Characterization Of The Function And Mechanism Of Ras In The Dimorphic Transition In The Yeast Yarrowia Lipolytica

The ability of many yeast species and some filamentous fungi to grow either in ovoid, single-celled yeast form or in long, multicellular filaments called hyphae or pseudohyphae, is known as dimorphism. Dimorphism is normally induced by several environmental factors, such as temperature, the availability of nitrogen source, and pH, and is thought to be important for cell survival in response to environmental changes. It is of importance to study the regulatory mechanism of fungal dimorphism because it fungal dimorphism can be viewed as a model of eukaryotic cell differentiation. In addition, it plays an important role in the pathogenesis of several pathogenic fungi.Dimorphism in yeast is regulated by a variety of molecules including Ras proteins, which are evolutionarily conserved GTP-binding proteins widespread in eukaryotes. The dimorphic yeast species Yarrowia lipolytica is distantly related to Saccharomyces cerevisiae. It can adopt yeast, pseudohyphae, and hyphae forms depending on growth conditions. We identified three Ras-coding genes in its genome, which were named YIRAS1, YIRAS2, and YIRAS3. In this thesis, we investigated the roles of these three YIRAS genes in dimorphic transition and the possible mechanism for their function.We explored the roles of three YIRAS genes in dimorphic transition by gene knock-out and gene overexpression. We found that YIRAS1and YIRAS2, two genes that encode two Ras proteins similar to the H-Ras of animal cells, play critical roles in dimorphic transition as deletion of either of them attenuated hyphal development. Between these two YIRAS genes, YIRAS2appears to be more important than YIRAS1because Ylras2A mutant showed a stronger defect in hyphal development than YlraslA mutant. YIRAS2also controls invasive growth in Y. lipolytica. In contrast, there is no significant role for YlRAS3, a gene whose coding product resembles mammalian K-Ras4B, in the control of dimorphic transition. These results demonstrate that the three YIRAS genes do not act equally in the regulation of dimorphic transition. We also generated double and triple YlrasA mutant strains and examined these strains for their ability to undergo filamentous growth. We found that the three YIRAS genes are dispensable for cell growth. This feature is different from what has been found in S. cerevisiae. Except that Ylrasl? Ylras3? cells underwent moderate hyphal growth, all other double or triple YlrasA mutants that harbor Ylras2? behaved like Ylras2? cells in dimorphic transition. This observation supports the notion that YIRAS2is the most important YIRAS gene for dimorphic transition. Overexpression experiments showed that YlRASl and Y1RAS2share overlapping functions since overexpression of YlRASl partially restored hyphal development to Ylras2? cells. We also observed that the phenotype of Ylcdc25cells resembles that of Ylrasl? Ylras2? Ylras3? cells. This finding supports the view that YlCdc25may function as the guanine-nucleotide-exchange factor (GEF) for the three YlRas proteins.We detected that the expression of YIRAS2was increased dramatically at the transcriptional level during yeast-to-hypha transition, consistent with a major role of YIRAS2in the regulation of dimorphic transition. Then, we investigated the roles of GTP/GDP cycling and membrane targeting in the function of YlRas2. We found that, like other yeast Ras proteins, YlRas2's function in filamentous growth only requires the active GTP-bound form of YlRas2. The rapid cycling of YlRas2between the GTP-and GDP-bound states seems not essential for its function in the control of filamentous growth, as the expression of both Ylras2G17V and Ylras2Q66L mutants restored filamentous growth to Ylras2? cells. The plasma membrane localization of YlRas2is important but not absolutely essential for its function in dimorphic transition, as an endomembrane-restricted YlRas2C245S mutant is partially function.We also investigated the mechanism by which YlRas2may regulate dimorphic transition. Like S. cerevisiae and C. albicans, there are two signaling pathways involved in dimorphic transition in Y. lipolytica, the MAPK and the cAMP-PKA pathway. YlSte20, YlSte11and YlKss1are the members of MAPK pathway. Gene knockout and overexpression experiments showed that all of them played positive roles in dimorphic transition in Y. lipolytica, and the signal was transducted via Ste20-Stell-Kssl kinase cascade. S. cerevisiae Ras2and C. albicans CaRasl are known to promote filamentous growth via the MAPK and the cAMP-PKA pathway. To test whether the same two pathways may play a role in Y. lipolytica, we overexpressed YIRAS2in two single mutants and one double mutant lacking YIKSS1and YITPK1, genes that encode the MAPK and the protein kinase A, respectively. We found that the overexpression of YlRas2enhanced filamentous growth in all three mutants, suggesting that both of the pathways may not be the major pathway regulated by YlRas2.To look for novel genes involved in YlRas2-controlled dimorphic transition, we screened for genes whose overexpression could restore hyphal development to Ylras2A cells. We have isolated several revertants. One of these mutants, mut5, was shown to have the gene HOY1overexpressed by TAIL-PCR. We confirmed that HOY1overexpression could restore hyphal development to Ylras2? cells. In addition, we identified that overexpression of MHYl, which encodes a transcription factor, exhibited an effect similar to the overexpression of HOY1. We speculate that Mhyl and Hoyl may function downstream of YlRas2and may constitute a new regulatory pathway in the control of dimorphic transition.