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Un mécanisme évolutif très important pour l'acquisition de nouvelles fonctions protéiques est la duplication génique. Les protéines paralogues résultantes subissent une divergence fonctionnelle permettant à leur gène d'être retenus dans le génome. Les événements de duplication étant à l'origine de l'expansion de la famille des domaines SH3, une compréhension de la divergence des propriétés de liaison de ceux-ci sur les protéines paralogues est essentielle pour saisir leur implication dans diverses fonctions cellulaires. La liaison des domaines SH3 peut être très spécifique ou elle peut reconnaître des groupes canoniques de peptides riches en proline. Ainsi, l'objectif de ce projet de maîtrise est d'améliorer notre compréhension sur l'évolution des propriétés de liaison des domaines SH3 portés par des paralogues. En utilisant des données sur les interactions physiques entre les protéines in vivo, nous avons établi que les domaines SH3 des myosines de type I de Saccharomyces cerevisiae, un organisme modèle, montraient une divergence fonctionnelle. Puis, les domaines SH3 ont été échangés entre les paralogues et les domaines ancestraux pré-duplications ont été insérés dans les paralogues. Cette expérience a révélé que le domaine SH3 présent au moment de la duplication montre le même profil d'interaction que les SH3 existants, mais que les SH3 plus anciens perdent graduellement leurs interactions. Ensuite, l'arrimage moléculaire des SH3s avec leurs peptides de liaison prédits ainsi que la caractérisation des interactions des SH3s libres montrent que l'affinité ne diminue pas avec le domaine ancestral. Ces résultats sont confirmés par le patron de PPIs des domaines SH3 libre de contexte protéique. Cela a permis de déterminer que la propriété de liaison n'est pas le facteur principal qui influence sur les interactions des domaines SH3 présent sur des paralogues, mais que c'est leur protéine hôte. Nos résultats s'accordent avec la recherche récente qui suggère que les domaines protéiques ne sont pas des éléments isolés dans une protéine, contrairement à la croyance répandue. / A very important evolutionary mechanism for the acquisition of new protein functions is gene duplication. The resulting paralogous proteins undergo functional divergence allowing them to be retained in the genome. Since duplication events are responsible for the expansion of the SH3 domain family, an understanding of the divergence of the binding properties of SH3 domains on paralogous proteins is essential to grasp their involvement in various cellular functions. The binding of SH3 domains can be very specific or it can recognize canonical groups of proline-rich peptides. The objective of this master project is to improve our understanding of the evolution of the binding properties of SH3 domains carried by paralogs. Using data on physical interactions between proteins in vivo, we established that the SH3 domains of the type I myosins from Saccharomyces cerevisiae, a model organism, show functional divergence. Then, the SH3 domains were exchanged between paralogs and the pre-duplication ancestral domains were inserted into the paralogs. This experiment showed that the SH3 domain at the time of duplication displays the same interaction profile as the extant SH3s, but, as the SH3s get older, they gradually lose their interactions. Next, molecular docking of the SH3s with their predicted binding peptides and characterization of the free SH3s PPIs shows that the affinity does not decrease with the ancestral domain. Those results are confirmed by the PPI network of the SH3 domains free of a protein context. These experiments determined that the host protein is the primary factor influencing paralogous SH3 domain interactions instead of the SH3 binding preferences. Our results are consistent with recent research suggesting that protein domains are not isolated elements in a protein, contrary to popular belief.

