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

Norihiro Nakamura,Aiko Hirata,Yoshinori Ohsumi,Yoh Wada

doi:10.1074/jbc.272.17.11344

Norihiro Nakamura, Aiko Hirata + Show 2 more

Open Access

https://doi.org/10.1074/jbc.272.17.11344

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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.

Highlights

The VAM2/VPS41 and VAM6/VPS39 were shown to encode hydrophilic proteins of 113 and 123 kDa, respectively
⌬vam2 or ⌬vam6 mutants showed similar phenotypes, i.e. the fragmented vacuolar morphology, delay in the proteinase A (PrA), proteinase B (PrB), and carboxypeptidase Y (CPY) processing, and absence of alkaline phosphatase (ALP) processing. ⌬vam2 ⌬vam6 double mutant exhibited the same phenotype as the mutants carrying single ⌬vam2 or ⌬vam6 mutation
Vam2/Vps41p and Vam6/Vps39p were predicted to be hydrophilic proteins; they associated with the sedimentable fractions

Summary

EXPERIMENTAL PROCEDURES

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). 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 ade ura leu vam2–3) and YN6 – 47D (MATa ade ura leu2-⌬1 vam6 –1) by the lithium acetate method [10, 11]. 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 3Ј untranslated region) to yield plasmid pYVQ213. 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

TABLE I Strains used in this study

RESULTS

DISCUSSION