Mss11p mediated regulation of transcription, pseudohyphal differentiation and flocculation in Saccharomyces cerevisiae

Franken, Jaco (Cornelius Jacobus) (2004-03)

Thesis (MSc)--University of Stellenbosch, 2004.

Thesis

ENGLISH ABSTRACT: In all cellular systems the ability to alter eellular programs in response to extracellular cues is essential for survival. This involves the integration of signals triggered by membrane bound receptors in order to adjust the expression of target genes and enzyme activities and consequently phenotypic outcome. The yeast Saccharomyces cerevisiae has evolved several adaptations, such as, sporulation and pseudohyphal differentiation, in order to survive changes in the surrounding environment. Pseudohyphal differentiation and the related phenotype, invasive growth, are proposed to be adaptations that enable the yeast to forage for scarce nutrients or escape from a detrimental environment. This dimorphic transition is associated with a change from the normal "yeast" form to a pseudohyphal form, which involves changes in budding pattern, cell-cycle progression, cellular elongation, and cell-eell and cell-substrate adherence. The outcome of these changes is elongated eells, which bud in a unipolar fashion and do not separate after budding to form chains of cells referred to as pseudohyphae. These pseudohyphae are able to penetrate the surface of agar containing growth medium, a process referred to as invasive growth. Nutrient-induced adaptations, such as pseudohyphal growth, have been extensively studied in S. cere visiae , and several factors have been implicated in the regulation thereof, many of which are part of specific signalling pathways. The most clearly defined are the filamentous growth specific MAP kinase cascade and the Gpa2p-cAMP-PKA pathway. MUC1/FL011, encoding a member of a family of cell wall associated proteins involved in cellcell/ cell-substrate adhesion, is regulated by these pathways and considered to be critical in the establishment of pseudohyphal differentiation and invasive growth. The promoter region of MUC1/FL011 represents one of the largest yeast promoters identified to date, with cis-acting elements present up to 2.4 kb upstream from the first coding triplet. The upstream regulatory region of MUC1/FL011 is almost identical to that of the STA2 gene, which encodes an extracellular glucoamylase required for the utilisation of extracellular starch. As suggested by the extent of homology between these two promoters, MUC1/FL011 and STA2 are co-regulated to a large degree and both require the same transcription factors. Mss11p plays a central role in the regulation of MUC1/FL011 and STA2 and consequently starch metabolism and pseudohyphaI differentiation. The regulation conferred by MSS11 on the transcriptional levels of MUC1/FL011 and STA2 also appears to be dependent on signals generated specifically in the presence of low nitrogen and glucose. Mss11p does not have significant homology to any other yeast protein, with the exception of limited homology to the transcriptional activator F108p. However, several distinctive domains have been identified in the MSS11 gene product. Firstly, Mss11p contains polyglutamine and poly-asparagine domains. It also contains a putative ATP- or GTP-binding domain (P-Ioop), commonly found in proteins such as kinases, ATPases or GTPases. Two short stretches close to the N-terminal, labelled H1 and H2, share significant homology to the transcriptional activator, F108p. Both the H2 domain and the extreme C-terminal of Mss11p are able to stimulate RNA polymerase II dependent transcription. Furthermore, the H1 domain together with the P-Ioop negatively regulates the activation potential of the H2 domain. This study presents further insight into the functioning of Mss11p and the involvement of the separate activation and regulatory domains in mediating transcriptional activation and pseudohyphal differentiation in response to nutrient limitation. Genetic interactions between Mss11p and other factors involved in the regulation of pseudohyphal growth and starch degradation were revealed, and specific regions of Mss11p were shown to be required by these factors in order to achieve their required function. In addition, results obtained in this study implicates Mss11p in the regulation of Ca2+-dependent flocculation and suggest that the FL01 gene is also regulated by Mss11p in this capacity.