Dissertation (PhD)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: Cells of the yeast Saccharomyces cerevisiae are able to change their morphological appearance in response to a variety of extracellular and intracellular signals. The processes involved in morphogenesis are well characterised in this organism, but the exact mechanism by which information emanating from the environment is integrated into the regulation of the actin cytoskeleton and the yeast cell cycle, is still not clearly understood. Considerable progress has, however, been made. The processes are investigated on various levels including: (i) the nature of the signals required to elicit a morphological adaptation, (ii) the mechanism by which these signals are perceived and transmitted to the nucleus for gene transcription regulation (signal transduction pathways), (iii) the role of the cytoskeleton, particularly actin, in morphogenesis, and (iv) the relationship between cell cycle regulators and factors required for alterations in cellular shape. The focus of this study was on elements involved in the regulation of one of these morphological processes, pseudohyphal formation, in S. cerevisiae. During pseudohyphal differentiation normal oval yeast cells become elongated and mother and daughter cells stay attached after cytokinesis to give rise to filaments. These filaments are able to penetrate the growth substrate, a phenomenon referred to as invasive growth. Actin remodelling is a prerequisite for the formation of elongated cells during pseudohyphal development and invasive growth. Its main contribution to this event is the directing of vesicles, containing cell wall constituents and enzymes, to specific sites of cell wall growth at the cell periphery. In order to fulfil this cellular function, actin is regulated on several levels. Signal transduction pathways that are activated in response to external nutritional signals play important roles in the regulation of the actin cytoskeleton during pseudohyphal differentiation. For this reason a literature review was compiled to introduce various aspects of actin-structure, the regulation of this structure and the functions actin performs during morphogenesis. The connection between signal transduction elements involved in morphological processes and actin remodelling is also reviewed. This study entailed the genetic analysis of numerous factors involved in the regulation of pseudohyphal differentiation, invasive growth and starch metabolism. Several transcriptional regulators playing a role in these phenomena were investigated. Apart from the transcription factors, which include Mss11p, Msn1p, Ste12p, F108p,Phd1p and Tec1p, additional elements ranging from transporters to G-proteins, were also investigated. Mutant strains deleted for one or more of these factors were constructed and tested to assess their abilities to form filaments that penetrate the growth substrate, and to utilise starch as a carbon source. Complex genetic relationships were observed for various combinations of these factors. Specifically, F108p,Msn1p and Ste12p were shown to act independently in controlling invasive growth and starch metabolism, suggesting that these factors are regulated by different signal transduction pathways. Mss11p, on the other hand, was found to play an indispensable role and seems to act as a downstream factor of Msn1 p, Fl08p, Ste12p and Tec1 p. The exception to this is Phd1 p, since multiple copies of PHD1 partially suppress the effect of a MSS11 deletion. The data suggests that Mss11 p functions at the confluence of several signalling pathways controlling the transcriptional regulation of genes required for invasive growth and starch degradation. Different nutritional signals were also found to differentially regulate specific signalling elements during the invasive growth response. For example, Tec1 p requires Msn1 p activity in response to growth on media containing a limited nitrogen source. This dependency, however, was absent when invasive growth was tested on glucose and starch media. Evidence was also obtained that confirmed the transcriptional co-regulation of MUC1 and STA2. MUC1 encodes a mucin-like protein that is required for invasive growth and pseudohyphal differentiation, whereas STA2 encodes a glucoamylase required for starch degradation. Unpublished results indicated that several transcriptional regulators of invasive growth also exert an effect on starch metabolism. The data generated during this study complemented and confirmed published results. It also contributed to the compilation of a more detailed model, integrating the numerous factors involved in these signalling processes. / AFRIKAANSE OPSOMMING: Saccharomyces cerevisiae