In this study, we compared the effects of altered membrane lipid composition on the localization of two membrane drug transporters from different superfamilies of the pathogenic yeast Candida albicans. We demonstrated that in comparison to the major facilitator superfamily multidrug transporter CaMdr1p, ATP-binding cassette transporter CaCdr1p of C. albicans is preferentially localized within detergent-resistant membrane (DRM) microdomains called 'rafts.' Both CaCdr1p and CaMdr1p were overexpressed as green fluorescent protein (GFP)-tagged proteins in a heterologous host Saccharomyces cerevisiae, wherein either sphingolipid (⌬sur4 or ⌬fen1 or ⌬ipt1) or ergosterol (⌬erg24 or ⌬erg6 or ⌬erg4) biosynthesis was compromised. CaCdr1p-GFP, when expressed in the above mutant backgrounds, was not correctly targeted to plasma membranes (PM), which also resulted in severely impaired drug resistance. In contrast, CaMdr1p-GFP displayed no sorting defect in the mutant background and remained properly surface localized and displayed no change in drug resistance. Our data clearly show that CaCdr1p is selectively recruited, over CaMdr1p, to the DRM microdomains of the yeast PM and that any imbalance in the raft lipid constituents results in missorting of CaCdr1p.

Candida albicans, the single most frequently isolated human fungal pathogen, was thought to be asexual until the recent discovery of the mating-type-like locus (MTL). Homozygous MTL strains were constructed and shown to mate. Furthermore, it has been demonstrated that opaque-phase cells are more efficient in mating than white-phase cells. The similarity of the genes involved in the mating pathway in Saccharomyces cerevisiae and C. albicans includes at least one gene (KEX2) that is involved in the processing of the ␣ mating pheromone in the two yeasts. Taking into account this similarity, we searched the C. albicans genome for sequences that would encode the ␣ pheromone gene. Here we report the isolation and characterization of the gene MF␣1, which codes for the precursor of the ␣ mating pheromone in C. albicans. Two active ␣-peptides, 13 and 14 amino acids long, would be generated after the precursor molecule is processed in C. albicans. To examine the role of this gene in mating, we constructed an mf␣1 null mutant of C. albicans. The mf␣1 null mutant fails to mate as MTL␣, while MTLa mf␣1 cells are still mating competent. Experiments performed with the synthetic ␣-peptides show that they are capable of inducing growth arrest, as demonstrated by halo tests, and also induce shmooing in MTLa cells of C. albicans. These peptides are also able to complement the mating defect of an MTL␣ kex2 mutant strain when added exogenously, thereby confirming their roles as ␣ mating pheromones.Candida albicans is the most common opportunistic fungal pathogen of humans and mostly infects immunosuppressed patients (3). It inhabits diverse niches, which include the gastrointestinal tract and the vagina, and causes infection of skin, mucous membranes, and the bloodstream. Since each of these sites of infection presumably requires differences in gene expression, a great deal of effort has been spent in looking at how such adaptation occurs in Candida. Much of this effort was predicated on the characterization of C. albicans as an asexual obligate diploid.That this fungus is asexual was disproved after the C. albicans genome sequencing project revealed the presence of sequences homologous to the Saccharomyces cerevisiae MAT (mating-type) loci. The C. albicans homologues of the MAT loci, the MTL (mating-type-like) loci, were found to be heterozygous in common laboratory strains tested (17). Strains homozygous for the MTL locus were generated either by a deletion strategy (18) or by loss, induced by growth on sorbose as the sole carbon source (19), of one homologue of chromosome 5, the site of the MTL loci (29). Strains thus constructed were able to mate both under laboratory conditions (29) and in the animal host (18). These studies set the stage for dissecting the mating pathway in C. albicans.Mating in fungi has been examined in both yeasts and molds of various sorts. Certain general parts of the process seem to hold across genera and indeed across the kingdom. These include dissimilar regulatory genes (mating-type loci), soluble phe...

dMitochondrial dysfunction in Candida albicans is known to be associated with drug susceptibility, cell wall integrity, phospholipid homeostasis, and virulence. In this study, we deleted CaFZO1, a key component required during biogenesis of functional mitochondria. Cells with FZO1 deleted displayed fragmented mitochondria, mitochondrial genome loss, and reduced mitochondrial membrane potential and were rendered sensitive to azoles and peroxide. In order to understand the cellular response to dysfunctional mitochondria, genome-wide expression profiling of fzo1⌬/⌬ cells was performed. Our results show that the increased susceptibility to azoles was likely due to reduced efflux activity of CDR efflux pumps, caused by the missorting of Cdr1p into the vacuole. In addition, fzo1⌬/⌬ cells showed upregulation of genes involved in iron assimilation, in iron-sufficient conditions, characteristic of iron-starved cells. One of the consequent effects was downregulation of genes of the ergosterol biosynthesis pathway with a commensurate decrease in cellular ergosterol levels. We therefore connect deregulated iron metabolism to ergosterol biosynthesis pathway in response to dysfunctional mitochondria. Impaired activation of the Hog1 pathway in the mutant was the basis for increased susceptibility to peroxide and increase in reactive oxygen species, indicating the importance of functional mitochondria in controlling Hog1-mediated oxidative stress response. Mitochondrial phospholipid levels were also altered as indicated by an increase in phosphatidylserine and phosphatidylethanolamine and decrease in phosphatidylcholine in fzo1⌬/⌬ cells. Collectively, these findings reinforce the connection between functional mitochondria and azole tolerance, oxidant-mediated stress, and iron homeostasis in C. albicans.

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triggers the induction of a number of nuclear genes, including CIT2 and DLD3, via activation of the transcriptional regulatory proteins Rtg1p and Rtg3p (4 -6). A major function of the Rtg1p/Rtg3p-regulated retrograde signaling pathway is to permit adequate synthesis of amino acids that rely on the tricarboxylic acid cycle for their production.