Visualization of Protein Compartmentation within the Plasma Membrane of Living Yeast Cells

Abstract

Different distribution patterns of the arginine/H؉ symporter Can1p, the H ؉ plasma membrane ATPase Pma1p, and the hexose transport facilitator Hxt1p within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using fluorescence protein tagging of these proteins. Although Hxt1p-GFP was evenly distributed through the whole cell surface, Can1p-GFP and Pma1p-GFP were confined to characteristic subregions in the plasma membrane. Pma1p is a well-documented raft protein. Evidence is presented that Can1p, but not Hxt1p, is exclusively associated with lipid rafts, too. Double labeling experiments with Can1p-GFP-and Pma1p-RFP-containing cells demonstrate that these proteins occupy two different nonoverlapping membrane microdomains. The size of Can1p-rich (Pma1p-poor) areas was estimated to 300 nm. These domains were shown to be stable in growing cells for >30 min. To our knowledge, this is the first observation of a cell polarization-independent lateral compartmentation in the plasma membrane of a living cell. INTRODUCTIONLipid rafts (detergent-resistant membranes, detergent-insoluble glycolipid-enriched microdomains, or glycolipid-enriched membranes) are dynamic assemblies enriched in sterols and sphingolipids occurring laterally distributed in the plasma membrane of most, if not all, eukaryotes (for reviews, see Simons and Ikonen, 1997;Brown and London, 1998). In mammalian cells cholesterol, sphingomyelin, and glycosphingolipids are the basic constituents of detergentresistant membranes (Rietveld and Simons, 1998), whereas in yeast these are ergosterol, inositolphosphoceramide, and its mannosylated derivatives (Kü bler et al., 1996;Bagnat et al., 2000). Existence of similar plasma membrane domains in plant cells can also be expected, although it has not been reported so far.A distinctive feature of lipid rafts is their insolubility in mild nonionic detergents (typically Triton X-100) at 4°C (Brown and Rose, 1992;Rietveld and Simons, 1998). Consequently, they are found floating in low-density fractions of solubilizates of mild detergent-treated membranes. Rafts obtained by this procedure selectively recruit specific membrane proteins, whereas others are excluded. The recognition of selective protein retention within the detergent-resistant domains led to a concept of lateral subcompartmentation within the plasma membrane (Simons and Ikonen, 1997;Brown and London, 1998).It was proposed that rafts form a platform for lipid (Simons and Ikonen, 2000) and protein sorting and trafficking (Simons and van Meer, 1988;Galbiati et al., 2001;Ikonen, 2001) and cell signaling (Field et al., 1997;Stauffer and Meyer, 1997;Simons and Toomre, 2000;Dykstra et al., 2001). Lipid rafts provide the cells also with a mechanism for functional and spatial control of exocytosis (Chamberlain et al., 2001) and are involved in immune cell activation (for reviews, see Dykstra et al., 2001;Galbiati et al., 2001;Katagiri et al., 2001). Accumulating evidence documents that compositionally distinct lipid microdomains are assembled ...

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).…”

“…The patchy pattern of Can1 distribution is stable for more than 90 min in growing yeast cells [17,18]. These structures thus clearly differ in both the size and the stability from mammalian lipid rafts, described as small entities (∼20-80 nm in diameter) freely diffusing in the plasma membrane [25,33].…”

“…The expression was performed under natural promoters. Details of the experimental procedure for Can1 [7] and Pma1 [17] visualization were published previously. Bar = 5 µm…”

“…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). The networklike compartment occupied by Pma1 is called membrane compartment of Pma1 (MCP) [17]. Spatiotemporal stability of MCC/MCP partitioning contradistinguishes these two membrane domains from other putative membrane compartments, such as transient sites of endocytosis [11] or movable domains postulated to specifically house the TORC2 complex [3].…”

“…The intracellular trafficking of all MCC-specific transporters recognized so far depends on ergosterol biosynthesis [9,17,24,32]. For Can1, in addition, the phosphatidyl ethanolamine was shown to be required for correct delivery of the permease to the plasma membrane [23].…”