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 Icb1-100 cells, defective in sphingoid base synthesis, Pma1 fails to oligomerize. At 30degreesC, Icb1-100 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 oligornerization 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 30degreesC in Icb1-100 cells. At 37degreesC in Icb1-100 cells, a more severe defect occurs in sphingoid base synthesis, and targeting of newly synthesized Pmal to the plasma membrane is impaired. These data indicate requirements for sphingolipids at three discrete stages: Pmal oligornerization at the ER, targeting to the plasma membrane, and stability at the cell surface.