S. cerevisiae have developed the ability to forage for nutrients when presented with conditions of starvation. This dimorphic adaptation is particularly noticeable when yeast are subject to nitrogen depravation and has been termed filamentous growth, as cells form filament-like projections away from the center of the colony. The regulation of this response is under the control of the well-characterized MAPK and cAMP pathways. Previous work showed that Cdk8p phosphorylated a key transcriptional activator of the filamentous response, Ste12p, and subsequently targeted the factor for degradation under conditions of limiting nitrogen. Data presented in this thesis suggests that Cdk8p is regulated by another kinase, Tpk2p. In vitro kinase assays demonstrate that Tpk2p directly phosphorylates Cdk8p on residue Thr37, leading to the destabilization of Cdk8p after growth for 4 hours in SLAD media. Lack of phosphorylation on Thr37 yields a hypo-hypofilamentous phenotype, whereas a phospho-mimic mutant, T37E displays a filamentous hyper-filamentous phenotype.

S. cerevisiae have developed the ability to forage for nutrients when presented with conditions of starvation. This dimorphic adaptation is particularly noticeable when yeast are subject to nitrogen depravation and has been termed filamentous growth, as cells form filament-like projections away from the center of the colony. The regulation of this response is under the control of the well-characterized MAPK and cAMP pathways. Previous work showed that Cdk8p phosphorylated a key transcriptional activator of the filamentous response, Ste12p, and subsequently targeted the factor for degradation under conditions of limiting nitrogen. Data presented in this thesis suggests that Cdk8p is regulated by another kinase, Tpk2p. In vitro kinase assays demonstrate that Tpk2p directly phosphorylates Cdk8p on residue Thr37, leading to the destabilization of Cdk8p after growth for 4 hours in SLAD media. Lack of phosphorylation on Thr37 yields a hypo-hypofilamentous phenotype, whereas a phospho-mimic mutant, T37E displays a filamentous hyper-filamentous phenotype.

S. cerevisiae have developed the ability to forage for nutrients when presented with conditions of starvation. This dimorphic adaptation is particularly noticeable when yeast are subject to nitrogen depravation and has been termed filamentous growth, as cells form filament-like projections away from the center of the colony. The regulation of this response is under the control of the well-characterized MAPK and cAMP pathways. Previous work showed that Cdk8p phosphorylated a key transcriptional activator of the filamentous response, Ste12p, and subsequently targeted the factor for degradation under conditions of limiting nitrogen. Data presented in this thesis suggests that Cdk8p is regulated by another kinase, Tpk2p. In vitro kinase assays demonstrate that Tpk2p directly phosphorylates Cdk8p on residue Thr37, leading to the destabilization of Cdk8p after growth for 4 hours in SLAD media. Lack of phosphorylation on Thr37 yields a hypo-hypofilamentous phenotype, whereas a phospho-mimic mutant, T37E displays a filamentous hyper-filamentous phenotype. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

xii, 41 p. : ill. / Cells convey information through signaling pathways. Distinct signaling pathways often rely on similar mechanisms and may even use the same molecules. With a variety of signals conveyed by pathways that share components, how does the cell maintain the integrity of each pathway? Budding yeast provides an example of multiple signaling pathways utilizing the same components to transduce different signals. The mating pathway, the high osmolarity glycerol (HOG) pathway and the filamentous growth (FG) pathway each respond to different environmental conditions and generate unique cellular responses. Despite the individuality of the pathways, they each contain a core group of the same signaling proteins. How does the cell generate a variety or responses utilizing the same group of proteins? Both the mating and HOG pathways utilize scaffolding factors that concentrate pathway components to the location of activation and in the case of the mating pathway alter the kinetics of the interaction. In addition, negative regulatory mechanisms operate in both the mating and HOG pathways. These negative regulatory mechanisms are understood in detail for the mating pathway but not for the HOG pathway. Mechanisms for providing specificity for the FG pathway are as yet unknown. The purpose of this work is to elucidate the mechanisms that provide specificity to the FG pathway. The search for specificity factors was done through both a random mutagenesis screen and a synthetic genetic array screen, looking for mutants in which activation of the FG pathway led to inappropriate activation of the HOG pathway. The random mutagenesis screen resulted in a large number of mutants that I organized into five complementation groups. The identity of the gene mutated in the largest complementation group was sought using a variety of methods including complementation with the yeast deletion collection and whole genome sequencing. A synthetic genetic array was screened as an alternative method to identify genes necessary for FG pathway specificity. These experiments have resulted in a list of candidate genes, but thus far have not yet led to any discernable mechanism for maintenance of FG pathway specificity. / Committee in charge: Karen Guillemin, Chairperson; George F. Sprague Jr., Advisor; Tom Stevens, Member; Tory Herman, Member; Diane Hawley, Outside Member

