A Genomic Screen for Yeast Vacuolar Membrane ATPase Mutants

Abstract

V-ATPases acidify multiple organelles, and yeast mutants lacking V-ATPase activity exhibit a distinctive set of growth defects. To better understand the requirements for organelle acidification and the basis of these growth phenotypes, ‫0074ف‬ yeast deletion mutants were screened for growth defects at pH 7.5 in 60 mm CaCl 2 . In addition to 13 of 16 mutants lacking known V-ATPase subunits or assembly factors, 50 additional mutants were identified. Sixteen of these also grew poorly in nonfermentable carbon sources, like the known V-ATPase mutants, and were analyzed further. The cwh36⌬ mutant exhibited the strongest phenotype; this mutation proved to disrupt a previously uncharacterized V-ATPase subunit. A small subset of the mutations implicated in vacuolar protein sorting, vps34⌬, vps15⌬, vps45⌬, and vps16⌬, caused both VmaϪ growth phenotypes and lower V-ATPase activity in isolated vacuoles, as did the shp1⌬ mutation, implicated in both protein sorting and regulation of the Glc7p protein phosphatase. These proteins may regulate V-ATPase targeting and/or activity. Eight mutants showed a VmaϪ growth phenotype but no apparent defect in vacuolar acidification. Like V-ATPase-deficient mutants, most of these mutants rely on calcineurin for growth, particularly at high pH. A requirement for constitutive calcineurin activation may be the predominant physiological basis of the VmaϪ growth phenotype.

A Genome-Wide Enhancer Screen Implicates Sphingolipid Composition in Vacuolar ATPase Function in Saccharomyces cerevisiae

“…Deletion of some trafficking-related genes has been shown to result in sensitivity to zinc, calcium, or alkaline conditions (Serrano et al 2004;Sambade et al 2005;Pagani et al 2007). Our strategy for screening effectively detected reduced VATPase activity levels to $10% of wild-type yeast (scored as ,50% of activity in the vma21QQ mutant strain).…”

“…It is therefore not surprising that we have identified additional genes involved in trafficking pathways that were not previously found in forward genetic screens. For instance, many of the vacuolar protein sorting (VPS) genes have not been identified in previous genomewide screens for Vma -phenotypes (Sambade et al 2005). The genetic relationship between these sorting pathways and vacuolar acidification most likely results from aberrant sorting of the V-ATPase.…”

“…Consequently, pkr1D cells score a partial Vma -phenotype while voa1D cells appear normal. Genetic screens in S. cerevisiae have been critical in identifying the components of the V-ATPase and its associated factors (Ohya et al 1991;Ho et al 1993;Sambade et al 2005). However, the most recently discovered V-ATPase assembly factor, Voa1p, was identified by proteomics and voa1D cells have no detectable growth phenotype (Ryan et al 2008).…”

“…Genome-wide screens for zinc or calcium (at pH 7.5) sensitivity have found genes that do not directly contribute to V-ATPase function yet show sensitivity to excess metals (Sambade et al 2005;Pagani et al 2007). An example is deletion of the vacuolar zinc transporter Zrc1p, which confers zinc sensitivity even though vacuolar acidification is normal (data not shown).…”

“…An example is deletion of the vacuolar zinc transporter Zrc1p, which confers zinc sensitivity even though vacuolar acidification is normal (data not shown). Also, a loss of the serine protease Kex2p results in both calcium and zinc sensitivity yet does not cause any defect in vacuolar acidification (Sambade et al 2005). Identifying which genetic pathways are directly linked to metal sensitivity independent of V-ATPase function will require further study.…”