Identification And Characterization Of Genes Regulating Bipolar Budding In The Yeast Yarrowia Lipolytica

The zygote cells form complex tissues and organs through several times division and differentiation during the embryonic development of plants and animals. To form the ordered structures the specific division site (or flat) should be along during cytokinesis. Therefore, it is important to choose the correct cell division plane during the embryonic development. In budding yeasts, the cell division plane is often not randomly chosen, but controlled by the spatial landmarks, left by the last cell cycle. The study on the composition and mechanism of the spatial landmarks is important for the understanding of how cell division is spatially controlled.In budding yeasts, the position of cytokinesis is directly affected by the bud site selection, which occurred shortly after the beginning of the cell cycle, because the position of a new budding site is also defines the future cell division plane. Thus we can study the pattern of bud site selection to understand how cells choose cytokinesis plane. Among many types of budding yeasts, bud site selection of Saccharomyces cerevisiae has been studied clearly, there are two patterns of bud site selection in this microorganism:axial and bipolar. Yarrowia lipolytica is also a budding yeast, which is far from S. cerevisiae in evolution. This species bud in a bipolar pattern only, which is different from S. cerevisiae. Here, we explored the molecular mechanism of the bipolar budding in Y. lipolytica.In S.cerevisiae, Rsrl and its regulators play a key role in bud site selection. We found the Rsrl’s homologue in Y.lipolytica and named it YlRsrl. We deleted YIRSR1and examined the budding pattern of Ylrsrl△cells. YlRsr1plays a prominent role in this bud-site selection process and its function in bud-site selection absolutely requires the cycling of YlRsr1between the GTP-and GDP-bound states, which is similar with Rsrl in S. cerevisiae. However, the bud site selection defect of Ylrsr1△cells (40%bipolar budding cells) was not as penetrant as that of S. cerevisiae rsrl△cells (less that5%bipolar budding cells), so there maybe some different between the bud site selection molecular regulatory pathway of the two yeasts. Other regulators may exist in Y lipolytica that function redundantly with YlRsrl in bud site selection. Interestingly, the Ras GTPase YlRas2is also involved in the control of bud site selection as Ylras2A cells were severely impaired in bipolar bud-site selection. But this defect is partly as about22%cells can still bud in bipolar way. Only when both YlRsrl and Y1Ras2lost their function in bud-site selection as the defect is completely (more than90%cells bud in random pattern). To our knowledge, this is the first demonstration of the involvement of Ras GTPase in yeast bud site selection. The GTP/GDP cycling and the plasma membrane localization of YlRas2play an important, but non-essential, role in YlRas2’s function in bud-site selection, which is different from Rsrl, suggesting that some differences exist in the way by which the two small GTPase act. Besides, we found YlRsrl is important for cell growth since the deletion of YIRSR1in cells lacking the Ras GTPase YlRas2caused lethality. The constitutively active YlRsr1Q63L mutants could function as the sole source of YlRsr1in supporting cell growth when YlRas2is absent. Therefore, the function of YlRsr1in regulating cell growth does not appear to require rapid GTP/GDP cycling of YlRsr1.The defect of bipolar budding in YlrsrlA and Ylras2△cells became less pronounced under hypha-inducing growth condition. We speculate that the increased fidelity in bud site selection may result from the alteration of cell polarity and cell morphogenesis, therefore, we explored whether YlRsr1is involved in this process. We show that YlRsr1plays an important role in cellular morphogenesis in Y. lipolytica. Cells deleted for YIRSR1exhibited impaired polarized growth during yeast-form growth, most YlrsrA cells were bigger than wild-type cells, contained two or more nuclei, and formed multiple buds or short cell chains. Pseudohyphal and hyphal development were also abnormal, Ylrsr1△cells formed markedly more pseudohyphae than wild-type cells did and the mother cells in both single cells and pseudohyphae were less elongated than those of wild-type cells. We also found the GTP/GDP cycling is not crucial for YlRsr1’s function in cellular morphogenesis as the constitutively active GTP-bound YlRsr1Q63L can rescue the morphological defect of Ylrs1△cells during YF and pseudohyphae growth.And then we identified the regulators of YlRsr1. Based on the amino acid sequence homology to S. cerevisae Bud2and the defect of bipolar budding in Ylbud2A cells, the GAP that helps YlRsr1hydrolyze GTP is likely to be YlBud2. The GEF for YlRsr1, which helps YlRsr1load GTP, however, is less clear. Our findings suggest that YlCdc25is likely to function partially as the GEF for YlRsr1based on the cell-enlargement phenotype of Ylcdc25 mutant cells and YlCdc25’s similar subcellular localization pattern with S. cerevisiae Bud5. However, the phenotypically of Ylcdc25mutant cells is different from the constitutively inactive Ylrsr1K16N mutant cells. Future investigation is needed to find the major GEF for YlRsrl.We also characterized the function of the potential bipolar budding landmark proteins in Y. lipolytica. Analysis of YlraxlA and Ylrax2△mutants’budding pattern indicates that both proteins are involved in selecting bud sites as a portion of cells buds in the unipolar way. Besides, Ylraxl△Ylrax2△mutant cells have the same defect with the two mutants in bud-site selection. In addition, YlRax2can localize at the distal pole as well as at the division site on both mother and daughter cells, localization to the division sites was persistent through multiple cell cycles. And the normal localize of YlRax2dependent on YlRaxl. Based on these results, we speculate that YlRaxl and YlRax2function together as the landmark proteins in regulating the bipolar budding pattern of Y. lipolytica. However, there may be other proteins play the similar functions to them. Another possible landmark protein YlBud8is not involved in selecting bud site but in the regulation of cell morphology, as the deletion of Y1BUD8promotes the pseudohyphae growth of cells.We tried to isolate new genes that involved in bud site selection through zeta element-mediated random insertion, but no intereating candidates were obtained, this work is still in progress.