Candida albicans est une levure pathogène opportuniste de l’homme. La capacité de C. albicans à changer de forme en réponse à des stimuli externes, passant d’une croissance bourgeonnante à filamenteuse, est associée à sa virulence. Cette morphogenèse requiert une réorganisation du cytosquelette d’actine et un trafic membranaire ciblé. Chez Saccharomyces cerevisiae, les petites protéines G de type Arf jouent un rôle important dans le trafic membranaire et la polarité cellulaire. Le rôle de ces protéines chez C. albicans est largement méconnu. C. albicans a 3 protéines Arf, Arf1-Arf3 et 2 Arf-like, Arl1 et Arl3. Nos résultats indiquent que seule Arf2 est nécessaire à la viabilité et à la résistance aux antifongiques, et qu’Arf2 et Arl1 sont critiques pour la croissance filamenteuse hyphale; le mutant arl1/arl1 en particulier forme des hyphes 2 fois plus courtes que la souche sauvage. Les mutants Δ/pTetARF2 et arl1/arl1 ont un défaut de virulence dramatique et ARL1 est particulièrement critique pour la candidose oropharyngée. Nos résultats indiquent que les défauts du mutant Δ/pTetARF2 seraient dus à une altération du Golgi, et ceux d’arl1/arl1 de l’incapacité de ce mutant à restreindre sa croissance à un site unique. Ce défaut de croissance polarisée du mutant arl1/arl1 n’est pas lié à la mislocalisation de son effecteur Imh1, ni à une misrégulation de la phosphatidylsérine flippase Drs2. Par contre, nos données suggèrent que le défaut de croissance hyphale de ce mutant résulterait d’une hypersécrétion. Cette étude nous a permis d’identifier Arf2 et Arl1 comme protéines clés du trafic membranaire, critiques pour la croissance filamenteuse et la virulence de C. albicans. / The human fungal pathogen Candida albicans switches from budding to filamentous growth. This dramatic morphogenesis is critical for its virulence and requires sustained polarized growth, via exocytosis and endocytosis, as well as reorganization of intracellular compartments. In the yeast Saccharomyces cerevisiae, Arf G-proteins and their regulators function at the interface of membrane traffic and cell polarity. The roles of this class of proteins during the transition to filamentous growth and virulence in C. albicans are largely unknown. In C. albicans there are 3 Arf proteins, Arf1-Arf3 and 2 Arf-like proteins, Arl1 and Arl3. Our results reveal that only Arf2 is required for viability and sensitivity to antifungal drugs and that both Arf2 and Arl1 are required for hyphal growth, with arl1/arl1 hyphal filaments being 2-fold shorter than that of the wild-type strain. Furthermore, both Δ/pTetARF2 and arl1/arl1 mutants have drastically reduced virulence, with ARL1 particularly critical for oropharyngeal candidiasis. We show that the defects in Δ/pTetARF2 is due to an alteration of Golgi integrity, while the defects in the arl1 mutant are likely to result from the inability of this mutant to restrict growth to a single site. Further analyses of the arl1/arl1 mutant revealed that this defect does not result from a misregulation of the GRIP-domain golgin coiled-coil tethering protein Imh1 nor of the phosphatidylserine flippase Drs2. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion. Together our work identifies Arf2 and Arl1 as key regulators of membrane traffic, critical for hyphal growth and virulence.

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