gisselle beskik oor die vermoë om hul morfologiese voorkoms in responstot 'n verskeidenheid van ekstrasellulêre en intrasellulêre seine te verander. Die prosesse betrokke by morfogenese is goed gekarakteriseerd in hierdie organisme, maar die presiese meganisme waardeur inligting vanuit die omgewing geïntegreer word in die reguleringvan die aktien-sitoskelet en die gisselsiklus, word nog nie ten volle verstaan nie. Aansienlike vordering in die verband is egter gemaak. Die prosesse word op verskeie vlakke ondersoek, insluitende: (i) die aard van die seine wat benodig word om 'n morfologiese aanpassing te inisïeer; (ii) die meganisme waardeur hierdie seine waargeneem en herlei word na die selkern vir die regulering van geen-transkripsie (seintransduksie paaie); (iii) die rol van die sitoskelet, spesifiek aktien, in morfogenese en (iv) die verhouding tussen selsiklusreguleerders en faktore wat benodig word vir verandering in selvorm. Hierdie navorsing fokus op elemente betrokke by die regulering van een van hierdie morfologiese prosesse in S. cerevisiae, naamlik pseudohife-vorming. Gedurende pseudohife-differensiëring neem tipiese ovaalvormige selle 'n verlengde voorkoms aan wat tot die vorming van filamente lei. Hierdie filamente is in staat om die groeisubstraat te penetreer, 'n verskynsel bekend as penetrasie-groei. Aktienherrangskikking is 'n voorvereiste vir die vorming van verlengde selle tydens pseudohife-ontwikkeling. Die hoofbydrae van aktien tot hierdie verskynsel is die oriëntering van uitskeidingsvesikels, wat selwandkomponente en ensieme bevat, na spesifieke areas van selwandgroei op die seloppervlak. Aktien word op verskeie vlakke gereguleer om hierdie sellulêre funksie te vervul. Seintransduksiepaaie wat geaktiveer word in respons tot ekstrasellulêre voedingsseine speel 'n belangrike rol in die regulering van die aktien-sitoskelet tydens pseudohife-differensiëring. Op grond hiervan is 'n literatuuroorsig saamgestel vir die bekendstelling van verskeie aspekte van aktienstruktuur, die regulering van hierdie strukture en die funksies wat deur aktien gedurende morfogenese vervul word. Die verband tussen seintransduksie-elemente betrokke by morfologiese prosesse en aktien herrangskikkingword ook behandel. Hierdie studie het die genetiese analisering van verskeie faktore betrokke by pseudohife-differensiëring, penetrasie-groei en styselmetabolisme, behels. Verskeie transkripsionele reguleerders wat In rol speel in hierdie prosesse was bestudeer. Buiten die transkripsiefaktore Mss11p, Msn1p, Ste12p, F108p,Phd1P en Tec1p, was addisionele faktore, wat gewissel het van transporters tot G-proteïene, ook ondersoek. Mutante-rasse met geendelesies vir een of meer van hierdie faktore is gekonstrueer en getoets om vas te stel hoe dit hul vermoë raak om penetrerende filamente te vorm, asook om te bepaal of stysel as koolstofbron gebruik kan word. Komplekse genetiese interaksies vir verskeie kombinasies van hierdie faktore is waargeneem. Dit was waargeneem dat F108p,Msn1p en Ste12p onafhanklik funksioneer tydens die regulering van penetrasie-groei en styselmetabolisme, wat impliseer dat hierdie faktore deur verskillende seintransduksiepaaie gereguleer word. Mss11 p word beskou as In onmisbare rolspeler in hierdie prosesse en dit kom voor asof hierdie protein as 'n stroom-af faktor is en vereis word vir die funksionering van Msn1p, F108p, Ste12p en Tec1p. Phd1p is egter 'n uitsondering, aangesien veelvuldige kopieë van PHD1 die effek van 'n MSS11-delesie gedeeltelik oorkom. Die data impliseer dat Mss11 p by die samevloei van verskeie seintransduksiepaaie, benodig vir die transkripsionele regulering van gene betrokke by penetrasie-groei en styselmetabolisme, funksioneer. Dit was ook waargeneem dat verskillende voedingsseine die faktore betrokke by die penetrasie-groeirespons differensieel reguleer. Tec1 p byvoorbeeld benodig Msn1paktiwitieit in respons tot groei op media met 'n beperkte stikstofbron. Hierdie afhanklike interaksie is egter afwesig wanneer penetrasie-groei bestudeer word op glukose- en styselmedia. Resultate wat die gesamentlike transkripsionele regulering van MUC1 en STA2 bevestig, is ook verkry. MUC1 kodeer vir 'n mukienagtige proteïen wat benodig word vir pseudohife-vorming en penetrasie-groei, terwyl STA2 kodeer vir 'n glukoamilase essensieël vir styselafbraak. Ongepubliseerde resultate dui daarop dat verskeie transkripsionele reguleerders van penetrasie-groei ook In effek uitoefen op styselmetabolisme. Die data wat gegenereer is tydens hierdie studie komplementeer en bevestig reeds gepubliseerde resultate. Dit het ook bygedra tot die samestelling van 'n gedetaileerde model wat die verskillende faktore, betrokke by hierdie seintransduksieprosesse, integreer.