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We report here that in addition to a cytoplasmic copper-zinc-containing superoxide dismutase (SOD) and a mitochondrial manganese-containing SOD, Candida albicans expresses a third SOD gene (SOD3). The deduced amino acid sequence contains all of the motifs found in previously characterized manganese-containing SODs, except the presence of a mitochondrial transit peptide. Recombinant Sod3p expressed and purified from Escherichia coli is a homotetramer with a subunit mass of 25.4 kDa. Mass absorption spectrometry detected the presence of both iron and manganese in purified Sod3p but, as determined by metal replacement experiments, the enzyme displays activity only when bound to manganese. Overexpression of SOD3 was shown to rescue the hypersensitivity to redox cycling agents of a Saccharomyces cerevisiae mutant lacking the cytoplasmic copper-zinc-containing SOD. Northern blot analyses showed that the transcription of SOD3 is induced neither by the transition from the yeast to the mycelial form of C. albicans nor by drug-induced oxidative stress. In continuous cultures, the expression of SOD3 was strongly stimulated upon the entry and during the stationary phase, concomitantly with the repression of SOD1. We conclude that Sod3p is an atypical cytosolic manganese-containing superoxide dismutase that is involved in the protection of C. albicans against reactive oxygen species during the stationary phase.

Manganese-dependent superoxide dismutase 2 (SOD2) in the mitochondria plays a key role in protection against oxidative stress. Here we probed the pathway by which SOD2 acquires its manganese catalytic cofactor. We found that a mitochondrial localization is essential. A cytosolic version of Saccharomyces cerevisiae Sod2p is largely apo for manganese and is only efficiently activated when cells accumulate toxic levels of manganese. Furthermore, Candida albicans naturally produces a cytosolic manganese SOD (Ca SOD3), yet when expressed in the cytosol of S. cerevisiae, a large fraction of Ca SOD3 also remained manganese-deficient. The cytosol of S. cerevisae cannot readily support activation of Mn-SOD molecules. By monitoring the kinetics for metalation of S. cerevisiae Sod2p in vivo, we found that prefolded Sod2p in the mitochondria cannot be activated by manganese. Manganese insertion is only possible with a newly synthesized polypeptide. Furthermore, Sod2p synthesis appears closely coupled to Sod2p import. By reversibly blocking mitochondrial import in vivo, we noted that newly synthesized Sod2p can enter mitochondria but not a Sod2p polypeptide that was allowed to accumulate in the cytosol. We propose a model in which the insertion of manganese into eukaryotic SOD2 molecules is driven by the protein unfolding process associated with mitochondrial import.

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a approximately 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.

A novel class of aspartic peptidases known as fungal yapsins, whose first member ScYps1p was identified more than a decade ago in Saccharomyces cerevisiae, is characteristically modified by the addition of a glycophosphatidylinositol moiety and has a preference for cleaving substrates C-terminally to mono- and paired-basic residues. Over the years, several other members, first in S. cerevisiae and then in other fungi, have been identified. The implication of fungal yapsins in cell-wall assembly and/or remodelling had been suspected for many years. However, it is only very recently that studies performed on S. cerevisae and Candida albicans have confirmed their importance for cell-wall integrity. Here, we review 16 years of research, covering all fundamental aspects of these unique enzymes, in an effort to track their functional significance. We also propose a nomenclature for fungal yapsins based on their sequence identity with the founding members of this family, the S. cerevisiae yapsins.

SummaryYeast cell wall assembly is a highly regulated and dynamic process. A class of cell surface aspartic peptidases anchored by a glycosylphosphatidylinositol (GPI) group, collectively known as yapsins, was proposed to be involved in cell wall construction. The Saccharomyces cerevisiae Yps1p, the prototypal yapsin, is processed internally within a loop region to produce an a/b two-subunit enzyme. Here we investigated the activation mechanism of GPI-anchored Yps1p and identified some of its substrates. We report that all activation steps of GPI-Yps1p take place at the cell surface and are regulated by the environmental pH. GPI-Yps1p is active in vivo at pH 6.0 and pH 3.0 and functions as a sheddase for a subset of GPI-anchored enzymes, including itself and the Gas1 glucanosyltransferase. Importantly, while native GPI-Yps1p weakly suppresses many phenotypes associated with the yeast kex2D mutant, loop mutants that interfere with conversion into the two-subunit enzyme restore the kex2D phenotypes to near wild type level. We propose that cleavage of this internal loop region plays an important regulatory function through stimulating its shedding activity. Collectively, our data provide a direct link between the pH regulation of yeast cell wall assembly and the activity of a yapsin.

The mycelial surface antigen recognized by monoclonal antibody (mAb) 4E1 has previously been shown to be present predominantly in the terminal third of the hyphal structures in Candida albicans. We report here the expression cloning of the corresponding gene (CSA1 ) by mAb 4E1‐coated magnetic beads sorting of Saccharomyces cerevisiae transformants expressing a C. albicans genomic library. The strategy is both highly selective and highly sensitive and provides an additional genetic tool for the cloning and characterization of C. albicans genes encoding surface proteins. CSA1 is an intronless gene encoding a 1203‐residue protein composed of repetitive motifs and domains. Northern analysis indicates that CSA1 is preferentially expressed during the mycelial growth phase, although a low level of CSA1 mRNA can be detected in the yeast form. As evidenced by indirect immunofluorescence microscopy with mAb 4E1, Csa1p is not randomly distributed over the surface of yeast cells, but localizes predominantly in the growing buds. This suggests that the distribution of Csa1p may be restricted to sites of cell surface elongation. Both heterozygous and homozygous C. albicans csa1Δ mutants are viable. Upon induction of mycelial growth, the number and size of hyphal structures derived from the mutants are similar to those observed in the parental wild‐type strain. The physiological role of Csa1p has yet to be determined. However, the presence in Csa1p of repeated cysteine‐rich hydrophobic domains with significant sequence similarity to motifs found in surface proteins (Ag2 and Pth11) from two distantly related fungal pathogens (Coccidioides immitis and Magnaporthe grisea respectively) suggests a common function in host interaction.

The peptide somatostatin exists as two different molecular species. In addition to the most common form, somatostatin‐14, there is also a fourteen amino acid N‐terminally extended form of the tetradecapeptide, somatostatin‐28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which upon cleavage generate either somatostatin‐14 or −28, respectively. In some species of fish two distinct, but homologous, precursors (prosomatostatin‐I and ‐II) give rise to somatostatin‐14 and −28, respectively. Whereas anglerfish prosomatostatin‐II was previously shown to release exclusively somatostatin‐28, the yeast Saccharomyces cerevisiae proteolytically matures the homologous prosomatostatin‐I precursor to somatostatin‐28 and −14 as well as to a lysine‐extended form of somatostatin‐14. The Kex2 endoprotease appears to be essential for the formation of lysine somatostatin‐14 and is involved either directly or indirectly in the release of mature somatostatin‐14. The isolation of yeast mutants defective in somatostatin‐28 expression (sex mutant) allowed the cloning of a non‐essential gene, which encodes an aspartyl protease, whose disruption severely affects the cleavage of mature somatostatin‐28 from both somatostatin precursors. We conclude that two distinct endoproteases, which demonstrate some cross specificity in vivo, are involved in the proteolytic maturation of prosomatostatin at mono‐ and dibasic processing sites in yeast.