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

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The yeast Saccharomyces cerevisiae is able to sense and respond to changes in its immediate environment. Information regarding the nutritional status of the extracellular environment is sensed by membrane receptor systems and relayed through signalling pathways to the nuclear interior, affecting the transcription of specific genes., Transcription factors, which function downstream of these signal transduction pathways, have to be transported into the nucleus after synthesis in the cytoplasm in order to regulate transcriptional events. Transport into the nucleus occurs in a tightly regulated manner at the nuclear pore complex, which is located in the nuclear membrane, and requires the recognition of transport signal sequences, which are present in the proteins that are to be transported. Signalling pathways control the nuclear accessibility of transcriptional regulators by modifying their respective signal sequences. In response to a limited availability of carbon or nitrogen, cells are able to change their morphology from a unicellular ovoid form to elongated cells attached to each other. This morphological change is associated with daughter cells that remain attached to their respective mother cells following unipolar budding, thus forming filamentous structures referred to as pseudohyphae. The regulation of the development of pseudohyphae is correlated with other physiological processes, such as starch degradation and the invasion of agar-containing media. Mss11p performs a central role in the regulation of the genes required for these processes and it has been shown to specifically regulate the expression of FL011, which encodes a cell surface protein critical for pseudohyphal development, and STA2, which encodes an extracellular glucoamylase functioning in the degradation of starch. The aim of this study was to characterise the functioning of Mss11p. Overexpression analysis indicates that Mss11p functions as an inducer of invasive growth, cell elongation and flocculation. Furthermore, MSS11 deletion improves biomass formation and suppresses the growth defect of yeast from a L:1278b genetic background transformed with the RAS2val19 allele on non-fermentable carbon sources. Biochemical analysis shows that Mss11p is a nuclear protein of approximately 97 kDa in apparent size that is maintained at relatively low levels in yeast. Finally, the data suggest a model in which Mss11p functions as a mediator of the transcriptional regulation of various genes. / AFRIKAANSE OPSOMMING: Die gis Saccharomyces cerevisiae is in staat om veranderinge in sy onmiddelike omgewing waar te neem en daarop te reageer. Inligting betreffende die beskikbaarheid van voedingstowwe in die omgewing word vanaf membraan reseptorsisteme deur middel van seintransduksiekaskades na die nukleus herlei, waar die transkripsie van spesifieke gene beïnvloed word. Transkripsie faktore wat stroom af van hierdie seintransduksie funksioneer, moet na die nukleus vervoer word na vervaardiging in die sitoplasma, om sodoende transkripsionele gebeurtenisse te reguleer. Die vervoer van faktore na die binnekant van die nukleus vind onder streng regulering plaas by die nukleêre porie kompleks, wat in die nukleêre membraan gesitueer is. Vervoer vind plaas deur middel van die herkenning van nukleêre lokaliseringsekwense wat in die proteïene wat vervoer word, teenwoordig is. Seintransduksiekaskades beheer die beskikbaarheid van proteïene tot die nukleus deur hulonderskeidelike nukleêre lokaliseringsekwense te modifiseer. Selle is in staat om hul morfologie te verander van 'n eensellige eliptiese vorm tot verlengde selle wat aan mekaar geheg bly in reaksie op die beperkende beskikbaarheid van koolstof of stikstof bronne. Hierdie morfologiese verandering word geassosieer met dogterselle wat ná monopolêre botselvorming aan hul moederselle geheg bly, en dus filamentagtige strukture vorm wat pseudohifes genoem word. Die regulering van die ontwikkeling van pseudohifes word gekorreleer met ander fisiologiese prosesse, soos styselafbraak en die penetrerende groei van selle op agar-bevattende media. Mss11p vervul 'n sentrale rol in die regulering van gene wat vir hierdie prosesse benodig word en reguleer die uitdrukking van FL011, wat kodeer vir 'n selwandproteïen wat krities is vir die ontwikkeling van pseudohifes, en STA2, wat kodeer vir 'n ekstrasellulêre glukoamilase wat vir die afbraak van stysel benodig word. Die doel van hierdie studie was om Mss11p-funksie te karakteriseer. Deur middel van oorproduksie is Mss11p as die induseerder van penetrerende groei, selverlenging en flokkulasie geïdentifiseer. Verder is bevind dat MSS11-delesie lei tot verhoogde biomassa formasie, en dat dieselfde delesie lei tot 'n oorkoming van 'n groeidefek van gis van die 2:1278b genetiese agtergrond wat met die RAS2val19aleel op nie-fermenteerbare koolstofbronne getransformeer is. Biochemiese analise dui daarop dat Mss11p 'n nukluêre proteïen is van ongeveer 97 kDa in oënskynlike grootte, wat teen lae vlakke in gis onderhou word. Die data stel 'n model voor waarin Mss11p as bemiddelaar optree vir die transkripsionele regulering van verskeie gene.

No description available.