Identifying the mechanisms of eukaryotic genome evolution by comparative genomics is often complicated by the multiplicity of events that have taken place throughout the history of individual lineages, leaving only distorted and superimposed traces in the genome of each living organism. The hemiascomycete yeasts, with their compact genomes, similar lifestyle and distinct sexual and physiological properties, provide a unique opportunity to explore such mechanisms. We present here the complete, assembled genome sequences of four yeast species, selected to represent a broad evolutionary range within a single eukaryotic phylum, that after analysis proved to be molecularly as diverse as the entire phylum of chordates. A total of approximately 24,200 novel genes were identified, the translation products of which were classified together with Saccharomyces cerevisiae proteins into about 4,700 families, forming the basis for interspecific comparisons. Analysis of chromosome maps and genome redundancies reveal that the different yeast lineages have evolved through a marked interplay between several distinct molecular mechanisms, including tandem gene repeat formation, segmental duplication, a massive genome duplication and extensive gene loss.

Signal recognition particle-dependent targeting of secretory proteins to the endoplasmic reticulum membrane is predominant in the yeast Yarrowia lipolytica. A conditional lethal mutant of the SCR2-encoded 7S RNA provided the first in vivo evidence for involvement of this particle in cotranslational translocation (He, F., Beckerich, J. M., and Gaillardin, C. M. (1992) J. Biol. Chem. 267, 1932Chem. 267, -1937. In order to identify partners of 7S RNA or signal recognition particle in their function, we selected synthetic lethal mutations with the 7S RNA mutation (sls). The SLS1 gene, cloned by complementation of the sls1 mutant growth defect, encodes a 426-amino acid polypeptide containing a NH 2 -terminal signal peptide and a COOH-terminal endoplasmic reticulum (ER) retention motif. The SLS1 gene product behaves as a lumenal protein of the ER. Sls1p was sedimented with membrane-rich organelles and was resistant to protease degradation without prior membrane solubilization. Immunofluorescence microscopy showed a typical endoplasmic reticulum perinuclear staining. Co-immunoprecipitation revealed that Sls1p resides close to the major translocation apparatus component, Sec61p. Deletion of the SLS1 gene led to a temperaturesensitive growth phenotype. Synthesis of several secretory proteins was shown to be specifically reduced in ⌬sls1 cells. We propose that Sls1p acts in the preprotein translocation process, interacting directly with translocating polypeptides to facilitate their transfer and/or help their folding in the ER.In order to enter the secretion pathway, secretory proteins of eukaryotic cells have to be transported across or inserted into the endoplasmic reticulum (ER) 1 membrane. To achieve this translocation step, secretory proteins must be specifically targeted to the translocation machinery in the ER membrane and be competent for crossing this membrane (2). In higher eukaryotes, the signal recognition particle (SRP) was shown to take part into these functions (3). SRP is composed of a single 7S RNA and six polypeptides (4). When the signal sequence of a nascent secretory polypeptide is extruded from the ribosome, it is first recognized by the nascent polypeptide associating complex (5), which allows specific binding of SRP. Interaction of SRP with the nascent chain-ribosome complex causes translational slow down. After binding of SRP to its membranebound receptor, SRP is displaced from the complex and the nascent chain is transferred to the translocation site where crossing takes place simultaneously to translation. As soon as the polypeptide emerges in the lumen of the ER, it interacts with various proteins for processing and folding. A somewhat different picture emerged from studies on the yeast Saccharomyces cerevisiae. Indeed, several secretory proteins in this yeast appeared to be transported post-translationally, both in vivo and in vitro (6, 7), and homologues of mammalian SRP components that have been identified in this yeast and function in translocation (8 -11) are not essential for cell viabil...

Glycosylphosphatidylinositol (GPI)-anchored proteins are an important class of cell wall proteins in Candida albicans because of their localization and their function, even if more than half of them have no characterized homolog in the databases. In this study, we focused on the IFF protein family, investigating their exposure on the cell surface and the sequences that determine their subcellular localization. Protein localization and surface exposure were monitored by the addition of a V5 tag on all members of the family. The data obtained using the complete proteins showed for Iff3 (or -9), Iff5, Iff6, and Iff8 a covalent linkage to the ␤-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOHlabile bonds. However, since some proteins of the Iff family were undetectable, we designed chimeric constructions using the last 60 amino acids of these proteins to test the localization signal. These constructions showed a ␤-1,6-glucan linkage for Iff1/Rbr3, Iff2/Hyr3, Iff4 and Iff7/Hyr4 C-terminal-Iff5 fusion proteins, and a membrane localization for the Iff10/Flo9 C terminus-Iff5 fusion protein. Immunofluorescence analyses coupled to these cell fraction data confirmed the importance of the length of the central serine/threonine-rich region for cell surface exposure. Further analysis of the Iff2/Hyr3 linkage to the cell surface showed for the first time that a serine/threonine central region of a GPI-anchored protein may be responsible for the disulfide and the NaOH bonds to the glucan and glycoproteins network and may also override the signal of the proximal site region.