AFRIKAANSE OPSOMMING: Die vermoë om sellulêre programme in reaksie op ekstrasellulêre seine te verander, is 'n essensiële vereiste vir alle sellulêre sisteme. Dit behels die integrasie van seine gegenereer deur membraan-gebonde reseptore om ekspressie van teikengene en ensiemaktiwiteite sodanig aan te pas dat gewenste fenotipise uitkomste bewerkstellig kan word. Die gis Saccharomyces cerevisiae het verskeie aanpassingsmeganismes ontwikkel, soos byvoorbeeld sporulasie en pseudohifeforming, om veranderinge in die omgewing te kan oorleef. Pseudohifevorming en die verwante fenotipe, penetrasiegroei, word beskou as aanpassings te wees wat die gis in staat stel om van 'n skadelike omgewing weg te kom, of dit in staat te stelom by skaars voedingstowwe uit te kom. Hierdie dimorfiese transisie word geassosieer met 'n verandering van die normale "gisvorm" tot pseudohifevorming wat veranderinge in die botpatroon, selsiklusprogressie, selverlenging, sel-sel en sel-substraat aanhegting behels. Die uitkoms van hierdie verandering is verlengde selle, wat unipolêr bot en nie van mekaar skei nie om sodoende kettings van selle te vorm en waarna verwys word as pseudohifes. Hierdie pseudohifes is ook in staat om die oppervlak van 'n agar bevattende groeimedium te penetreer, 'n proses waarna verwys word as penetrasiegroei. Aanpassings soos pseudohitevorminq is die afgelope dekade intensief nagevors, en verskeie faktore en seintransduksienetwerke is in die regulering daarvan geïmpliseer. Onder hierdie seintransduksienetwerke is die bes gedefiniëerde paaie die filamentasie-spesifieke MAP-kinasekaskade en die Gpa2p-cAMP-PKA pad. MUC1/FL011 kodeer vir 'n lid van 'n geenfamilie wat met sel-sel/sel-substraat aanhegting geasosieer word en dit word deur hierdie seintransduksie netwerke gereguleer. MUC1/FL011 word as essensieel vir pseudohife vorming beskou. MUC1/FL011 word gereguleer deur die grootste gispromoter wat tot op hede geïdentifiseer is, met cis-werkende elemente so ver as 2.4 kb stroom-op van ATG. Die MUC1/FL011 promoter is feitlik identies tot die van die STA2-geen, wat kodeer vir 'n ekstrasellulêre glukoamilase wat die gis in staat stelom ekstrasellulêre stysel te benut. Weens die homologie tussen die twee promoters, word MUC1/FL011 en STA2 tot In groot mate ge-koreguleer en beide benodig dieselfde transkripsiefaktore. Mss11p speel In sentrale rol in die regulering van MUC1/FL011 en STA2 en dus ook in die regulering van pseudohifevorming en styselmetabolisme. Die regulering wat deur Mss11p of MUC1/FL011 en STA2 uitgeofen word, blyk verder onderhewig te wees aan seine wat gegenereer word spesifiek in die teenwoordigheid van lae konsentrasies glukose en stikstof. Mss11p het nie betekenisvolle homologie met enige ander gisproteïen nie, behalwe vir beperkte homologie met die tranksripsionele aktiveerder F108p. Verskeie onderskeidbare domeine is egter in die MSS11 geenproduk teenwoordig. Eerstens, Mss11p bevat kenmerkende poliglutamien en poli-asparagien domeine. Verder bevat Mss11p ook In voorspelde ATP- of GTP-bindings domein (P-Ius), wat algemeen in proteïene soos kinases, ATPasaes en GTPases voorkom. Twee kort areas naby die N-terminaal, aangedui as H1 en H2, het betekenisvolle homologie met die transkripsiefaktor F108p. Beide die H2 domein en die ektreme C-terminaal van Mss11p is in staat om RNA polimerase " afhanklike transkripsie te stimuleer. Verder het die H1-domein in samewerking met die P-Ius In negatiewe uitwerking op die aktiveringspotensiaal van die H2-domein. Hierdie studie bied verdere insig tot die werking van Mss11p en die betrokkenheid van die verskeie aktiverings- en reguleringsdomeine by die oemiddetlinq van transkripsionele aktivering en pseudohifevorming in reaksie op beperking van voedingstowwe. Genetiese interaksies tussen Mss11p en ander faktore betrokke met die regulering van pseudohifevorming en styselafbraak is in hierdie studie aangetoon. Voorts is daar ook gewys dat spesifieke areas van Mss11p benodig word deur hierdie faktore om hulle biologiese funksie uit te oefen. Daar is ook In rol vir Mss11p in die regulering van Ca2+-afhanklike flokkulasie aangetoon en daar is bewys dat die FL01 geen deur Mss11p benodig word om hierdie effek uit te oefen.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/50032
This item appears in the following collections: