Half-life of the plasma membrane ATPase and its activating system in resting yeast cells

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...

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“…Wild-type Pma1 at the cell surface is normally long-lived (6). In lcb1-100 cells, wild-type Pma1 reaches the plasma membrane ( Fig.…”

Section: Resultsmentioning

confidence: 93%

“…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).…”

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confidence: 99%

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

Wang

Chang

2002

Proc. Natl. Acad. Sci. U.S.A.

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“…At the plasma membrane, wild-type Pma1 is a paradigm of a stable membrane protein with a half-life of Ϸ11 h (9). Unlike other cell surface proteins, the endocytosis of which is signaled by ubiquitination, Pma1 is not ubiquitinated (10), and whether cell surface Pma1 undergoes recycling has not been established.…”

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confidence: 99%

A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface

Gong

Chang

2001

Proc. Natl. Acad. Sci. U.S.A.

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Pma1 is a plasma membrane H ؉ -ATPase whose activity at the cell surface is essential for cell viability. In this paper we describe a temperature-sensitive pma1 allele, pma1-10 (with two point mutations in the first cytoplasmic loop of Pma1), in which the newly synthesized mutant protein fails to remain stable at the cell surface at 37°C. Instead, Pma1-10 appears to undergo internalization for vacuolar degradation in a manner dependent on End4, Vps27, Doa4, and Pep4. By contrast with wild-type Pma1, mutant Pma1-10 is hypophosphorylated and fails to associate with a Triton-insoluble fraction at 37°C, suggesting failure to enter lipid rafts. Kinetic analysis reveals that, at the permissive temperature, newly synthesized Pma1-10 acquires Triton-insolubility before becoming stabilized. We suggest that phosphorylation and lipid raft association may play important roles in maintaining protein stability at the plasma membrane.

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“…Actually a certain inactivation of this enzyme has been reported in conditions similar to the ones used in this work [24]. To check this possibility we investigated the behaviour of ATPase during our experiments.…”

Section: Resultsmentioning

confidence: 60%

Turnover of the K⁺ transport system in Saccharomyces cerevisiae

1991

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The stability of the K' transport system in Sacchurornyces cerevisiae has been studied upon inhibition of protein synthesis with cycloheximide. Addition of the antibiotic gave rise lo an inactivation of this transport. This activation followed first-order kinetics and was stimulated by the presence of a fermentable substrate. A half-life of about 4 h could be calculated in the presence of glucose. The results indicate that, similarly to sugar carriers, K' transport system is less stable than the bulk of proteins of this organism.

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Half-life of the plasma membrane ATPase and its activating system in resting yeast cells

Cited by 74 publications

References 19 publications

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

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

A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface

Turnover of the K⁺ transport system in Saccharomyces cerevisiae

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Half-life of the plasma membrane ATPase and its activating system in resting yeast cells

Cited by 74 publications

References 19 publications

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

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

A mutant plasma membrane ATPase, Pma1-10, is defective in stability at the yeast cell surface

Turnover of the K+ transport system in Saccharomyces cerevisiae

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Turnover of the K⁺ transport system in Saccharomyces cerevisiae