Diss. (sammanfattning) Stockholm : Stockholms universitet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Cornell University, May, 2007. / Vita. Includes bibliographical references.

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.

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.

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.

Vesicular trafficking, the movement of vesicles between organelles and the plasma membrane for secretion, consists of multiple highly regulated processes. Many protein families function as specificity and regulatory determinants to ensure correct vesicle targeting and timing of trafficking events. The SNARE proteins dock and fuse vesicles to their target membranes. Sec1/Munc18 (SM) proteins regulate membrane fusion through interactions with the SNAREs—SM proteins have been shown to act as both inhibitors and stimulators of SNARE assembly and membrane fusion. However, the details of these SM protein functions are not understood. Constructing a model of SM protein function has been challenging due to the various modes of interactions reported between SM proteins and their SNAREs. SM proteins interact with their cognate SNAREs and SNARE complexes through several distinct modes. The most conserved mode is an interaction with the syntaxin N-peptide; other modes of binding, such as the syntaxin closed conformation, are hypothesized to be specific for specialized cell types. In order to elucidate the general function of SM proteins, I investigated the function of the endosomal SM protein Vps45p by analyzing its interactions with its cognate syntaxin Tlg2p and its role in SNARE assembly. I had two main hypotheses: that the Tlg2p N-peptide does not solely mediate the interaction between Vps45p and Tlg2p; and that Vps45p functions to stimulate SNARE complex assembly. I systematically mapped the interaction between Vps45p and Tlg2p using various Tlg2p truncations containing the different domains of Tlg2p and discovered a second binding site on Tlg2p that corresponds to the closed conformation. The neuronal SM-syntaxin pair interacts in a similar manner, indicating that this interaction mode is conserved. To characterize the closed conformation binding mode further, and determine its relationship to the N-peptide binding mode, I developed a quantitative fluorescent electrophoretic mobility shift assay. Results indicate that these two sites do not bind simultaneously and that the N-peptide binding modulates the closed conformation affinity. Furthermore, I monitored the effect of Vps45p on SNARE complex assembly using size exclusion chromatography. Under the conditions tested, Vps45p did not appear to stimulate SNARE complex assembly. The work presented here addresses several puzzling issues in the field and significantly contributes to the construction of a new mechanistic model for SM protein function. In this new model, the SM protein is recruited to the membrane by its interaction with the syntaxin N-peptide. The SM protein then binds the syntaxin closed conformation thus inhibiting SNARE complex assembly. Upon dissociation of the SM protein from the closed conformation, an event perhaps regulated by the SM protein, syntaxin opens and interacts with the other SNAREs to form a SNARE complex. Fusion ensues, stimulated by the SM protein.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 106-116.