title: Identification and characterization of a drug-sensitive strain enables puromycin-based translational assays in Saccharomyces cerevisiae.
abstract: Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C-terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6∆, pdr1∆ and pdr3∆ (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin-based assays in yeast, which will enable new avenues of inquiry into protein production and maturation.
authors.affiliation: Institute for Systems Biology, Seattle, WA, USA; Molecular and Cellular Biology Program, University of Washington, Seattle, WA, USA.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Cary
authors.forename: Sung Hwan
authors.initials: SH
authors.lastname: Yoon
authors.forename: Cecilia Garmendia
authors.initials: CG
authors.lastname: Torres
authors.forename: Kathie
authors.initials: K
authors.lastname: Wang
authors.forename: Michelle
authors.initials: M
authors.lastname: Hays
authors.forename: Catherine
authors.initials: C
authors.lastname: Ludlow
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
authors.forename: Aimée M
authors.initials: AM
authors.lastname: Dudley
mesh_terms: D000935:Antifungal Agents / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D011691:Puromycin / Q000494:pharmacology*; D012270:Ribosomes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D029701:Saccharomyces cerevisiae Proteins; D011691:Puromycin
keywords: ERG6; PDR1; PDR3; p-bodies; puromycin; translation