Abbreviations used in this paper: LiAc, lithium acetate; MCC, membrane compartment of Can1; mRFP, monomeric red fl uorescent protein.

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 plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping subcompartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H þ -symporters, not however of the Sur7 protein.Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).