Tesis sobre el tema "Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Signal Transduction"

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2007.
Title from electronic title page (viewed Apr. 25, 2008). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry and Biophysics." Discipline: Biochemistry and Biophysics; Department/School: Medicine. On title page, [beta] appears as Greek character.

In response to external stimuli, many intracellular signaling proteins undergo dynamic changes in localization to the plasma membrane. Using the Saccharomyces cerevisiaemating pathway as a model, I investigated the molecular interactions that govern plasma membrane localization of signaling proteins, and how the plasma membrane compartmentalization of a signaling complex influences the overall signaling behavior of the pathway. Signaling proteins often consist of multiple interaction domains that collectively dictate their localization and function. Ste20 is a p21-activated kinase (PAK) that functions downstream of the Rho-type GTPase Cdc42 to activate several mitogen-activated protein (MAP) kinase pathways in budding yeast, including the mating pathway. I identified a short domain in Ste20 that directly binds to membrane lipids via electrostatic interaction. A mutation in this domain abolishes both the localization and function of Ste20. Thus, the previously known Cdc42 binding is necessary but not sufficient; instead, direct membrane binding by Ste20 is also critical. By replacing this domain with heterologous membranebinding domains, I demonstrated that phospholipid specificity is not essential in vivo. Functionally important short membrane-binding domains were also found in the Cdc42 effectors Gic1 and Gic2, indicating that generic membrane binding can work in concert with the CRIB domain to regulate activation of Cdc42 targets. These results underscore the importance of cooperation between protein-protein and protein-membrane interaction in achieving proper localization of signaling proteins at the cell cortex. At the system level, MAP kinase cascades can be graded or switch-like. The budding yeast mating pathway exhibits a graded response to increasing levels of pheromone. Previously the scaffold protein Ste5 was hypothesized to contribute to this graded response. To test this idea, I activated the pathway in a variety of ways and measured the response at the single cell level. I found that the graded response is not perturbed by the deletion of negative regulators of the pathway whereas the response became switch-like when the pathway was activated by a crosstalk stimulus that bypasses the upstream components. Interestingly, activation of the pathway in the cytoplasm using the graded expression of MAPKKK resulted in an ultrasensitive response. In contrast, activation of the pathway at the plasma membrane using the graded expression of membranetargeted active pathway components remained graded. In these settings, the scaffold protein Ste5 increased ultrasensitivity when limited to the cytosol; however, if Ste5 was allowed to function at the plasma membrane, signaling was graded. The results suggest that, in the mating pathway, the inherently ultrasensitive MAPK cascade is converted to a graded system by the scaffoldmediated assembly of signaling complexes at the plasma membrane. Therefore, the plasma membrane localization of Ste5 helps shape the input-output properties of the mating MAPK pathway in a manner that is suitable for the biology of mating. Taken together, this thesis underscores the importance of plasma membrane localization during mating pathway signaling in yeast. The examples described here provide further appreciation of how multiple interaction domains can function together to achieve specific targeting of the signaling proteins, as well as advances in understanding the role of scaffold proteins in modulating signaling behavior to promote graded signaling at the plasma membrane.

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.

Thesis (MSc)--Stellenbosch University, 2003.
ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity.
AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit.

本文データは平成22年度国立国会図書館の学位論文(博士)のデジタル化実施により作成された画像ファイルを基にpdf変換したものである
Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第7406号
農博第990号
新制||農||762(附属図書館)
学位論文||H10||N3152(農学部図書室)
UT51-98-G335
京都大学大学院農学研究科食品工学専攻
(主査)教授 木村 光, 教授 天知 輝夫, 教授 江﨑 信芳
学位規則第4条第1項該当

