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

Abstract

The proton-pumping H ؉ -ATPase, Pma1p, is an abundant and very long lived polytopic protein of the yeast plasma membrane. Pma1p constitutes a major cargo of the secretory pathway and thus serves as a model to study plasma membrane biogenesis. Pma1p associates with detergent-resistant membrane domains (lipid "rafts") already in the ER, and a lack of raft association correlates with mistargeting of the protein to the vacuole, where it is degraded. We are analyzing the role of specific lipids in membrane domain formation and have previously shown that surface transport of Pma1p is independent of newly synthesized sterols but that sphingolipids with C26 very long chain fatty acid are crucial for raft association and surface transport of Pma1p (Gaigg, B., Timischl, B., Corbino, L., and Schneiter, R. (2005) J. Biol. Chem. 280, 22515-22522). We now describe a more detailed analysis of the function that sphingolipids play in this process. Using a yeast strain in which the essential function of sphingolipids is substituted by glycerophospholipids containing C26 very long chain fatty acids, we find that sphingolipids per se are dispensable for raft association and surface delivery of Pma1p but that the C26 fatty acid is crucial. We thus conclude that the essential function of sphingolipids for membrane domain formation and stable surface delivery of Pma1p is provided by the C26 fatty acid that forms part of the yeast ceramide.

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

“…Remarkably, analysis of Pma1 stability in this suppressor strain revealed that newly synthesized ATPase is stably delivered to the cell surface and that it acquires detergent resistance [34]. Shortening the C26 fatty acid on these suppressor lipids by means of an elo3D mutation, however, neutralized the suppressor activity of these lipids, resulting in the rapid turnover of Pma1 and the newly synthesized Pma1 failed to acquire detergent resistance [34]. These results thus strongly indicate that lipids containing C26 fatty acids, either ceramide or glycerophospholipid bound are important for stable biogenesis and raft association of Pma1.…”

“…These C26-containing PIs thus replace the essential function of sphingolipids and structurally and functionally mimic sphingolipids [33]. Remarkably, analysis of Pma1 stability in this suppressor strain revealed that newly synthesized ATPase is stably delivered to the cell surface and that it acquires detergent resistance [34]. Shortening the C26 fatty acid on these suppressor lipids by means of an elo3D mutation, however, neutralized the suppressor activity of these lipids, resulting in the rapid turnover of Pma1 and the newly synthesized Pma1 failed to acquire detergent resistance [34].…”

The lateral compartmentation of the yeast plasma membrane

“…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).…”

The role of lipids in the biogenesis of integral membrane proteins

“…These C26-containing PIs thus replace the essential function of sphingolipids and structurally and functionally mimic sphingolipids (Lester et al 1993). Remarkably, analysis of Pma1 stability in this suppressor strain revealed that newly synthesized ATPase acquires detergent resistance and that it is stably delivered to the cell surface (Gaigg et al 2006). Shortening the C26 fatty acid on these suppressor lipids by means of an elo3Δ mutation, however, neutralized the suppressor activity of these lipids and resulted in the rapid turnover of the newly synthesized Pma1 (Gaigg et al 2006).…”