Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1.
The plasma membrane H(+)-ATPase, Pma1, is an essential and long-lived integral membrane protein. Previous work has demonstrated that the Pma1-D378N mutant is a substrate for endoplasmic reticulum (ER)-associated degradation and causes a dominant negative effect on cell growth by preventing ER export of wild-type Pma1. We now show that Pma1-D378N is ubiquitylated, and it heterooligomerizes with wild-type Pma1, resulting in ubiquitylation and ER-associated degradation of wild-type Pma1. In temperature-sensitive lcb1-100 cells, defective in sphingoid base synthesis, Pma1 fails to oligomerize. At 30 degrees C, lcb1-100 is a suppressor of pma1-D378N because wild-type Pma1 fails to heterooligomerize with Pma1-D378N; wild-type Pma1 moves to the cell surface, indicating that oligomerization is not required for delivery to the plasma membrane. Even in the absence of Pma1-D378N, wild-type Pma1 is ubiquitylated and it undergoes internalization from the cell surface and vacuolar degradation at 30 degrees C in lcb1-100 cells. At 37 degrees C in lcb1-100 cells, a more severe defect occurs in sphingoid base synthesis, and targeting of newly synthesized Pma1 to the plasma membrane is impaired. These data indicate requirements for sphingolipids at three discrete stages: Pma1 oligomerization at the ER, targeting to the plasma membrane, and stability at the cell surface.
Wang
Qiongqing
Q
Departments of Anatomy and Structural Biology and Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Chang
Amy
A
D015153:Blotting, Western; D002455:Cell Division; D002462:Cell Membrane / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect; D005690:Galactose / Q000494:pharmacology; D007700:Kinetics; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism; D015003:Yeasts
D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases; D005690:Galactose