Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface – ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes – MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

The vacuole of the fungus Saccharomyces cerevisiae has been a seminal model for lysosomal trafficking, biogenesis, and function. Our laboratory uncovered 15 deletion mutants associated with these processes utilizing a genome‐wide immunodetection screen of a MAT‐α deletion strain library. This study focuses on confirmation of the l5 mutants for defects at the endosome & vacuole interface (env mutants) and characterization of four uncharacterized env mutants : env6Δ, env7Δ, env8Δ, and env9Δ. Microscopic, biochemical, and bioinformatics techniques were used to characterize the four mutants with respect to vacuole morphology, growth under various conditions, and gene homology. env6Δ has fragmented vacuoles with acidification defects, exhibits severe growth sensitivities, and the deletion is a dubious open reading frame (ORF). env7Δ exhibits normal vacuolar morphology, no severe growth sensitivities, and ENV7 encodes a putative serine/threonine kinase with 29% identity to human STK16 kinase. env8Δ has fragmented vacuoles, no severe growth sensitivities, and the ORF encodes a highly conserved protein with possible involvement in membrane insertion of tail‐anchored proteins. env9Δ has vacuoles with MVBs, and exhibits no severe growth sensitivities. Complementation studies of the four mutants following reintroduction of each ORF are currently in progress. Funding was provided by NSF‐RUI and NIH‐AREA grants.