DOMINANT LETHAL MUTATIONS IN THE PLASMA-MEMBRANE H-ATPASE GENE OF SACCHAROMYCES-CEREVISIAE()

The plasma membrane H+-ATPase of Saccharomyces cerevisiae is an essent ial protein that is required to establish cellular membrane potential and maintain a normal internal pH. An Asp-378 to Asn substitution at t he residue phosphorylated during catalysis is dominant lethal when the pma1-D378N mutation is expressed along with a wild-type plasmamembran e H+-ATPase (PMA1) gene. Several mutations in the first two putative t ransmembrane domains are also dominant lethal. However, these dominant lethal mutants often appear to be innocuous, because they are frequen tly lost by gene conversion to the wild-type sequence during the proce ss of introducing the mutant sequence and subsequently removing the wi ld-type gene. Loss of the mutation by gene conversion does not occur w hile introducing recessive lethal mutations. Cells carrying the wild-t ype PMA1 gene on the chromosome and a dominant lethal mutation under t he control of a GAL1 promoter on a centromere containing plasmid exhib it a galactose-dependent lethality. Indirect immunofluorescence staini ng using anti-Pma1 antibodies shows that induction of dominant lethal PMA1 mutations leads to the accumulation of a number of intensely stai ning cytoplasmic structures that are not coincident with the nucleus a nd its immediately surrounding endoplasmic reticulum. These structures also accumulate the endoplasmic reticulum protein Kar2. Expression of the dominant lethal protein also prevents transport of the wild-type ATPase to the plasma membrane.