The intracellular space of eukaryotic cells is subdivided into functionally distinct membrane-enclosed organelles. Regulation of these intracellular membranes requires an intricate network of specialized lipids and proteins that maintain organellar integrity and mediate transport between organelles. Proteins of the sorting nexin (SNX) family are membrane-binding regulators of transport events within the endomembrane system. The endomembrane system includes organelles associated with endocytic, secretory and degradative processes in the cell. The aims of this thesis were to functionally characterize SNX18 and SNX33, members of the SNX9-subfamily of sorting nexins, and to elucidate the role of SNX18 in autophagy. We demonstrated that all three proteins in the SNX9-family are capable of both membrane binding and remodeling, and interact with the membrane scission enzyme dynamin. We found that SNX18 localizes to endosomal structures in the endomembrane system, together with several identified factors previously described as regulators of endosomal transport. These results indicate that SNX18 mediates budding of membrane carriers in endosomal trafficking. In addition to this, knockdown of SNX18 in cultured cells was found to inhibit autophagy. Autophagy is a catabolic process by which cells degrade and recycle cellular components. It is a cellular response to various stress conditions such as oxidative stress, nutrient deprivation and infections. The components destined for degradation by autophagy are sequestered into a double-membrane structure called the autophagosome in which they are delivered to the lysosome. SNX18 interacts directly with proteins connected to autophagosome formation. Moreover, we demonstrated that the membrane-remodeling capability of SNX18 is a prerequisite for autophagosome formation. Taken together, our results lead to the conclusions that SNX18 remodels cellular membranes during formation of carriers for endosomal transport and that it is a positive regulator of autophagy and autophagosome formation.

Mitochondria are dynamic; they alter their shape through fission, fusion and budding of vesicles. Mitochondrial vesicles serve as a quality control mechanism enabling these organelles to rid themselves of damaged lipids and proteins. Dysregulation in mitochondrial dynamics and quality control have been linked to Parkinson’s Disease, making the identification of molecules requisite for these processes a priority. We identified the endocytic protein, Sorting nexin 9 (Snx9) through a genome wide siRNA screen for genes which substantially alter mitochondrial morphology and therefore are important for its maintenance. In this work, the role of Snx9 in mitochondrial morphology is examined. Ultrastructural imaging of mitochondria within cells silenced for Snx9 revealed unbudded vesicles along a hyperfused mitochondrial reticulum suggesting a role for Snx9 in the release of these vesicles. The vesicular profiles contained concentric membranous whorls enriched for neutral lipids. Localization studies suggest the Parkinson’s disease genes, Parkin and Vps35 localize to the unbudded profiles.

Mitochondria are dynamic; they alter their shape through fission, fusion and budding of vesicles. Mitochondrial vesicles serve as a quality control mechanism enabling these organelles to rid themselves of damaged lipids and proteins. Dysregulation in mitochondrial dynamics and quality control have been linked to Parkinson’s Disease, making the identification of molecules requisite for these processes a priority. We identified the endocytic protein, Sorting nexin 9 (Snx9) through a genome wide siRNA screen for genes which substantially alter mitochondrial morphology and therefore are important for its maintenance. In this work, the role of Snx9 in mitochondrial morphology is examined. Ultrastructural imaging of mitochondria within cells silenced for Snx9 revealed unbudded vesicles along a hyperfused mitochondrial reticulum suggesting a role for Snx9 in the release of these vesicles. The vesicular profiles contained concentric membranous whorls enriched for neutral lipids. Localization studies suggest the Parkinson’s disease genes, Parkin and Vps35 localize to the unbudded profiles.

Mitochondria are dynamic; they alter their shape through fission, fusion and budding of vesicles. Mitochondrial vesicles serve as a quality control mechanism enabling these organelles to rid themselves of damaged lipids and proteins. Dysregulation in mitochondrial dynamics and quality control have been linked to Parkinson’s Disease, making the identification of molecules requisite for these processes a priority. We identified the endocytic protein, Sorting nexin 9 (Snx9) through a genome wide siRNA screen for genes which substantially alter mitochondrial morphology and therefore are important for its maintenance. In this work, the role of Snx9 in mitochondrial morphology is examined. Ultrastructural imaging of mitochondria within cells silenced for Snx9 revealed unbudded vesicles along a hyperfused mitochondrial reticulum suggesting a role for Snx9 in the release of these vesicles. The vesicular profiles contained concentric membranous whorls enriched for neutral lipids. Localization studies suggest the Parkinson’s disease genes, Parkin and Vps35 localize to the unbudded profiles.