The yeast SKN7 gene encodes a transcription factor that is involved in a variety of processes in cell physiology including cell wall synthesis, cell cycle progression, and oxidative stress resistance. Using a transcriptional reporter-based system, it has been demonstrated that Skn7p is regulated by the two-component osmosensor Sln1p in a manner that requires the phosphorelay molecule Ypd1p, but not the response regulator Ssk1p. Consistent with its regulation by an osmosensor, Skn7p is involved in negative regulation of the osmoresponsive HOG MAP kinase cascade. Cells lacking SKN7 and the protein serine/threonine phosphatase encoded by PTC1 are severely disabled for growth, and hyperaccumulate intracellular glycerol. The growth defect of skn7Delta ptc1Delta mutants can be bypassed by overexpression of specific phosphatase genes, or by deletion of the HOG MAP kinase pathway-encoding genes PBS2 or HOG1.
MID2 was isolated in a screen designed to identify upstream regulators of Skn7p. Mid2p is an extensively O-mannosylated protein that is localized to the plasma membrane. Mutants with defective beta-1,6-glucan synthesis grow more quickly when MID2 is absent. Conversely, MID2 is essential for viability in cells lacking FKS1, the gene encoding the primary catalytic subunit of beta-1,3-glucan synthase. mid2Delta mutants are resistant to calcofluor white, a drug that interferes with cell wall chitin synthesis, while cells overexpressing MID2 are supersensitive to the drug. mid2Delta mutants have a significant reduction in stress-induced chitin synthesis, while cells overexpressing MID2 hyperaccumulate cell wall chitin. Consistent with a proposed role in sensing and responding to cell wall stress, high copy expression of specific components of the cell wall integrity MAP kinase cascade suppress various mid2Delta phenotypes, and Mid2p is essential for full activation of the Mpk1p MAP kinase during various cell wall stress and morphogenic conditions.
Observations from genetic and biochemical experiments suggest that Mid2p is a regulator of the small G-protein encoded by RHO1. Deletion of MID2 is lethal to mutants lacking the Rho1p GEF Rom2p, but suppresses the low temperature growth defect of mutants lacking the Rho1p GAP Sac7p. Conversely, high copy expression of MID2 is a strong suppressor of mutants lacking TOR2, an upstream activator of Rom2p, but is toxic to sac7Delta mutants. High copy expression of MID2 causes increased GEF activity towards Rho1p. Mid2p appears to act in parallel to Rom1p and Rom2p in promoting GDP-GTP exchange for Rho1p in a mechanism that is not yet understood.

L'ubiquitylation est une modification post-traductionnelle impliquée dans de nombreux processus cellulaires qui nécessite une régulation fine. La protéine Cdc48, qui appartient à la famille des ATPases AÂA (ATPase associated with various activities), participe à cette régulation. En effet, il s'agit d'une chaperonne dépendante de l'ubiquitine, essentielle à la viabilité cellulaire, qui est capable de réguler le devenir de protéines ubiquitylées, directement ou indirectement via des cofacteurs. Cdc48 est impliquée dans la régulation de nombreuses fonctions biologiques et contribue à l'homéostasie cellulaire en participant à la dégradation de protéines via le système ubiquitine/ protéasome et l'autophagie. Lors de ma thèse, je me suis intéressée à la caractérisation de nouvelles fonctions de la protéine Cdc48 chez la levure S. Cerevisiae. Dans un premier temps, j'ai participé à la description d'une nouvelle fonction de Cdc48 et son cofacteur Ufd3, deux nouveaux partenaires du complexe de déubiquitylation Ubp3-Bre5, dans la ribophagie, un processus d'autophagie sélective permettant la dégradation vacuolaire des ribosomes matures en condition de carence nutritionnelle. Dans un deuxième temps, je me suis intéressée au rôle de Cdc48 dans le noyau et plus précisément à la chromatine. Cette étude a mis en évidence que la protéine Cdc48 est recrutée sur les gènes activement transcrits, et qu'elle régule le processus d'ubiquitylation de l'histone H2B sur certains gènes. Nous avons donc caractérisé une nouvelle fonction de la protéine Cdc48 qui serait un régulateur important de Pubiquitylation de l'histone H2B, une marque d'histone dont la fonction est encore mal comprise
The ubiquitylation is a post-translational modification involved in numerous cellular processes and needs to be tightly regulated. The protein Cdc48, which belongs to the AAA ATPase family (ATPase associated with various activities), contributes to this regulation. Indeed, it is a ubiquitin-1 selective chaperone, essential for cell viability, which is capable of regulating the fate of ubiquitylated proteins directly or indirectly via specific cofactors. Cdc48 is involved in the regulation of a large array of biological processes and contributes notably to cellular homeostasis by participating in protein degradation via the ubiquitin/proteasome System and the autophagic pathway. During my PhD, I focused on the characterization of new fonctions of the protein Cdc48 in I the yeast S. Cerevisiae. At first, I participated in the description of a new function of Cdc48 and its cofactor Ufd3, two new partners of the deubiquitylation complex Ubp3-Bre5, in the ribophagy, a selective autophagy process induced by nutrient starvation that allows the vacuolar degradation of mature ribosomes. In a second time, I became interested in the role of Cdc48 in the nucleus and more specifically at the chromatin. This study showed that the protein Cdc48 is recruited to actively transcribed genes and that it regulates the process of the ubiquitylation of the histone H2B on certain I genes. We therefore characterized a novel function of the protein Cdc48, which would be an important regulator of the ubiquitylation of H2B, a histone mark whose function is still poorly understood

