Evidence for Coupled Biogenesis of Yeast Gap1 Permease and Sphingolipids: Essential Role in Transport Activity and Normal Control by Ubiquitination

Abstract

Current models for plasma membrane organization integrate the emerging concepts that membrane proteins tightly associate with surrounding lipids and that biogenesis of surface proteins and lipids may be coupled. We show here that the yeast general amino acid permease Gap1 synthesized in the absence of sphingolipid (SL) biosynthesis is delivered to the cell surface but undergoes rapid and unregulated down-regulation. Furthermore, the permease produced under these conditions but blocked at the cell surface is inactive, soluble in detergent, and more sensitive to proteases. We also show that SL biogenesis is crucial during Gap1 production and secretion but that it is dispensable once Gap1 has reached the plasma membrane. Moreover, the defects displayed by cell surface Gap1 neosynthesized in the absence of SL biosynthesis are not compensated by subsequent restoration of SL production. Finally, we show that down-regulation of Gap1 caused by lack of SL biogenesis involves the ubiquitination of the protein on lysines normally not accessible to ubiquitination and close to the membrane. We propose that coupled biogenesis of Gap1 and SLs would create an SL microenvironment essential to the normal conformation, function, and control of ubiquitination of the permease. INTRODUCTIONBiological membranes were long seen as a "fluid mosaic" of proteins simply dispersed in a homogeneous lipid bilayer (Singer and Nicholson, 1972). This view contrasts with current models of the plasma membrane that take into account the heterogeneity of lipids in length and structure and the very high density of intrinsic proteins, ϳ30,000 per m 2 (Jacobson et al., 2007). These models also integrate the important notion of lateral heterogeneity, i.e., that the plasma membrane contains domains of various lipid compositions and physical properties. Among these domains, lipid rafts have been the subject of many studies. Although the definition of rafts is still debated (Pike, 2006), they are generally viewed as small domains of various sizes (ϳ10 -200 nm in diameter) that are formed by the dynamic assembly of sphingolipids with sterols (Simons and Ikonen, 1997;Jacobson et al., 2007). These domains selectively include or exclude proteins, and they are thought to exist in a liquid-ordered phase distinct from the bulk liquid-disordered phase of the membrane (Brown and London, 1998). Their tightly packed state makes them more resistant to solubilization by nonionic detergents such as Triton X-100, so that they yield detergent-resistant membranes (DRMs) after detergent extraction in the cold (London and Brown, 2000). Despite the limitations of detergent-dependent methods and the caution that must be exerted when interpreting the resulting data, the isolation of DRMs remains a useful tool for investigating protein-lipid interactions and membrane domains (Brown and London, 1998;Shogomori and Brown, 2003;Lichtenberg et al., 2005). A complementary approach consists in visualizing raft components directly in the plasma membrane of living cells. In Saccharomyces ...

The lateral compartmentation of the yeast plasma membrane

“…As described below, several other integral proteins localize to MCC. A number of other plasma membrane proteins, such as hexose trasporter Hxt1 or general amino acid permease Gap1, distribute homogeneously in the membrane [13,17]. Finally, it has been shown that the predominant yeast plasma membrane protein H + -ATPase, Pma1, strictly avoids MCC patches and resides in the area in between them ( Figure 1C).…”

“…Specific lipid requirements were also reported for other plasma membrane proteins. The plasma membrane targeting of Gap1 (normally homogeneously distributed) and of Pma1 (MCP-resident) is impaired in cells defective in long-acyl chains biosynthesis [6,13]. Long chain bases also regulate the phosphorylation of the primary components of the eisosome, Pil1 (phosphorylation inhibited by long chain bases) and Lsp1 (long chain bases stimulate phosphorylation).…”

Thematic Review Series: Sphingolipids. New insights into sphingolipid metabolism and function in budding yeast

“…Another plasma membrane protein, the general amino acid permease Gap1, depends in various ways upon sphingolipids (102). Gap1 synthesized in lcb1-100 cells at a restrictive temperature is transported to the plasma membrane but is not incorporated into detergent-resistant lipid rafts, lacks amino acid transport activity, and is rapidly endocytosed in a ubiquitin-mediated manner.…”

Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

“…Permease misfolding has also been illustrated in the case of the Gap1 permease synthesized under conditions inhibiting sphingolipid biogenesis, and this causes its Ub-dependent downregulation (12). Inhibition by rapamycin of TORC1, as well as various stress conditions likely reducing TORC1 activity, also promotes Gap1 downregulation.…”