The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A sitedirected mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.To initiate their pathway, secretory proteins are first targeted to the endoplasmic reticulum (ER) 1 membrane in eukaryotic organisms. In mammalian cells, a cytoplasmic particle (the Signal Recognition Particle or SRP) first recognizes these proteins when the signal peptide emerges from the ribosome and then ensures their delivery to the ER membrane through its interaction with the SRP receptor (1). SRP binding to the nascent chain-ribosome complex causes a translational pause that is released upon docking of SRP (2). As translation resumes, the complex is transferred to the translocation site where crossing through the ER membrane takes place. Sec61␣, one of the three polypeptides of the translocation pore, is a polytopic membrane protein; the two others, Sec61␤ and ␥, span the ER membrane once. Three to four units of this heterooligomer are needed to form the aqueous pore (3). In vitro reconstitution experiments, using purified mammalian components, show that the SRP receptor and the Sec61 complex are sufficient to achieve translocation of some preproteins, while the TRAM protein is required for translocation of other preproteins (4). In vivo, many other soluble or membrane proteins could be involved in this process to adjust the translocation rate to cell growth.In the model yeast, Saccharomyces cerevisiae, components involved in SRP-dependent targeting are not essential, and several secretory proteins were shown to cross the ER membrane post-translationally (5, 6). This translocation mode relies both on cytosolic chaperones whose binding delays preproteins folding (7) and on membrane proteins that ensure specific insertion of secretory proteins at the translocation site. A heptameric complex containing the trimeric Sec61 complex and four other polypeptides, Sec62p, Sec63p, Sec71p, and Sec72p, allows in vitro post-translational translocation of several pr...

Candida albicans is an opportunistic pathogen. It adheres to mammalian cells through a variety of adhesins that interact with host ligands. The spatial organization of these adhesins on the cellular interface is however poorly understood, mainly because of the lack of an instrument able to track single molecules on single cells. In this context, the atomic force microscope (AFM) makes it possible to analyze the force signature of single proteins on single cells. The present study is dedicated to the mapping of the adhesive properties of C. albicans cells. We observed that the adhesins at the cell surface were organized in nanodomains composed of free or aggregated mannoproteins. This was demonstrated by the use of functionalized AFM tips and synthetic amyloid forming/disrupting peptides. This direct visualization of amyloids nanodomains will help in understanding the virulence factors of C. albicans.

The core component of the translocation apparatus, Sec61p or α, was previously cloned in Yarrowia lipolytica. Using anti-Sec61p antibodies, we showed that most of the translocation sites are devoted to co-translational translocation in this yeast, which is similar to the situation in mammalian cells but in contrast to the situation in Saccharomyces cerevisiae, where post-translational translocation is predominant. In order to characterize further the minimal translocation apparatus in Y. lipolytica, the β Sec61 complex subunit, Sbh1p,was cloned by functional complementation of a Δsbh1,Δ sbh2 S. cerevisiae mutant. The secretion of the reporter protein is not impaired in the Y. lipolytica sbh1 inactivated strain. We screened the Y. lipolytica two-hybrid library to look for partners of this translocon component. The ER-membrane chaperone protein, calnexin, was identified as an interacting protein. By a co-immunoprecipitation approach, we confirmed this association in Yarrowia and then showed that the S. cerevisiae Sbh2p protein was a functional homologue of YlSbh1p. The interaction of Sbh1p with calnexin was shown to occur between the lumenal domain of both proteins. These results suggest that theβ subunit of the Sec61 translocon may relay folding of nascent proteins to their translocation.

Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae α-1,6-mannosyltransferases Anp1p and Och1p, designated YlAnl1p and YlOch1p, are described. In order to confirm the function of the Y. lipolytica proteins, including the previously isolated YlMnn9p, in the N-glycosylation pathway, a phenotypic analysis of the disrupted strains ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9 and ΔYlmnn9ΔYloch1 was performed. Disruption of the YlMNN9, YlANL1 and YlOCH1 genes caused an increased sensitivity to SDS, compatible with a glycosylation defect, and to Calcofluor White, characteristic of cell-wall defects. Moreover, Western-blot analysis of a heterologous glycosylated protein confirmed a direct role of YlMnn9p and YlAnl1p in the N-glycosylation process. These mutant strains, ΔYlmnn9, ΔYlanl1, ΔYloch1, ΔYlanl1ΔYlmnn9 and ΔYlmnn9ΔYloch1 may thus be used to establish a model for the Y. lipolytica N-linked glycosylation pathway.