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Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

A conserved ambient pH signal transduction pathway has been evidenced in both ascomycetous yeasts and filamentous fungi, called the Rim or Pal pathway, respectively. However, closely related PalC orthologues are found only in Yarrowia lipolytica and in filamentous fungi, where the Rim9p/Pall factor has a much longer C-terminal tail than in other yeasts. We show here that, like Aspergillus nidulans pall mutants, a Ylrim9[DELTA] mutant has a less extreme phenotype than other mutants of the pathway, whereas rim9 mutants in Saccharomyces cerevisiae and Candida albicans reportedly exhibit a tight Rim phenotype. Deletion of the long C-terminal tail of YIRim9p/ Pall had no phenotypic effect on ambient pH signalling. We also show that the Y. lipolytica PalC orthologue, named YIRim23p, is absolutely required for the alkaline pH response. Its only interactant identified in a genome-wide two-hybrid screen is YISnfT/Vps32p, confirming the link between the Rim and the Vps pathways. YIRim13p and YIRim20p both interact with YISnf7/ Vps32p but not with YIRim23p. The long C-terminal tail of YIRim9p/Pall interacts neither with YIRim23p nor with YISnf7/Vps32p. These results show that YIRim23p is a bona fide component of the Rim pathway in Y. lipolytica and that it participates in the complexes linking pH signalling and endocytosis.

Ambient pH signalling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Insertional mutagenesis in the yeast Yarrowia lipolytica identified two components of the endosome-associated ESCRT-I complex involved in multivesicular body (MVB) vesicle formation, YIVps28p and YIVps23p. They were shown to be required at alkaline pH, like Rim factors, for transcriptional activation of alkaline-induced genes and repression of acid-induced genes. The constitutively active YlRIM101-1119 allele, which suppresses the pH-signalling defects of Ylrim mutations, also suppresses Ylvps defects in pH response, but not in endocytosis. The contribution of the ESCRT-III component Snf7p could not be assessed due to the essential nature of this component in Y. lipolytica. Unlike Rim factors, YIVps4p, a component of the MVB pathway acting downstream from ESCRT complexes, seems not to be required for the alkaline response. In Y. lipolytica, all vps mutations including those affecting YIVPS4, affected growth at acidic pH, a feature not exhibited by Ylrim mutations. These results suggest that Rim and Vps pathways cooperate in ambient pH signalling and that this relation is conserved across the full range of hemiascomycetous yeasts.

Author(s): Bernard Dujon (corresponding author) [1]; David Sherman [5, 6]; Gilles Fischer [1]; Pascal Durrens [6, 7]; Serge Casaregola [8]; Ingrid Lafontaine [1]; Jacky de Montigny [9]; Christian Marck [10]; [...]

Research has been conducted on the glycosylphosphatidylinositol (GPI)-anchoring, a mechanism for attaching proteins to cell surface. The role of the Gpi7p side chain in anchoring of the GPI-modified proteins which target cell walls has been investigated and the results are presented.

Depending on the pH of the growth medium, the yeast Yarrowia lipolytica secretes an acidic protease or an alkaline protease, the synthesis of which is also controlled by carbon, nitrogen, and sulfur availability, as well as by the presence of extracellular proteins. Previous results have indicated that the alkaline protease response to pH was dependent on YlRim101p, YlRim8p/YlPalF, and YlRim21p/YlPalH, three components of a conserved pH signaling pathway initially described in Aspergillus nidulans. To identify other partners of this response pathway, as well as pH-independent regulators of proteases, we searched for mutants that affect the expression of either or both acidic and alkaline proteases, using a YlmTn1-transposed genomic library. Four mutations affected only alkaline protease expression and identified the homolog of Saccharomyces cerevisiae SIN3. Eighty-nine mutations affected the expression of both proteases and identified 10 genes. Five of them define a conserved Rim pathway, which acts, as in other ascomycetes, by activating alkaline genes and repressing acidic genes at alkaline pH. Our results further suggest that in Y. lipolytica this pathway is active at acidic pH and is required for the expression of the acidic AXP1 gene. The five other genes are homologous to S. cerevisiae OPT1, SSY5, VPS28, NUP85, and MED4. YlOPT1 and YlSSY5 are not involved in pH sensing but define at least a second protease regulatory pathway.

Anti-Ras single-chain FV antibody fragments are constructed from secretion vectors of the two yeast species. Results show that both species secrete soluble and functional anti-Ras single-chain Fv antibody fragments and possess the potential for industrial scale production of this recombinant product.

Research has been conducted on the yeast Yarrowia lipolytica. The integrative vectors for gene expression in Y. lipolytica which carry long terminal repeat of the Y. lipolytica retrotransposon have been developed and described.

Research describes six morphogenetic genes in Yarrowia lipolytica involved in hyphal formation and its filamentous morphology. Results show that a Ras-cyclic AMP pathway forms the hyphae and processing abnormalities of the extracellular and the parietal proteins lead to morphogenetic defects.

Researchers describe an autocloning method that produces multiple copies of the gene for extracellular lipase in Yarrowia lipolytica. These strains could produce 40 times more lipase per liter than strains containing only one copy of the gene. This makes them a promising source of lipase for industrial use, waste treatment, and pancreatic insufficiency treatment.

Results indicate that LIP2 gene of Yarrowia lipolytica encodes a 334-amino-acid precursor protein which is subsequently processed by endoprotease into lipase which is extracellularly secreted.