Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 79-82).

As critical regulators of vesicular trafficking, Rab proteins comprise the largest GTPase family, with thirty-eight functionally distinct members and another twenty isoforms in the human genome. Activated Rab GTPases interact with effector proteins involved in vesicle formation, transport, tethering, docking and fusion. The specificity of Rab interactions with effectors and regulatory factors plays a central role with respect to the fidelity of membrane trafficking. Rab recognition determinants and the mechanisms underlying interactions with structurally diverse regulatory factors and effectors are complex and poorly understood. Using Rab5 mediated endocytic transport as a model system, the work described in this thesis provides insight into the structural basis underlying the interaction of effectors and regulatory factors with Rab GTPases. In addition, structural and biochemical approaches have been used to define how specific Rab5 interacting proteins function in the endocytic and recycling pathways. These results establish novel structural and functional concepts that can be tested using family wide analyses of Rab GTPase recognition determinants and regulatory roles in the cell.

Chez les eucaryotes, les RNA Binding Proteins (RBP) s’associent aux transcrits pour former des Particules Ribo-Nucléoprotéiques (mRNP) dynamiques, dont la localisation et la composition sont déterminantes pour la maturation, l’export, la stabilité et la traduction des ARNm. Les protéines à motif LA sont des protéines de liaison à l’ARN, présentes chez plusieurs centaines d’espèces eucaryotes, qui se répartissent en 5 sous-familles : LA authentiques, LARP1, 4, 6 et 7. Les membres de ces sous-familles partagent des caractéristiques évolutives, des domaines additionnels et des fonctions conservés. Mes travaux de thèse ont contribué à l’étude fonctionnelle de protéines LARP6 chez Arabidopsis thaliana. On sait, à l’heure actuelle, que chez Manduca sexta et plusieurs espèces de vertébrés, LARP6 est impliquée dans la régulation de la différenciation cellulaire. Chez l’Homme, elle agit en tant que RBP pour coordonner la traduction des ARNm codant les sous-unités du collagène de type I. Les plantes vasculaires ont la particularité de contenir plusieurs protéines LARP6, classées en trois groupes évolutifs. Chez A. thaliana, l’unique représentant de chaque sous-famille semble s’être spécialisé. D’ailleurs, les protéines LARP6b et c ont des profils d’expression mutuellement exclusifs, où LARP6c est présente dans le pollen et LARP6b est ubiquitaire mais absente du gamétophyte mâle. Nous avons axé notre travail sur la protéine LARP6c et démontré qu’elle est cytoplasmique et impliquée, dans le pollen, dans le contrôle de la quantité d’ARNm codant des acteurs du transport vésiculaire. Les ARNm identifiés comme cibles potentielles de LARP6c codent, eux aussi, des facteurs impliqués dans le transport ; et possèdent dans leur 5’-UTR deux motifs qui pourraient permettre leur co-régulation par fixation de RBP. La délétion de LARP6c, affecte la capacité du tube pollinique à se diriger vers l’ovule suggérant un défaut de communication; ce qui est cohérent avec la dérégulation des ARNm codant des acteurs de la sécrétion/réception de signaux extracellulaires. Nous proposons que LARP6c intervient, dans le pollen, en tant que protéine de mRNP et co-régule la traduction et/ou la stabilité de transcrits codant des acteurs des voies de communications dépendantes de la sécrétion et de l’endocytose, et intervenant dans les échanges mâle/femelle / In eucaryotes, RNA Binding Proteins (RBP) associate with transcripts to form dynamic Ribo-Nucleoprotein Particles (mRNP), whose localization and composition are determinant for mRNA maturation, export, stability and translation. LA motif proteins are RNA binding proteins, found in several hundred eucaryotic species, which fall in 5 sub-families: genuine LA, LARP1, 4, 6 and 7. Members of these subfamilies share conserved evolutionary history, additional motifs and functions. My thesis work contributed to deciphering the functional properties of the Arabidopsis thaliana LARP6 proteins. Currently, we know that in Manduca sexta and many vertebrates species LARP6 is implicated in the regulation of cellular differentiation. In humans, it acts as an RBP to coordinate the translation of mRNA coding for type I collagen subunits. Vascular plants differ in possessing many LARP6 proteins classified in three evolutionary groups. In A. thaliana, the unique member of each subfamily seems to be specialized. LARP6b and c proteins present mutually exclusive expression profiles, with LARP6c only present in pollen and LARP6b ubiquitously expressed except in the male gametophyte. We mostly focused our work on LARP6c and showed it to be cytoplasmic and implicated in controlling the level of mRNAs encoding vesicular transport actors in pollen tubes. Putative identified LARP6c mRNA baits also encode proteins involved in transport and share two motifs in their 5’-UTR that could allow their co-regulation via RBP binding. LARP6c deletion induces deficiencies in pollen tube guidance towards the ovule, suggesting a communication default. This is consistent with the deregulation of mRNA coding for extra-cellular signal secretion/reception actors. We propose that LARP6c acts as an mRNP protein in pollen and co-regulates translation and/or stability of mRNA coding for actors of communication pathways depending on secretion and endocytosis; hence acting on male/female exchanges.

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicles are targeted to sites of exocytosis on the plasma membrane in part by a conserved multi-subunit protein complex termed the exocyst. In addition to tethering vesicles to the plasma membrane, the exocyst complex and components therein may also add a layer of regulation by directly controlling assembly of the SNARE complex, which is required for membrane fusion, as well as other regulatory factors such as Sec1p. In the past, we have shown that Sec6p interacts with Sec9p in vivo and that that interaction retards binary SNARE complex formation in a SNARE assembly assay. Though many interactions have been mapped using in vitro methods, confirming them in vivoand placing them into the context of a complete model that accounts for all observed interactions (and lack of interactions) has proven difficult. In order to address these problems, I have studied the interactions between Sec6p and other factors involved in exocytosis at the plasma membrane via in vivo methods. My hypothesis was that Sec6p interaction with Sec9p and subsequent inhibition of SNARE complex assembly in vitro was an intermediate state and Sec6p was part of a set of cofactors that accelerated SNARE complex assembly in vivo. To test this hypothesis I showed that the interaction between the plasma membrane t-SNARE Sec9p and the yeast exocyst subunit Sec6p can be observed in vivoand designed point mutations to disrupt that interaction. Interestingly, I also showed that Sec6p:Sec9p interaction involves the free pool of Sec6p rather than the exocyst bound fraction of Sec6p. Point mutations in the N-terminal domain of Sec6p result in temperature sensitive growth and secretion defects, without loss of Sec6p-Sec9p interaction. However, at the non-permissive temperature, the exocyst subunits Sec5p, Sec10p and Sec15p are mislocalized and are absent from the exocyst complex. The resulting subcomplex, containing Sec3p, Sec8p, Exo70p and Exo84p, remains stably assembled and localized at sites of polarized secretion. This subcomplex is likely due to disruption of interaction between Sec6p and Sec5p, and may be similar to that observed at restrictive temperatures in the sec6-54temperature sensitive mutant. Additionally, one of the sec6 temperature sensitive mutants displays a loss of binding to the yeast regulatory protein Sec1p. In vitro binding studies indicate a direct interaction between Sec1p and the free pool of the wild-type Sec6p protein, suggesting close interplay between Sec6p and Sec1p in the regulation of SNARE complexes. A coherent model which incorporates all these interactions has continued to be elusive. However, the results I have found do suggest several hypotheses which should prove testable in the future.

