Cell polarity is a fundamental feature of eukaryotic cells. The intracellular trafficking of proteins between cellular compartments and the cytoskeleton regulates the establishment and maintenance of cell polarity. These events are largely regulated by the Ras superfamily of small GTPases. Rabs (Ras-related in brain) and Arfs (ADP-ribosylation factor), subfamilies of the Ras superfamily, play a major role in coordinating vesicle formation and in mediating vesicle association with cytoskeletal components for transport of vesicles to their correct cellular compartment or membrane domain. Although these GTPases mediate membrane trafficking, how they interact with each other and effector proteins to participate in vesicle formation and transport at the trans-Golgi network (TGN) remains poorly understood. The TGN is a major sorting station for biosynthetic cargo molecules (i.e. apical and basolateral) into distinct carriers for delivery to their correct acceptor compartment. We have analyzed the role of Rab14 at the Trans-Golgi Network (TGN), apical endosomes, and in vesicle formation at the TGN by overexpressing wild type and mutant forms of Rab14 in polarized and non-polarized cells. We localized Rab14 to a domain of the TGN distinct from that of the TGN/basolateral protein, TGN38. Overexpression of inactive Rab14 causes the TGN to expand and mislocalizes the apical membrane protein VIP/MAL to the lateral membrane. Furthermore, inactive Rab14 colocalizes with Arf1-GDP at the TGN and increases the amount of COPI at the TGN. These results suggest that Rab14 is involved in the trafficking of proteins from the TGN to apical endosomes and modulates vesicle formation at the TGN by regulating Arf1 activation and COPI localization to distinct domains of the TGN. Thus, Rab14 defines a new role for COPI-mediated vesicle formation at the TGN.

Lesions in the human Lyst gene are associated with the lysosomal disorder Chediak Higashi Syndrome. The absence of Lyst causes the formation of enlarged lysosome related compartments in all cells. This defect results in severe immunodeficiency, neurological dysfunction, and ultimately in death. Despite decades of research, the mechanism for how these enlarged compartments arise is not well established. Two opposing models have been proposed for Lyst function. The fission model describes Lyst as a positive regulator of fission from lysosomal compartments, while the fusion model identifies Lyst as a negative regulator of fusion between lysosomes. To date, a consensus on which model is correct has not been reached. This thesis details my investigation of Lyst function using Dictyostelium discoideum. To establish a definitive model for the function of the Dictyostelium Lyst ortholog, LvsB, we used assays that distinguish between defects in vesicle fusion versus fission. We compared the phenotype of cells defective in LvsB with that of two known fission defect mutants ([mu]3 and WASH null mutants). The temporal localization characteristics of the post-lysosomal marker vacuolin, as well as vesicular acidity and fusion dynamics of LvsB null cells are distinct from those of both fission defect mutants. These distinctions are predicted by the fusion defect model and implicate LvsB as a negative regulator of vesicle fusion. This work also presents evidence that LvsB antagonizes the function of two fusion regulatory proteins, Rab14 and dLIP5. The Dictyostelium Rab14 GTPase is known to stimulate lysosome fusion, and here we implicate dLIP5 as a promoter of Rab14 activity. Constitutive activation of Rab14 increases vesicle fusion in wild type cells but not in dLIP5 mutant cells. Thus, Rab14 activity is dependent on dLIP5. Additionally, the aberrant vesicle morphology and fusion phenotypes of LvsB mutant cells are suppressed by expression of dominant inactive Rab14 or disruption of dLIP5. This suppression suggests that LvsB antagonizes Rab14 activity to negatively regulate vesicle fusion. These studies validate the fusion model for LvsB function and provide new insights into the relationships that dictate vesicle fusion regulation. By extension, we propose that Lyst negatively regulates vesicle fusion by antagonizing the activity of a RabGTPase. / text