Proteomic Analysis of Rta2p-Dependent Raft-Association of Detergent-Resistant Membranes in Candida albicans

In Candida albicans, lipid rafts (also called detergent-resistant membranes, DRMs) are involved in many cellular processes and contain many important proteins. In our previous study, we demonstrated that Rta2p was required for calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Here, we found that Rta2p was co-localized with raft-constituted ergosterol on the plasma membrane of C. albicans. Furthermore, this membrane expression pattern was totally disturbed by inhibitors of either ergosterol or sphingolipid synthesis. Biochemical fractionation of DRMs together with immunoblot uncovered that Rta2p, along with well-known DRM-associated proteins (Pma1p and Gas1p homologue), was associated with DRMs and their associations were blocked by inhibitors of either ergosterol or sphingolipid synthesis. Finally, we used the proteomic analysis together with immunoblot and identified that Rta2p was required for the association of 10 proteins with DRMs. These 5 proteins (Pma1p, Gas1p homologue, Erg11p, Pmt2p and Ali1p) have been reported to be DRM-associated and also that Erg11p is a well-known target of azoles in C. albicans. In conclusion, our results showed that Rta2p was predominantly localized in lipid rafts and was required for the association of certain membrane proteins with lipid rafts in C. albicans

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

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

Characterization of AnNce102 and its role in eisosome stability and sphingolipid biosynthesis

The plasma membrane is implicated in a variety of functions, whose coordination necessitates highly dynamic organization of its constituents into domains of distinct protein and lipid composition. Eisosomes, at least partially, mediate this lateral plasma membrane compartmentalization. In this work, we show that the Nce102 homologue of Aspergillus nidulans colocalizes with eisosomes and plays a crucial role in density/number of PilA/SurG foci in the head of germlings. In addition we demonstrate that AnNce102 and PilA negatively regulate sphingolipid biosynthesis, since their deletions partially suppress the thermosensitivity of basA mutant encoding sphingolipid C4-hydroxylase and the growth defects observed upon treatment with inhibitors of sphingolipid biosynthesis, myriocin and Aureobasidin A. Moreover, we show that YpkA repression mimics genetic or pharmacological depletion of sphingolipids, conditions that induce the production of Reactive Oxygen Species (ROS), and can be partially overcome by deletion of pilA and/or annce102 at high temperatures. Consistent with these findings, pilA " and annce102 " also show differential sensitivity to various oxidative agents, while AnNce102 overexpression can bypass sphingolipid depletion regarding the PilA/SurG foci number and organization, also leading to the mislocalization of PilA to septa.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Eisosome Organization in the Filamentous AscomyceteAspergillus nidulans▿†

Eisosomes are subcortical organelles implicated in endocytosis and have hitherto been described only in Saccharomyces cerevisiae. They comprise two homologue proteins, Pil1 and Lsp1, which colocalize with the transmembrane protein Sur7. These proteins are universally conserved in the ascomycetes. We identify in Aspergillus nidulans (and in all members of the subphylum Pezizomycotina) two homologues of Pil1/Lsp1, PilA and PilB, originating from a duplication independent from that extant in the subphylum Saccharomycotina. In the aspergilli there are several Sur7-like proteins in each species, including one strict Sur7 orthologue (SurG in A. nidulans). In A. nidulans conidiospores, but not in hyphae, the three proteins colocalize at the cell cortex and form tightly packed punctate structures that appear different from the clearly distinct eisosome patches observed in S. cerevisiae. These structures are assembled late during the maturation of conidia. In mycelia, punctate structures are present, but they are composed only of PilA, while PilB is diffused in the cytoplasm and SurG is located in vacuoles and endosomes. Deletion of each of the genes does not lead to any obvious growth phenotype, except for moderate resistance to itraconazole. We could not find any obvious association between mycelial (PilA) eisosome-like structures and endocytosis. PilA and SurG are necessary for conidial eisosome organization in ways that differ from those for their S. cerevisiae homologues. These data illustrate that conservation of eisosomal proteins within the ascomycetes is accompanied by a striking functional divergence