Mechanism and regulation of import and degradation of cytosolic proteins in the lysosome/vacuole

Protein degradation is an essential process in cells serving to eliminate abnormal proteins, regulate protein activities and provide amino acids during starvation for the synthesis of critical proteins. Protein degradation is regulated by hormones and nutrients, and increases when cells are deprived of serum. The increase in degradation is accompanied by the formation of autophagic vacuoles containing sequestered cytoplasm and organelles. Biochemical and morphological evidence indicates that starvation induced protein degradation occurs in lysosomes. This process has been studied by red-cell mediated microinjection to introduce radiolabeled proteins into cultured fibroblasts. Degradation of RNase a is enhanced in response to serum deprivation. The sequence required for RNase a degradation has been localized to a short peptide (lys-phe-glu-arg-gln). Antibodies raised against this pentapeptide precipitate 25–30% of cytosolic proteins. These proteins are selectively degraded when cells are deprived of serum. An hsc73 isozyme specifically recognizes the peptide sequence. Degradation of RNase A by isolated lysosomes is stimulated by ATP and hsc73 protein. We have extended our studies from mammalian lysosomes to the yeast vacuole. Catabolic inactivation of fructose 1,6-bisphosphatase (FBPase) is mediated by vacuolar degradation of FBPase in response to glucose. Degradation of FBPase is dependent on the PEP4 gene whose product is required for the activities of several vacuolar proteases. Immunofluorescence and cell fractionation experiments further indicate that FBPase is redistributed from the cytosol to the vacuole in response to glucose. The identification of such an import process defines a new protein targeting pathway in yeast. Degradation of FBPase requires protein synthesis. Vacuolar targeting of FBPase also requires the early part of the secretory pathway. We propose that a receptor protein is synthesized in response to glucose. This protein traverses the early part of the secretory pathway en route to the va-cuole. The presence of such a factor on the vacuolar membrane is required for import of FBPase to occur.