Regulation of Cytosol to Lysosome Protein Targeting and Degradation Pathway

A novel pathway of targeting cytosolic proteins to lysosomes fordegradation has been established in our laboratory. Fructose-1,6-bisphosphatase (FBPase), the key gluconeogenic enzyme, is induced when yeast cells are grown in poor carbon sources. FBPase is rapidly degraded in the yeast lysosome (vacuole) when glucose-starved cells are replenished with fresh glucose. The glucose-induced degradation of FBPase is physiologically important. It eliminates proteins that are no longer needed and prevents energy futile cycles which may be hazardous to cells. Recent evidence indicates that FBPase is imported first into a novel class of small vesicles prior to uptake by the vacuole. The FBPase-associated vesicles have been purified to near homogeneity. These vesicles are distinct from all the known intracellular organelles examined. In order to identify molecules involved in the vesicle trafficking pathway, we have isolated several vid mutants defective in FBPase degradation. We cloned the VID24 gene which encodes a novel protein of 41 kD. In response to glucose, Vid24p is induced and localized to the FBPase-containing vesicles as a peripheral protein. Vid24p binding to the vesicles is critical for FBPase targeting from the vesicles to the vacuole. We have recently sequenced several VID genes and plan to study the expressions, distributions and functions of the Vid proteins. We have also developed an in vitro assay that faithfully reproduces FBPase import into the intermediate vesicles and the vacuole using permeabilized cells. The import is stimulated by cytosol and ATP. The import is a saturable process and shows substrate specificity. Our long term goals are to study how FBPase is imported into the vesicles and how FBPase is delivered from the vesicles to the vacuole for degradation.