A dominant negative mutant of PMA1 interferes with the folding of the wild type enzyme.
Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H (+)-ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co-expressed with the wild type allele, both proteins are retained in the ER and degraded. We have used a PMA1-D378T dominant lethal allele to analyse the mechanism underlying the retention of the wild type enzyme by the dominant negative mutant. A genetic screen was performed for isolation of intragenic suppressors of PMA1-D378T allele. This analysis pointed to transmembrane helix 10 (TM10) as an important element in the establishment of the dominant lethality. Deletion of the TM10 was able to suppress not only the PMA1-D378T but all the dominant lethal alleles tested. Biochemical analyses suggest that dominant lethal proteins obstruct, through TM10, the correct folding of the wild type enzyme leading to its retention and degradation by ERAD.
Eraso
Pilar
P
Departamento de Bioquímica and Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Arturo Duperier, 4, 28029 Madrid, Spain.
Mazón
María J
MJ
Portillo
Francisco
F
D000483:Alleles; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005799:Genes, Dominant; D005804:Genes, Lethal; D016153:Genes, Suppressor; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017510:Protein Folding; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D029701:Saccharomyces cerevisiae Proteins; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases