SUMMARY<p><p>Gap1, the general amino acid permease of Saccharomyces cerevisiae, is a plasma membrane protein which is synthesized and most active under conditions of poor nitrogen supply. Under these conditions, the role of Gap1 is to scavenge external amino acids to be used as nitrogen sources or directly as building blocks for protein synthesis. Gap1 is a member of the Yeast Amino Acid Transporter (YAT) family, a family of amino acid transporters highly conserved in bacteria and fungi. <p>The intracellular trafficking of Gap1 has been the subject of intense investigation as well as the role of lipids (in particular sphingolipids) in its activity and folding. These studies have all been carried out in the cellular context using versatile yeast genetics as exploratory tools. While such in vivo investigations allow to identify physiologically relevant features, they do not provide the details to understand the molecular basis of these phenomena. In order to decipher the molecular features responsible for biological functions, physiological analysis must be combined with biochemical, biophysical and structural studies which typically will be performed on isolated and purified proteins.<p>Our work during this study fits in this trans-disciplinary approach that aims at understanding different properties of the protein: (I) how sphingolipids modulate the activity of Gap1, (II) what part of the protein, and more specifically, what residues, are implicated in its regulation and (III) what are the molecular determinants of the multi-specificity of Gap1.<p>At the beginning of this work, Gap1 had not been previously produced and purified. During this thesis we have identified suitable expression and purification strategies for Gap1 and initiated first characterization studies. In the process, we have developed a new expression system in S. cerevisiae based on the nitrogen catabolite repression. <p>The capacity of this system to express other proteins was successfully tested for two other yeast transporters (Mep2 and Uga4) and for two human proteins: MD-2, a soluble protein and Vglut1, a vesicular transporter.<p>Therefore, we propose our expression design as a viable alternative to existing systems of production in yeast and as a valuable tool to be tested when starting the expression of a new target. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Des études ont montré que la substitution, par le tourteau de canola (TC), d’autres sources protéiques augmente la production laitière des vaches d’en moyenne 1 kg/j. Par contre, les prédictions des protéines métabolisables (PM), en utilisant le modèle du NRC (2001), laissent penser que l'inclusion de TC diminue l’approvisionnement en PM pour les vaches. Une différence majeure entre le tourteau de soya (TS) et le TC est la valeur de la digestibilité intestinale estimée dans le modèle du NRC (2001): la digestibilité du TC est de 75 % contre 93 % pour le TS. Cela pourrait être dû à une sous-estimation de la digestibilité avec la technique de sachets mobiles. Ainsi, notre objectif était d'évaluer la digestibilité des protéines non dégradées dans le rumen par la méthode conventionnelle des sachets mobiles et par l’enrichissement avec le¹⁵N. Quatre vaches munies d’une fistule ruminale et d’une canule duodénale ont été nourries avec : deux sources de tourteau de canola, une commerciale (TCC) et l’autre marquée avec ¹⁵N(TCM), et du tourteau de soya (TS). Les résultats montrent qu’il n’y a aucune différence significative dans la dégradabilité effective de la protéine brute entre le TS, le TCC et le TCM (63,5 %, 62,8 % et 63,1 %, erreur standard moyen (ESM) = 2,47, respectivement). Pour la digestibilité intestinale de la protéine brute, aucunedifférence n’a été observéeentre les deux méthodes citées pour le TCM (76,1 %, et 70,04 %, ESM = 0,2, respectivement). Par contre, les résultats indiquent un effet significatif de la taille des pores des sachets sur la digestibilité intestinale. Comme nos résultats montrent qu’il n’y a pas une sous-estimation de la digestibilité, plusieurs questions se posent sur le profil des acides aminés du TC qui pourrait être responsable de la réponse positive sur la production laitière de substitution d’autres sources protéiques. / Many studies have shown that substituting canola meal (CM) to other protein sources in dairy cows, increases milk production average by 1 kg/d. Contrariwise, the predictions of metabolizable protein, using the model of the NRC (2001), showed that the inclusion of CM decreases the predicted availability of metabolizable protein for cows. A major difference between soybean meal (SBM) and CM is the value of the estimated intestinal digestibility in the model of NRC (2001): the intestinal digestibility of CM is 75% against 93% for SBM. This could be caused by an underestimation of the intestinal digestibility with the mobile bags technique. Then, the goal of this study was to evaluate intestinal digestibility of the undegraded protein by the conventional method of mobile bags and ¹⁵N method. Four cows equipped with ruminal fistula and duodenal cannula were fed with: two sources of canola, commercial canola meal (CCM), and the other canola meal labelled with¹⁵N (LCM), and a commercial soybean meal (SBM). Results showed that there is no significant difference in the effective degradability of CP for SBM, CCM and LCM (63.5%, 62.8% and 63.1%, standard error of the mean (SEM) = 2.47, respectively). For intestinal digestibility of the crude protein CP, there was no difference between the method of mobile bags and ¹⁵N method for the LCM (76.1%, 70.04%, SEM= 0.2, respectively). Contrariwise, the results indicate a significant effect of bag pore size on the intestinal digestibility. As our results show that there is no underestimation of digestibility, several questions arise on the amino acid profile of the CM that may be responsible for the positive response in milk production of substituting CM to other protein sources.