Biochemical and molecular confirmation of candidate Endosome and Vacuole interface (env) mutants derived from a genomic screen and characterization of four novel env deletion mutants: env6 ‐ env9

Abstract

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.