, the staining on the vacuolar membrane became stronger; however, the strength of the signals from the spots and the numbers of the spots per cells were essentially unchanged (Fig. 8, D-F). We also tried to determine the subcellular localization of a GFP-Vam2p fusion protein; however, we could not detect signals of the GFP-Vam2p although the expression of the GFP-VAM2 fusion gene complemented the defective vacuolar assembly in the Delta vam2 mutant cells (data not shown).


Fig. 8. Localization of GFP-Vam6p by a laser scanning confocal microscope. Exponentially growing cells of NNY60L (Delta vam6) harboring pGFP-VAM6 (low copy plasmid, panels A-C) and NNY60L harboring pGFP-VAM6M (multi-copy plasmid, panels D-F) were fixed and viewed under a confocal laser scanning microscope. The DIC images (panels A and D), the GFP images (panels C and F), and overlaid images (panels B and E) are shown. The scale bar indicates 5 µm.
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DISCUSSION

Vacuolar assembly is a complex process; genetic analyses toward understanding the molecular basis of the vacuolar biogenesis and morphogenesis have identified that the function of over 50 members of the VPS, PEP, VAM (1, 4, 5, 21, 22), and others (8, 23-25) are required for the vacuolar assembly in yeast cells. The VAM2 gene was identified multiple times by mutant screenings with different strategies. vps41 mutants exhibit mislocalization of CPY, and cvt8 was shown to be defective in cytosol-vacuolar transport of a vacuolar protein aminopeptidase I. The VAM6 gene has been also known as the VPS39 and CVT4 (5, 6, 26). In this study, we characterized the VAM2/VPS41/CVT8 and VAM6/VPS39/CVT4 genes and their gene products.

Delta vam2 or Delta vam6 mutants showed similar phenotypes, i.e. the fragmented vacuolar morphology, delay in the PrA, PrB, and CPY processing, and absence of ALP processing. Delta vam2 Delta vam6 double mutant exhibited the same phenotype as the mutants carrying single Delta vam2 or Delta vam6 mutation (data not shown). We noted that not only the phenotype of the vam2 and vam6 null mutations but also their gene products shared some similar charcteristics. Vam2/Vps41p and Vam6/Vps39p were predicted to be hydrophilic proteins; however, they associated with the sedimentable fractions. The association of Vam2/Vps41p and Vam6/Vps39p to the sedimentable materials was disrupted by high salt and urea but not by detergent, implicating that protein-protein interaction is responsible for this association. Vam2/Vps41p and Vam6/Vps39p are found in a large protein complex. Furthermore, Vam2/Vps41p and Vam6/Vps39p were interacting physically as they could be co-immunoprecipitated after chemical cross-linking. Most parts of Vam2/Vps41p and Vam6/Vps39p were fractionated in the P13 fraction where the vacuolar membranes were enriched (17), and the GFP-Vam6p are localized to the vacuolar membranes. We have not succeeded in confirming the subcellular localization of Vam2/Vps41p by cytological observations; however, we speculate that Vam2/Vps41p is co-localized to Vam6/Vps39p because of the physical interaction shown by co-sedimentation and co-immunoprecipitation analyses. The mutant phenotypes and the subcellular localizations of Vam2p and Vam6p, together, strongly indicate that Vam2/Vps41p and Vam6/Vps39p execute their function at the same, late step in the Golgi-vacuolar membrane trafficking.

The fragmented vacuolar morphology of the class II vam mutants raised two possibilities for their function. The class II VAM gene products may be required for the assembly of the vacuolar compartments, or alternatively, the cells cannot maintain the large vacuoles from fragmentation without the function of the class II VAM genes (1, 3). Yeast cells lacking Vam2/Vps41p or Vam6/Vps39p display the characteristic fragmented vacuolar morphology. The Delta vam2 and Delta vam6 cells, as well as the other class II vam mutants, also exhibited a significant delay in the processing of CPY and the accumulation of precursor form of ALP. The defective protein processing suggests that the function of VAM2/VPS41 and VAM6/VPS39 are required for the assembly of mature large vacuoles. However, we cannot exclude a possibility that they also participate in the maintenance of the morphology of prominent vacuoles from the fragmentation. Yeast vacuole as well as plant vacuoles are known to be the sites of huge accumulations of various primary and secondary metabolites (27, 28). The electrochemical potential differences across the vacuolar membranes for the small or ionic molecules give a physical force, i.e. osmotic pressures onto the membranes. Therefore, a certain mechanism must prevent rupture and/or deformation of the vacuolar membranes from the stress caused by the electrochemical gradients across the membranes (29-31). Loss of this function may result in the similar phenotypes to those of the class II vam mutations, i.e. fragmented vacuolar morphology.

The GFP-Vam6p is predominantly localized to the vacuolar membranes. The nature of this condensed localization of GFP-Vam6p remains to be clarified. In the case of the plasma membrane, Golgi-derived secretory vesicles fuse at the specialized regions of the plasma membrane. This fusion and subsequent deposition of materials at the specialized portion of the plasma membrane are in part responsible for oriented bud growth (32, 33). We recently found that Vam3p, the other member of the class II VAM gene products also showed patched localization on the vacuolar membranes. Vam3p associates to the vacuolar membranes, and it is a syntaxin-related molecule and has an essential function in the vacuolar assembly.2 The condensed localization of certain VAM gene products on the vacuolar membranes may suggest that some regions on the vacuolar membranes are specialized for accepting the Golgi or prevacuolar-derived membrane flows although further biochemical and morphological characterizations of the vacuolar membranes are required for confirming this speculation.


FOOTNOTES

*   This study was supported by Grants-in-Aid for Scientific Research in the priority areas of "Bioenergetics" and "The Molecular Basis of Flexible Organ Plans in Plants" from the Ministry of Education, Science and Culture of Japan (to Y. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Present address: Dept. of Cell Biology, National Institute for Basic Biology, Myodaiji, Okazaki, Japan.
par    To whom correspondence and requests for reprints should be addressed. Tel./Fax: 81-3-5454-6648; E-mail: cywada{at}komaba.ecc.u-tokyo.ac.jp.
1   The abbreviations used are: HA, influenza hemagglutinin; ADH, alcohol dehydrogenase; ALP, vacuolar alkaline phosphatase; CPY, carboxypeptidase Y; DTSSP, dithiobis(sulfosuccinimidyl propionate); GFP, Aequorea victoria green fluorescent protein; MmAb, mouse monoclonal antibody; PrA, proteinase A; PrB, proteinase B; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride.
2   Y. Wada, N. Nakamura, Y. Ohsumi, and A. Hirata, submitted for publication.

ACKNOWLEDGEMENTS

We thank Yasuhiro Anraku for providing the yeast genomic library and the HA-encoding DNA fragment and Yasuo Niwa for the GFP(S65T) construct. We also thank Tom H. Stevens for the vps39 and vps41 mutant strains and valuable discussions. We also thank Akihiko Nakano for helpful advice and discussions throughout this study.


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