Sphingoid base synthesis is required for oligomerization and cell surface stability of the yeast plasma membrane ATPase, Pma1

Abstract

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°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°C in lcb1-100 cells. At 37°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.T he plasma membrane ATPase, encoded by PMA1, is a polytopic membrane protein whose essential physiological function is to pump protons out of the cell (1). Because Pma1 activity is critical for generating a membrane potential and regulating cytoplasmic pH, it is not surprising that numerous mechanisms exist to regulate Pma1 function at the plasma membrane (1), and to ensure efficient Pma1 delivery to the cell surface by way of the secretory pathway. Export of newly synthesized Pma1 from the endoplasmic reticulum (ER) is facilitated by a specialized COPII coat component, Lst1 (2, 3). En route to the plasma membrane, Pma1 associates with ergosterol-and sphingolipid-enriched microdomains called lipid rafts, which have been suggested to play a role in Pma1 targeting to the cell surface (4, 5). Pma1 association with lipid rafts is disrupted in temperature-sensitive lcb1-100 cells defective in sphingoid base synthesis (4). On arrival at the plasma membrane in wild-type cells, Pma1 remains stable with a half-life of Ϸ11 h (6). By contrast with cell surface proteins that undergo rapid turnover, Pma1 is not detectably ubiquitylated (7). The parameters that maintain Pma1 longevity are currently unknown, although a temperature-sensitive pma1 mutant with increased turnover at the plasma membrane has been recently characterized, which may provide insight into cell surface stability (8).Because of its critical enzymatic function, PMA1 has been the focus of extensive mutagenesis studies (9). Many of these pma1 mutants are targeted for ER-associated degradation (ERAD) (10), making them a valuable resource for genetic approaches to understanding ER quality control (11). Of these mutants, Pma1-D378N is th...

Lipid-dependent surface transport of the proton pumping ATPase: A model to study plasma membrane biogenesis in yeast

“…Formation of mixed oligomers between the normal and mutant proteins could lead to the recognition of the entire complex as misfolded and subject to ER quality control. Such a model is supported by the observation that assembly of the multimeric Pma1 complex is dependent on long-chain base synthesis and that a block in long-chain base synthesis relieves the dominant lethal phenotype of a Pma1 mutant [27].…”

“…The fact that these lipids affect detergent solubility of newly synthesized Pma1 already upon ER exit would indicate that lipids and protein may already assemble at their site of synthesis and are then co-transported to the surface [4]. A failure to properly assemble this proteinelipid complex results either in a diversion of surface destined vesicle to the vacuole, a failure in stabilization of the complex upon arrival at the plasma membrane, or both [18,19,27].…”

Very Long-chain Fatty Acid-containing Lipids rather than Sphingolipids per se Are Required for Raft Association and Stable Surface Transport of Newly Synthesized Plasma Membrane ATPase in Yeast

“…A relationship between Pma1p biogenesis and lipid synthesis is indicated by the observations that long-chain base or ceramide synthesis is required for oligomerization and raft association of Pma1p in the ER (2,7). Oligomerization of Pma1p, however, is not required for ER export or surface delivery but might be important for stabilization of the protein at the cell surface (2,7).…”

Sphingolipids and membrane biology as determined from genetic models

“…In addition, in the yeast mutant pma 1-7, in which lipids rafts are perturbed, newly synthesized Pma 1p is mistargeted to the endosomal system for degradation instead of being delivered to the plasma membrane. These studies suggest a model in which the targeting of Pma 1p to the plasma membrane requires its association with lipid rafts, although additional parameters appear to influence it as well [97,98] In yeast, sphingolipids are also required for GPI-anchored proteins to associate stably with the membrane. In the lcb1-100 mutant line, GPI-anchored proteins fail to associate with the membrane, behaving instead like peripheral membrane proteins.…”