| To survive extreme environmental conditions, pathological situations, and in response to certain developmental programs, eukaryotic organisms employ a degradative process called autophagy. This pathway targets cytosolic components and organelles for destruction in the vacuole/lysosome through engulfment by double-membrane vesicles that form in the cytosol. Despite our increased understanding of the significance of this pathway to eukaryotic development and disease, and the fact that many specific genetic components have been identified, the molecular mechanisms leading to the formation of these double-membrane vesicles remains largely unknown. Many advances have been made with the discovery of a pathway closely related to autophagy in the yeast Saccharomyces cerevisiae. This pathway, termed the Cytoplasm to Vacuole Targeting (Cvt) pathway, is a biosynthetic pathway to target residential vacuolar hydrolases properly; however, despite its metabolic role, it is both genetically and molecularly related to the catabolic autophagy pathway.;This thesis examines the function of several molecular components involved in these pathways in S. cerevisiae. Chapters 2 and 3 describe the characterization of ATG23, a previously unidentified gene required for Cvt and efficient autophagy. Atg23 displays functional and physical interactions with Atg9, a protein of unknown function. This work has described new functions for five Atg proteins, as well as a previously unidentified step in Cvt and autophagy. In chapter 4, I present analysis of the Atg12-Atg5-Atg16 protein complex, whose function was previously unknown. I provide evidence consistent with the hypothesis that these proteins form a coat complex around the double membrane vesicle. This includes fluorescent microscopy images showing the Atg16-containing protein complex forming a structure around a growing vesicle. This is the first report of visualizing vesicle formation in vivo by fluorescence microscopy in S. cerevisiae. This technique can be expanded to visualize many Atg proteins during the formation of double membrane vesicles.;These discoveries have advanced our understanding of these two pathways. There are many remaining questions, however; Chapter V describes work that remains to be done to advance on these discoveries. |
