title: Yeast mutants deficient in ER-associated degradation of the Z variant of alpha-1-protease inhibitor.
abstract: Saccharomyces cerevisiae mutants deficient in degradation of alpha-1-proteinase inhibitor Z (A1PiZ) have been isolated and genetically characterized. Wild-type yeast expressing A1PiZ synthesize an ER form of this protein that is rapidly degraded by an intracellular proteolytic process known as ER-associated protein degradation (ERAD). The mutant strains were identified after treatment with EMS using a colony blot immunoassay to detect colonies that accumulated high levels of A1PiZ. A total of 120,000 colonies were screened and 30 putative mutants were identified. The level of A1PiZ accumulation in these mutants, measured by ELISA, ranged from two to 11 times that of A1PiZ in the parent strain. Further studies demonstrated that the increased levels of A1PiZ in most of the mutant strains was not the result of defective secretion or elevated A1PiZ mRNA. Pulse chase experiments indicated that A1PiZ was stabilized in several strains, evidence that these mutants are defective in ER-associated protein degradation. Genetic analyses revealed that most of the mutations were recessive, approximately 30% of the mutants characterized conformed to simple Mendelian inheritance, and at least seven complementation groups were identified.
authors.affiliation: Department of Biology, College of Arts and Science, University of Nevada, Reno 89557, USA. mccracke@cmb.unr.edu
authors.forename: A A
authors.initials: AA
authors.lastname: McCracken
authors.forename: I V
authors.initials: IV
authors.lastname: Karpichev
authors.forename: J E
authors.initials: JE
authors.lastname: Ernaga
authors.forename: E D
authors.initials: ED
authors.lastname: Werner
authors.forename: A G
authors.initials: AG
authors.lastname: Dillin
authors.forename: W E
authors.initials: WE
authors.lastname: Courchesne
mesh_terms: D009154:Mutation; D012331:RNA, Fungal / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D000515:alpha 1-Antitrypsin / Q000235:genetics; D019896:alpha 1-Antitrypsin Deficiency
chemical_list: C489019:A1PiZ protein, S cerevisiae; D012331:RNA, Fungal; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D000515:alpha 1-Antitrypsin