No description available.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Le transport intracellulaire du cholestérol et sa distribution correcte au niveau des différentes membranes sont essentiels pour assurer de nombreuses fonctions cellulaires. Malgré l’importance de ce transport les mécanismes de sa régulation restent encore mal connus. L’objectif de cette thèse était de mieux caractériser les acteurs du transport intracellulaire du cholestérol. Dans ce contexte, nous nous sommes intéressés à deux acteurs de ce transport : la dynamine et les Rab GTPases. Dans la première partie de la thèse nous avons utilisé le dynasore, un inhibiteur pharmacologique de la dynamine pour étudier le rôle de la dynamine dans le contrôle du transport endolysosomal dans les cellules HeLa et les macrophages humains. Nous avons ainsi confirmé le rôle de la dynamine dans la sortie du compartiment endolysosomal et la régulation de l’homéostasie du cholestérol. Dans la deuxième partie de la thèse, nous avons étudié le rôle de Rab7 et de Rab9 dans le transport du cholestérol en utilisant la technique d’ARN interférence ainsi que l’expression de mutants dominant négatifs. Nous avons montré qu’en plus de son rôle classique dans les étapes tardives du transport du cholestérol, Rab7 contrôle les étapes précoces du transport endosomal. Enfin, nous avons évalué le rôle de Rab7 dans notre modèle de macrophages humains surchargés. Nous avons mis en évidence un effet limité de l’inactivation de Rab7 sur le contrôle de l’homéostasie du cholestérol mais à l’inverse un effet majeur pour l’efflux du cholestérol vers l’apo AI. En conclusion, notre étude a permis de mieux caractériser le transport vésiculaire du cholestérol et de démontrer son importance dans la régulation de l’homéostasie intracellulaire en cholestérol. Nos résultats permettent également d’établir le rôle critique de Rab7 dans le trafic des LDL au niveau des endosomes précoces. / Intracellular transport of cholesterol and its distribution within cellular membranes are essential to maintain correct cellular functions. Despite the importance of this transport, mechanisms that regulate cholesterol transport still poorly defined. The objectives of this thesis were to better characterize the actors of intracellular cholesterol trafficking. In this context, we focused our interest on two known actors of intracellular transport : dynamin and Rab GTPases. In the first part of this thesis, we used dynasore, a pharmacological dynamin inhibitor, to study the role of dynamin in the control of endolysosomal transport in HeLa cells and human macrophages. We confirmed the role of dynamin in endolysosomal sorting and cholesterol homeostasis regulation. In the second part of this thesis, we studied the role of Rab7 and Rab9 in the regulation of cholesterol transport using RNA interference and dominant negative mutants. We showed that in addition to it classical role in late steps of cholesterol transport, Rab7 controls also early steps of endosomal trafficking. Finally, we evaluated the role of Rab7 in our model of loaded human macrophages. We showed a weak impact of Rab7 inactivation on cholesterol homeostasis but a major effect on cholesterol efflux to apo AI. In conclusion, in this study we have better characterized the vesicular transport of cholesterol and demonstrated its importance in cholesterol intracellular homeostasis. Our results also establish that Rab7 plays a critical role in the sorting of LDL at the early endosome.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 209-224.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Not embargoed. Vita. Bibliography: 229-249.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and to the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multi-subunit tethering complexes and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in other trafficking pathways. Contrary to these other pathways, our lab previously suggested that the exocyst subunit Sec6, a component of the exocytosis-specific tethering complex, inhibited Sec9:Sso1 SNARE complex assembly due to interactions in vitro with the SNARE protein Sec9 (Sivaram et al., 2005). My goal for this project was to test the hypothesis that Sec6 inhibited SNARE complex assembly in vivo. I therefore chose to generate Sec6:Sec9 loss-of-binding mutants, and study their effect both in vitro and in vivo. I identified a patch of residues on Sec9 that, when mutated, are sufficient to disrupt the novel Sec6-SNARE interaction. Additionally, I found that the previous inhibitory role for Sec6 in SNARE assembly was due to a data mis-interpretation; my re-interpretation of the data shows that Sec6 has a mild, if any, inhibitory effect on SNARE assembly. My results suggest a potential positive role for Sec6 in SNARE complex assembly, similar to the role observed for other tether-SNARE interactions.