Lysosomes are responsible for the degradation of long-lived cytosolic proteins via a chaperone mediated autophagy pathway or by a macroautophagy pathway. The lysosomal protein LAMP-2 is involved in the autophagy degradation pathway. When this gene was deleted in mice, cardiomyopathy resembling Danon's Disease was found. Furthermore, autophagy may play some role in cancer, since the cancer suppressor gene beclin 1 is homologous to the autophagy gene APG6. Therefore, defects in lysosomal degradation may lead to abnormal cell growth and metabolic disorders. The long-term goals of our research are to study how cytosolic proteins are targeted to lysosomes/vacuoles for degradation. We have utilized the key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) as a marker protein to study this pathway in S. cerevisiae. FBPase is targeted from the cytosol to a novel class of vesicle (Vid vesicle) when S. cerevisiae are shifted from low glucose media to high glucose media. FBPase is then targeted to the vacuole for degradation. VID genes regulate the FBPase degradation pathway and are classified into two categories. Class A VID genes regulate FBPase import into Vid vesicles, while Class B VID genes control the fusion of Vid vesicles with the vacuole. We propose to (1) study how Class A genes regulate FBPase import. We will focus on VID21, VID27 and VID28, since their precise roles in FBPase import have not been determined and their gene products have not been characterized. (2) We will examine how FBPase is imported into Vid vesicles. We hypothesize that FBPase import is mediated by a transport machinery on Vid vesicles. We propose to purify and identify Vid vesicle proteins that interact with FBPase and test their functions in the import process. (3) Finally, we will determine whether a SNARE mediated fusion machinery is involved in the fusion of Vid vesicles with the vacuole. We will also determine how Class B genes regulate the fusion process.
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