À l’interface entre la cellule et le milieu extérieur, les protéines membranaires ont un rôle indispensable au fonctionnement cellulaire. Elles jouent un grand nombre de fonctions et peuvent être impliquées dans des pathologies graves. La connaissance de ces protéines membranaire peut permettre de mieux comprendre de nombreux phénomènes biologiques et pathologiques et leur localisation, accessible, en fait de bonne cibles thérapeutique. La connaissance de la structure des protéines apporte une quantité d’information très importante sur leur fonctionnement. À ce jour si plusieurs dizaines de milliers de structures de protéines solubles ont pu être résolues, moins de deux cents structures de protéines membranaires sont connues. Cette différence provient des difficultés à produire des protéines membranaires pures et stables en quantités nécessaires à la détermination des structures tridimensionnelles. Les conditions d’expression des protéines membranaires est très variables en fonction de la protéine. Notre équipe de recherche s’intéresse au développement de stratégies pour l’expression hétérologue et la purification de protéines membranaires impliquées dans des pathologies humaines dans le but d’en étudier la structure et les relations structurefonction et par une approche protéine. Dans cette thèse sont présentés les résultats obtenus sur l’expression et la purification des récepteurs humains de la voie de signalisation Hedgehog, Patched et Smoothened, dont le disfonctionnement est mis en cause dans de nombreux cancers mais également dans des maladies neuro-dégénératives. Ces protéines ont pu être exprimées dans différents systèmes hétérologues : les levures Saccharomyces cerevisiae et Pichia pastoris et dans les Cellules S2 de drosophile. Nous avons pu mettre en évidence que Smoothened était exprimé sous sa forme native dans les levures et qu’il était possible de la purifier et la produire dans l’optique d’une étude structurale à partir de Pichia pastoris (de Rivoyre et al, FEBS letters, 2005). Patched et Smoothened ont également pu être exprimé stablement et fonctionnellement par des clones de cellules S2. Ce système qui permet la purification de ces protéines s’est également avéré très intéressant pour une étude comparative du fonctionnement des protéines humaines et des protéines de drosophile (de Rivoyre et al, JBC, 2006)
Membrane-bound proteins play a significant role in cell function due to their position in between the cell and the external medium. These proteines are for this reason, involved in a number of human diseases. Knowing membrane-bound proteins will allow us to better understand several biological and pathophysiological functions. Furthermore, their localisations make them interesting therapeutic targets. Knowing the structure of proteins gives a tremendous amount of information on their functions. To date, even if several thousands structures of soluble proteins have been solved; only less than two hundred structures of membrane-bound proteins are known. This important difference is partly due to the fact that membrane-bound proteins are difficult to obtain pure in a stable conformation in order to determine their three-dimensional structures. Recombinant expression of membrane-bound proteins has a high variability depending on the nature of the protein studied. Our research team is therefore interested in the development of recombinant expression and purification strategies of membrane-bound proteins involved in numerous human diseases, in order to study their functions but also to establish structurefunction relationships. My thesis work has focused on the expression and the purification of human receptors of the Hedgehog pathway, Patched and Smoothened. Alteration of these two proteins is known to be involved in numerous cancers but also in some neurological diseases. These two proteins have been expressed in two different systems : yeast cells Saccharomyces cerevisiae and Pichia pastoris and also in drosophila cells S2. We have been able to show that the protein Smoothened is expressed in Pichia pastoris in its native conformation in yeast cells an dit is therefore possible to purify it in order to perform structural studies. Patched and Smoothened have also been stably and functionaly expressed in S2 cells. This system is also interesting to perform comparative studies between drosophila and human proteins

