Multiparametric imaging of adhesive nanodomains at the surface of Candida albicans by atomic force microscopy.

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.

Unexpected role for a serine/threonine-rich domain in the Candida albicans Iff protein family.

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 ß-1,6-glucan network but, remarkably, showed that Iff2/Hyr3 was linked through disulfide bridges or NaOH-labile 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 ß-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 ω site region.

Development of a Vector Set for High or Inducible Gene Expression and Protein Secretion in the Yeast Genus Blastobotrys.

Converting lignocellulosic biomass into value-added products is one of the challenges in developing a sustainable economy. Attempts to engineer fermenting yeasts to recover plant waste are underway. Although intensive metabolic engineering has been conducted to obtain Saccharomyces cerevisiae strains capable of metabolising pentose sugars mainly found in hemicellulose, enzymatic hydrolysis after pretreatment is still required. Blastobotrys raffinosifermentans, which naturally assimilates xylose and arabinose and displays numerous glycoside hydrolases, is a good candidate for direct and efficient conversion of renewable biomass. However, a greater diversity of tools for genetic engineering is needed. Here, we report the characterisation of four new promising promoters, a new dominant marker, and two vectors for the secretion of epitope tagged proteins along with a straightforward transformation protocol. The TDH3 promoter is a constitutive promoter stronger than TEF1, and whose activity is maintained at high temperature or in the presence of ethanol. The regulated promoters respond to high temperature for HSP26, gluconeogenic sources for PCK1 or presence of xylose oligomers for XYL1. Two expression/secretion vectors were designed based on pTEF1 and pTDH3, two endogenous signal peptides from an α-arabinanase and an α-glucuronidase, and two epitopes. A heterologous α-arabinoxylan hydrolase from Apiotrichum siamense was efficiently secreted using these two vectors.

Genome evolution in yeasts.

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.

Exon junction complex components Y14 and Mago still play a role in budding yeast.

Since their divergence from Pezizomycotina, the mRNA metabolism of budding yeasts have undergone regressive evolution. With the dramatic loss of introns, a number of quality control mechanisms have been simplified or lost during evolution, such as the exon junction complex (EJC). We report the identification of the core EJC components, Mago, Y14, and eIF4A3, in at least seven Saccharomycotina species, including Yarrowia lipolytica. Peripheral factors that join EJC, either to mediate its assembly (Ibp160 or Cwc22), or trigger downstream processes, are present in the same species, forming an evolutionary package. Co-immunoprecipitation studies in Y. lipolytica showed that Mago and Y14 have retained the capacity to form heterodimers, which successively bind to the peripheral factors Upf3, Aly/REF, and Pym. Phenotypes and RNA-Seq analysis of EJC mutants showed evidence of Y14 and Mago involvement in mRNA metabolism. Differences in unspliced mRNA levels suggest that Y14 binding either interferes with pre-mRNA splicing or retains mRNA in the nucleus before their export and translation. These findings indicate that yeast could be a relevant model for understanding EJC function.

New components of Yarrowia lipolytica Golgi multi-protein complexes containing the alpha-1,6-mannosyltransferases YlMnn9p and YlAnl1p.

This paper reports the identification and the characterization of two new components of Yarrowia lipolytica Golgi multi-protein complexes. Blast analysis on the Y. lipolytica complete genome allowed us to find a new alpha-1,6-mannosyltransferase, YlAnl2p, which displays an overall identity of 59% and shares a Golgi cellular localization with the previously described YlAnl1p. Moreover, YlAnl2p was shown to directly interact with YlMnn9p using the two-hybrid system suggesting that the two proteins form a second Golgi sub-complex. In order to further elucidate the composition of the Y. lipolytica Golgi complexes containing alpha-1,6-mannosyltransferases, as M-Pol complexes in Saccharomyces cerevisiae, two-hybrid screens were performed using either YlMnn9p or YlAnl1p as bait. A specific partner of YlAnl1p, named YlAni1p was identified. The two proteins were shown to co-localize and co-precipitate in Y. lipolytica. YlAni1p, which displays a coiled-coil domain as Golgin, and YlAnl1p could be involved in the Golgi apparatus maintenance in the yeast Y. lipolytica.

Rbt1 protein domains analysis in Candida albicans brings insights into hyphal surface modifications and Rbt1 potential role during adhesion and biofilm formation.

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 ß-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.

Characterization of Ire1 in the yeast Yarrowia lipolytica reveals an important role for the Sls1 nucleotide exchange factor in unfolded protein response regulation.

Following endoplasmic reticulum (ER) stress, eukaryotic cells trigger a conserved signal transduction pathway called the unfolded protein response (UPR) that regulates the ER's capacity to perform protein folding according to cellular demand. In Saccharomyces cerevisiae, the UPR is initiated by Ire1, a type I transmembrane serine/threonine kinase/endoribonuclease, that senses unfolded protein levels within the ER in collaboration with the ER Hsp70-family member, BiP/Kar2. Here, we report on the characterization of the Yarrowia lipolytica Ire1 ortholog. Our results show that Sls1, a nucleotide exchange factor for BiP, has important functions in regulating ER stress and the interaction of BiP and Ire1. They suggest that Sls1 regulates this interaction, by stimulating the conversion of BiP from the ADP-bound to the ATP-bound state, which favors its interaction with Ire1. Moreover, we identified known and new partners for Ire1 using the Tandem Affinity Purification (TAP) approach.

Identification and characterization of two alpha-1,6-mannosyltransferases, Anl1p and Och1p, in the yeast Yarrowia lipolytica.

In this study, the identification and characterization of the Yarrowia lipolytica homologues of Saccharomyces cerevisiae alpha-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 Delta Ylmnn9, Delta Ylanl1, Delta Yloch1, Delta Ylanl1 Delta Ylmnn9 and Delta Ylmnn9 Delta Yloch1 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, Delta Ylmnn9, Delta Ylanl1, Delta Yloch1, Delta Ylanl1 Delta Ylmnn9 and Delta Ylmnn9 Delta Yloch1 may thus be used to establish a model for the Y. lipolytica N-linked glycosylation pathway.

Sbh1p, a subunit of the Sec61 translocon, interacts with the chaperone calnexin in the yeast Yarrowia lipolytica.

The core component of the translocation apparatus, Sec61p or alpha, 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 beta Sec61 complex subunit, Sbh1p, was cloned by functional complementation of a Deltasbh1, Deltasbh2 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 the beta subunit of the Sec61 translocon may relay folding of nascent proteins to their translocation.