title: Loss of the homotypic fusion and vacuole protein sorting or golgi-associated retrograde protein vesicle tethering complexes results in gentamicin sensitivity in the yeast Saccharomyces cerevisiae.
abstract: Gentamicin continues to be a primary antibiotic against gram-negative infections. Unfortunately, associated nephro- and ototoxicity limit its use. Our previous mammalian studies showed that gentamicin is trafficked to the endoplasmic reticulum in a retrograde manner and subsequently released into the cytosol. To better dissect the mechanism through which gentamicin induces toxicity, we have chosen to study its toxicity using the simple eukaryote Saccharomyces cerevisiae. A recent screen of the yeast deletion library identified multiple gentamicin-sensitive strains, many of which participate in intracellular trafficking. Our approach was to evaluate gentamicin sensitivity under logarithmic growth conditions. By quantifying growth inhibition in the presence of gentamicin, we determined that several of the sensitive strains were part of the Golgi-associated retrograde protein (GARP) and homotypic fusion and vacuole protein sorting (HOPS) complexes. Further evaluation of their other components showed that the deletion of any GARP member resulted in gentamicin-hypersensitive strains, while the deletion of other HOPS members resulted in less gentamicin sensitivity. Other genes whose deletion resulted in gentamicin hypersensitivity included ZUO1, SAC1, and NHX1. Finally, we utilized a Texas Red gentamicin conjugate to characterize gentamicin uptake and localization in both gentamicin-sensitive and -insensitive strains. These studies were consistent with our mammalian studies, suggesting that gentamicin toxicity in yeast results from alterations to intracellular trafficking pathways. The identification of genes whose absence results in gentamicin toxicity will help target specific pathways and mechanisms that contribute to gentamicin toxicity.
authors.affiliation: Department of Medicine, Division of Nephrology, and the Indiana Center for Biological Microscopy, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122, USA.
authors.forename: Mark C
authors.initials: MC
authors.lastname: Wagner
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Molnar
authors.forename: Bruce A
authors.initials: BA
authors.lastname: Molitoris
authors.forename: Mark G
authors.initials: MG
authors.lastname: Goebl
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D027682:Cation Transport Proteins / Q000032:analysis / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005839:Gentamicins / Q000633:toxicity*; D006056:Golgi Apparatus / Q000737:chemistry*; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones; D021381:Protein Transport / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; D005839:Gentamicins; D008565:Membrane Proteins; D018832:Molecular Chaperones; C492183:NHX1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017923:Sodium-Hydrogen Exchangers; C080045:VPS16 protein, S cerevisiae; C092934:VPS45 protein, S cerevisiae; D033921:Vesicular Transport Proteins; C077019:ZUO1 protein, S cerevisiae