C Terminus of Nce102 Determines the Structure and Function of Microdomains in the Saccharomyces cerevisiae Plasma Membrane

Abstract

The plasma membrane of the yeast Saccharomyces cerevisiae contains stably distributed lateral domains of specific composition and structure, termed MCC (membrane compartment of arginine permease Can1). Accumulation of Can1 and other specific proton symporters within MCC is known to regulate the turnover of these transporters and is controlled by the presence of another MCC protein, Nce102. We show that in an NCE102 deletion strain the function of Nce102 in directing the specific permeases into MCC can be complemented by overexpression of the NCE102 close homolog FHN1 (the previously uncharacterized YGR131W) as well as by distant Schizosaccharomyces pombe homolog fhn1 (SPBC1685.13). We conclude that this mechanism of plasma membrane organization is conserved through the phylum Ascomycota. We used a hemagglutinin (HA)/Suc2/His4C reporter to determine the membrane topology of Nce102. In contrast to predictions, its N and C termini are oriented toward the cytosol. Deletion of the C terminus or even of its last 6 amino acids does not disturb protein trafficking, but it seriously affects the formation of MCC. We show that the C-terminal part of the Nce102 protein is necessary for localization of both Nce102 itself and Can1 to MCC and also for the formation of furrow-like membrane invaginations, the characteristic ultrastructural feature of MCC domains.Stable lateral domains coexist within the plasma membrane of the yeast Saccharomyces cerevisiae. Nce102, a protein originally thought to be involved in nonclassical export (6) and more recently in sensing sphingolipids (10), is the main organizer of one type of these domains, termed MCC (membrane compartment of Can1) (25). MCC consists of evenly distributed, isolated patches enriched in sterols and specific proteins (15,16,25,26). We showed that MCC-specific proton symporters accumulate in these patches in a reversible, membrane potential-dependent manner. This Nce102-mediated transient MCC accumulation plays a key role in the turnover of the transporters (16). Each MCC patch is accompanied by an eisosome, a cytosolic complex located directly beneath the membrane (36).In an early freeze-etching study, Moor and Mühlethaler (28) demonstrated that the yeast plasma membrane contains numerous furrow-like invaginations. Recently, MCC patches were identified with these plasma membrane structures, and Nce102 was shown to be necessary for furrow formation. On the ultrastructural level, the MCC patches of nce102⌬ cells appeared as flat, smooth, elongated areas within an otherwise particle-rich plasma membrane (32).There is now increasing evidence that cytosolic Pil1, a primary component of eisosomes, is a prerequisite for MCC patch formation. It marks the sites where Nce102 and the MCCspecific transporters will subsequently accumulate (16,23,29). Data published so far do not indicate a direct involvement of cytoskeletal components in this process (26). Accordingly, markers of classical endocytosis, which are coupled to the cortical patches of actin, were localized outside the MCC ...

Formation and stability of eisosomes in the filamentous fungus Ashbya gossypii

“…Homologues of the main components, Pil1 and Lsp1, are conserved, but genes encoding other potential eisosome components were apparently lost. Several functionally partially redundant components, such as Slm1 and Slm2 (Fadri et al, 2005), Pkh1 and Pkh2 (Casamayor et al, 1999;Luo et al, 2008;Walther et al, 2007), and Nce102 and Fhn1 (Loibl et al, 2010), are encoded by only one gene in A. gossypii. Likewise only a single syntenic gene is present in the A. gossypii genome for the fairly uncharacterized twin genes YMR031c and YKL050c, and YMR086w and YKL105c (Deng et al, 2009;Frohlich et al, 2009).…”

“…The transmembrane protein ScNce102 colocalizes with eisosomes and is important to maintain eisosome density, most likely by controlling the plasma membrane organization through sphingolipid signaling (Frohlich et al, 2009). The S. cerevisiae genome carries a duplicated copy of the ScNCE102 gene, called ScFHN1, which, when overexpressed, can complement the phenotype of a ScNCE102 deletion (Loibl et al, 2010). The A. gossypii genome encodes only one homologue and a deletion mutant shows normal polar growth efficiency, and normal eisosome and actin patch distribution (Fig.…”

Topological and Mutational Analysis of Saccharomyces cerevisiae Fks1

“…This method has been used for topological analysis of numerous membrane proteins, including Pmt1 (43), Stt3 (22), Nce102 (27), and Arv1 (47). We combined this method with in silico analyses to generate a model of S. cerevisiae Fks1 topology.…”

Eisosome Ultrastructure and Evolution in Fungi, Microalgae, and Lichens

“…Of the two CAN1 homologues in C. reinhardtii, AOC5 is strongly upregulated in eisosome-free vegetative cells with nitrogen starvation (77), as are other arginine transporters, whereas AOC6 is expressed at constant low levels (ϳ10 reads per kilobase per million mapped reads); expression levels in eisosome-forming C. reinhardtii zygotes have not been evaluated. Sur7p fails to colocalize with eisosomes in S. pombe, whereas Nce102p/Fhn1 is important for their integrity (43), as is also the case in S. cerevisiae (31). Sequences homologous to Sur7p and to the conserved Marvel domain of Nce102p were found in fungi and metazoa but not in any algae.…”

“…Hence, the current yeast model (25,26) proposes that Pil1p and Lsp1p, which contain membrane curvatureinducing BAR domains (6,27,28), together with Seg1p (29,30), form a submembrane complex (6) reportedly influenced by Pil1p phosphorylation (25); this complex then either creates or associates with the MCC domains, presumably inducing an inward curvature. The MCC protein Nce102p has also been implicated in generating curvature (5,31).…”