UC San Diego

Eukaryotic cells maintain a complex organization of vesicular transport pathways through which integral membrane proteins and lipids travel. This allows the cell to maintain the proper functional composition of various intracellular membrane-bound compartments and communication with the external environment via the plasma membrane.

I have studied two facets of vesicular trafficking. Chapter 1 describes the process of vesicle budding from the Golgi and its relationship to the unique morphology of the Golgi. Chapter 2 discusses the regulation of endocytosis and an attempt to find a target for a ubiquitin-specific protease necessary for endocytosis in yeast.

The mechanism underlying the Golgi's flattened structure and its relation to secretory function is an intriguing question in cell biology. Golgi membranes, from yeast to humans, are enriched in phosphatidylinositol-4-phosphate (PtdIns(4)P). In a lipid binding screen, GOLPH3 was identified as a novel PtdIns(4)P-binding protein that depends upon PtdIns(4)P for its Golgi localization. GOLPH3 binds MYO18A, connecting the Golgi to F-actin, thus transmitting a tensile force necessary for Golgi budding and morphology. The same process generating the extended Golgi ribbon observed by fluorescence microscopy and the flattened form observed by electron microscopy drives the formation of transport vesicles.

Integral plasma membrane proteins are down-regulated by endocytosis followed by trafficking to the lysosome for degradation. Can1p is a yeast arginine permease at the plasma membrane and undergoes such regulation. Canavanine is a toxic arginine analog that enters the cell through Can1p. A screen of viable yeast deletion mutants to identify genes which when deleted result in hypersensitivity to canavanine was conducted to identify novel components involved in down-regulation and internalization of plasma membrane proteins. Three genes identified were ubiquitin-specific proteases. By visual screening, deletion of UBP3 (UBiquitin-specific Protease 3) was observed to stabilize Can1p-GFP and other endocytic cargoes at the plasma membrane. Rescue experiments show that Ubp3p catalytic activity is necessary for endocytosis. Biochemical experiments indicate that Ubp3p is in a complex with Bre5p, and that Bre5p is also necessary for plasma membrane protein internalization. In conclusion, Ubp3p and its co-factor Bre5p are responsible for deubiquitination and positive regulation of an unidentified protein involved in endocytosis.