Tagging Morphogenetic Genes by Insertional Mutagenesis in the Yeast Yarrowia lipolytica

Abstract

The yeast Yarrowia lipolytica is distantly related to Saccharomyces cerevisiae, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on environmental conditions. Previous results have indicated that the STE and RIM pathways, which mediate cellular switching in other dimorphic yeasts, are not required for Y. lipolytica morphogenesis. To identify the pathways involved in morphogenesis, we mutagenized a wild-type strain of Y. lipolytica with a Tn3 derivative. We isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven genes homologous to S. cerevisiae CDC25, RAS2, BUD6, KEX2, GPI7, SNF5, and PPH21. We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature and to Calcofluor white. Chitin staining was used to detect defects in their cell walls. Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in Y. lipolytica and that perturbations in the processing of extracellular, possibly parietal, proteins result in morphogenetic defects.

GPI7 Involved in Glycosylphosphatidylinositol Biosynthesis Is Essential for Yeast Cell Separation

“…Mutations in GPI7 also affect cell wall anchorage in S. cerevisiae and Candida albicans (9). GPI7 is involved in chlamydospore formation, budding patterns, and cell shape in C. albicans (10) and invasive growth in the dimorphic yeast Yarrowia lipolytica (11). However, how GPI7 is involved in these phenomena is still unknown.…”

Inactivation of Kex2p Diminishes the Virulence of Candida albicans

“…In fungi, kexins cycle between the outer face of the Golgi and the prevacuolar compartment where they process proteins involved in maintenance and remodeling of the cell wall (4,5), proteins associated with the formation of aerial hyphae (6), ␣-type mating prepropheromones (7), killer toxins (7) , zymogens of secreted proteinases (8 -10), lipases (11), polysaccharide-degrading enzymes (12,13), and themselves (14). Deleting the kexin of the yeast Yarrowia lipolytica abolishes the formation of hyphae (15). kex2-null mutants of the yeast Saccharomyces cerevisiae are viable but exhibit conditional morphological abnormalities (16), defective vacuolar proton-translocating V-ATPase activity (17), cold-sensitive growth (16), and a partial defect in meiosis (genome-www.stanford.edu/Saccharomyces).…”

Insertional Mutagenesis in the n -Alkane-Assimilating Yeast Yarrowia lipolytica : Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization

“…Alternatively, a restriction enzyme was used that cuts in the polylinker of the MTC (Fig. 1, HindIII to EcoRI), followed by amplification of the left and right borders as described previously for Tn3 insertion in Y. lipolytica (26). The PCR product was sequenced using the same primers.…”

“…2), left and right borders were amplified with the primer pairs Ap1-Zeta1 and Ap1-Zeta3, respectively. All amplifications were performed on a Perkin-Elmer thermal cycler 2400 with the Expand long-template PCR system (Roche Diagnostics GmbH) as previously described (26). PCR fragments were sequenced directly after gel purification on an ABI373 DNA sequencer according to the manufacturer's instructions (Perkin-Elmer Biosystems).…”

“…To circumvent these difficulties, we first developed a transposon tagging approach to identify genes involved in HC utilization (18), leading to the characterization of PEX10 (J.-M. Nicaud, unpublished data), which is involved in peroxisome biogenesis. However, identification of the tagged genes was plagued by a high level of nonhomologous integration (26).…”