Compartment acidification is required for efficient sorting of proteins to the vacuole in Saccharomyces cerevisiae.

D.J Klionsky,H Nelson,N Nelson

doi:10.1016/s0021-9258(19)50747-5

D.J Klionsky, H Nelson + Show 1 more

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https://doi.org/10.1016/s0021-9258(19)50747-5

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Abstract

The vacuole of the yeast Saccharomyces cerevisiae contains a proton-translocating ATPase that acidifies the vacuolar lumen and generates a pH gradient across the vacuole membrane. We have investigated the role of compartment acidification of the vacuolar system in the sorting of vacuolar proteins. Strains with chromosomal disruptions of the genes encoding the A, B, or c subunit of the vacuolar ATPase are unable to acidify their vacuoles. These vat mutant strains accumulate and secrete precursor forms of the soluble vacuolar hydrolases carboxypeptidase Y and proteinase A. The kinetics of secretion suggests that missorting occurs in the Golgi complex or in post-Golgi vesicles. The presence of mature forms of the vacuolar proteins within the cell indicates that vat mutations do not cause defects in zymogen processing. Precursor forms of the membrane-associated vacuolar hydrolase alkaline phosphatase are also accumulated in vat mutant cells but to a lesser extent, suggesting that sorting of vacuolar membrane proteins is less sensitive to changes in the lumenal pH. A similar type of missorting defect can be induced in wild-type cells at pH 7.5. These results indicate that acidification of the vacuolar system is important for efficient sorting of proteins to the vacuole.

Highlights

The vacuole of the yeast Saccharomyces cerevisiae man et al, 1989) and may serve as a trigger for the autoacticontains a proton-translocating ATPase thatcidifies vation of proteinase A (Woolford et al, 1986), a model based the vacuolar lumen and generatepsHagradient across in part on the pH-dependent autocatalysis of the homologous the vacuole membrane
Phosphatase are accumulated in vat mutant cells There are some discrepancies concerning the imporbut to a lesserextent, suggesting that sortingof vacu- tance of compartment acidification inprotein sorting
We have found that mutants that ardeefective in compartment acidification are able to process precursor vacuolar proteins, but exhibit significant missorting defects

Summary

EXPERIMENTAL PROCEDURES

Strains and Plasmids-The Saccharomycescerevisiae yeast strains used were W303-1B (MATa ku hk3 d e 2 trpl urd') and W303-1B with LEU2 chromosomal disruptions in the VATA,VATB, or VATc gene (uatA-Al,uatB-Al, or uatc-Al) (Nelson and Nelson, 1990; Noumi et al, 1991).The VATA gene encoding subunit A of the yeast vacuolar ATPase was cloned by amplification from genomic DNA of yeast strain W303 by PCR (Fig. 1). PCR was performed with yeast DNA, a primer (200 pmol) containing a BamHI site and corresponding to the sense strand at positions 736-760 of the TFPl gene, and an antisense primer (200 pmol) corresponding to positions 1777-1797 This generated a fragment encodingthe amino-terminal portionof the VATA gene (encompassing amino acids 1-284), whichis located 5' to the50-kDa protein coding sequence. + with yeast DNA, a 41-nucleotide primer (200 pmol) corresponding to the sense strand a t positions 1778-1797 3160-3180, and an antisense primer (200 pmol) corresponding to positions 4430-4452 and containing an NdeI site This produced a fragment encoding the carboxyl-terminal half of the VATA gene (coding for amino acids 285-617), located 3' to the gene for the 50-kDa protein. Cells were allowedto continue growingat 30 "Cfor designated lengths of time after the pHshift prior to theaddition of radioactive label

RESULTS

I vafA vafB vafc

DISCUSSION