Exposure to external stimuli promotes a variety of cellular responses including changes in morphology, gene expression and cell division status. These responses are promoted by signaling pathways composed of modules that are conserved from lower to higher eukaryotes. In Saccharomyces cerevisiae response to the external stimuli provided by mating pheromone is governed by the pheromone response pathway. This pathway is composed of a G protein coupled receptor/heterotrimeric G protein (Gαβγ) module and a MAP kinase cascade. Activation of this pathway allows the heterotrimeric G protein βγ dimer (Gβγ) to recruit polarity proteins to promote changes in cell morphology and to activate signaling through the MAP kinase cascade. Here we investigate the regulation of these pheromone-induced responses. We first examine how an asymmetric polarization response is generated. Normally, a gradient of pheromone serves as a spatial cue for formation of a polarized mating projection, but cells can still polarize when pheromone is present uniformly. Here we show that an intact receptor/Gαβγ module is required for polarization in response to both a gradient and uniform concentration of pheromone. Further investigation into regulation of Gβγ by Gα revealed that the two interaction interfaces between Gα and Gβ have qualitatively different roles. Our results suggest that one interface controls signaling whereas the other governs coupling to the receptor. Overall our results indicate that communication between the receptor and Gαβγ is required for proper polarization. We then examine how G1 CDKs regulate MAP kinase signaling. Response to pheromone is restricted to the G1 stage of the cell cycle. Once cells commit to a round of division they become refractory to mating pheromone until that round of division is complete. One contributor to this specificity involves inhibition of signaling through the MAP kinase cascade by G1 CDKs, but it was not known how this occurs. Here, we show that the MAP kinase cascade scaffold Ste5 is the target of this inhibition. Cln/CDKs inhibit signaling by phosphorylating sites surrounding a small membrane-binding domain in Ste5, thereby disrupting the membrane localization of Ste5. Furthermore, we found that disrupting this regulation allows cells to arrest at an aberrant non-G1 position. Our findings define a mechanism and a physiological benefit for restricting pheromone-induced signaling to G1. This thesis describes findings related to generation of an asymmetric polarization response, heterotrimeric G protein function, and coordination of differentiation signaling with cell division status. Lessons learned here might be applicable to the regulation of polarization and differentiation responses in other systems as the signaling modules are conserved.

