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

Abstract

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.

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

“…Pma1-10 is hypophosphorylated and fails to associate with detergent insoluble domains at the non-permissive temperature. Indicating that phosphorylation and lipid raft association may play important roles in maintaining protein stability at the plasma membrane [19,20]. This view is consistent with the fact that disruption of membrane microdomains at the plasma membrane by incubation with the anti-cancer drug edelfosine, an alkylglycerophosphocholine, disrupts raft association of plasma membrane localized Pma1 and induces its vacuolar turnover [21].…”

“…Turnover of Pma1 in elo3D is dependent on ongoing endocytosis, indicating that the protein reaches the plasma membrane first, but that it fails to become stabilized there and instead is endocytosed and delivered to the vacuole for degradation [29]. Thus the wild-type ATPase in the elo3D mutant background behaves similarly as the conditional Pma1-10 allele with regard to its increased turnover that correlates with a failure to acquire detergent resistance [19].…”

“…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

“…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). Raft association of Pma1p, on the other hand, is required for surface delivery and subsequent stabilization of the protein (7)(8)(9).…”

The role of lipids in the biogenesis of integral membrane proteins

“…The fact that these lipids affect detergent solubility of newly synthesized Pma1 already upon ER exit would indicate that lipids and protein already assemble at their site of synthesis and are then cotransported to the surface (Lee et al 2002). A failure to properly assemble this protein lipid complex results 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 (Bagnat et al 2001;Gong and Chang 2001;Wang and Chang 2002).…”