Les glycosylphosphatidylinositols (GPIs) sont des modifications glycolipidiques permettant d'ancrer certaines protéines eucaryotes secrétées à la surface cellulaire. Les ancres GPI partagent la même sructure centrale très conservée protéine-PEthN-6Manα1-2Man&l-6Manαl-4GlcNH2&l-6inositol-PO4-lipide, synthétisée en plus de 12 étapes enzymatiques. Au moins une protéine a déja été identifiée pour chacune de ces étapes, à l'exception de l'addition du second mannose. Une stratégie bioinformatique m'a permis d'identifier une protéine non caractérisée, Gpi 18p, responsable de l'ajout de Man2. Des cellules mutantes où l'expression de Gpi 18p est bloquée accumulent Man1 (EthNP)-GPI indiquant que l'addition d'EthN-P aurait lieu plus tôt que préalablement supposé dans la biosynthèse GPI. J'ai étendu mes recherches sur les modifications des glycanes GPI aux champignons pathogènes ou intéressants sur le plan évolutif. Mon travail a contribué à la caractérisation génétique de la fonction de Smp3p chez l levure à fission Schizasaccharomyces pombe chez lequel l'addition d'un quatrième mannose s'est avérée obligatoire. Enfin, j'ai pu initialement caractérisé les GPIs chez le champignon pathogène Cryptococcus neoformans, par prédiction bioinformatique. La fonction des enzymes de la voie de biosynthèse des GPIs ainsi identifiées, a été testée chez les mutants gpi de S. Cerevisiae. Un système acellulaire utilisant les membranes de C. Neoformans a permis de visualiser des intermédiaires GPI. Nos données préliminaires suggèrent que les glycanes GPI pourraient être structurellement moins complexes que ceux de S. Cerevisiae et des mammifères de part l'absence des ramifications EthN-Ps et Man-4
Glycosylphosphatidylinositols (GPIs) are essential glycolipid modification of certain eukaryotic secretory proteins. Their primary function is to anchor a protein to the surface of a cell. However, GPIs have also been implicated in many important cellular processes such as cell adhesion, fungal pathogenesis, and cell wall formation. All GPI anchors share a highly conserved core structure of protein-CO-PEthN-6Manα1-2Manα1-6Manα1-4GlcNH2αl-6myo-inositol-PO4-lipid which is synthesized in the endoplasmic reticulum. In both S. Cerevisiae and humans, the GPI biosynthesis pathway involves more than ten enzymatic steps for which at least one protein has been identified except for addition of the second mannose. I devised a bioinformatics-based strategy to identify a previously uncharacterized protein, Gpi 18p, which is responsible for addition of intermediate lipid having an EthN-P side-branch indicating that EthN-P addition to GPI glycans may occur earlier in GPI biosynthesis than previously thought. . .

Cotranslational translocation is initiated by targeting of a ribosome-bound nascent polypeptide chain (RNC) to the endoplasmic reticulum (ER) membrane. The targeting reaction is coordinated by the signal recognition particle (SRP) through its interaction with the RNC and the membrane-bound SRP receptor (SR). A vacant translocon is a prerequisite for the subsequent nascent chain release from SRP-SR-RNC complex. It has been proposed that the protease-accessible cytosolic domains of the Sec61p complex play an important role in posttargeting steps by providing the binding site for the ribosome or interacting with the SR to initiate the signal sequence releasing. In this study, we have investigated the detailed mechanism that allows transfer of the ribosome-nascent chain (RNC) from the SRP-SR complex to the translocon using yeast S. cerevisiaeas the model system. Point mutations in cytoplasmic loops six (L6) and eight (L8) of yeast Sec61p cause reductions in growth rates and defects in translocation of nascent polypeptides that utilize the cotranslational translocation pathway. Sec61 heterotrimers isolated from the L8 sec61 mutants have a greatly reduced affinity for 80S ribosomes. Cytoplasmic accumulation of protein precursors demonstrates that the initial contact between the large ribosomal subunit and the Sec61 complex is important for efficient insertion of a nascent polypeptide into the translocation pore. In contrast, point mutations in L6 of Sec61p inhibit cotranslational translocation without significantly reducing the ribosome binding activity, indicating that the L6 and L8 sec61mutants impact different steps in the cotranslational translocation pathway. An interaction between the signal recognition particle receptor (SR) and the Sec61 complex has been proposed to facilitate transfer of the ribosome-nascent chain (RNC) complex to an unoccupied translocon. The slow growth and cotranslational translocation defects caused by deletion of the transmembrane span of yeast SRβ (srp102pΔTMD) are exaggerated upon disruption of the SSH1 gene, which encodes the pore subunit of a cotranslational translocation channel. Disruption of the SBH2 gene, which encodes the β-subunit of the Ssh1p complex, likewise causes a synthetic growth defect when combined with srp102pΔTMD. The in vivo kinetics of translocon gating by RNCs were slow and inefficient in the ssh1Δ srp102pΔTMD mutant. A critical role for translocon β-subunits in SR recognition is supported by the observation that deletion of both translocon β-subunits causes a block in the cotranslational targeting pathway that resembles elimination of either subunit of the SR, and could be partially suppressed by expression of carboxy-terminal Sbh2p fragments.