EXPRESSION OF RAT CATHEPSIN-D CDNA IN SACCHAROMYCES-CEREVISIAE - IMPLICATIONS FOR INTRACELLULAR TARGETING OF CATHEPSIN-D TO VACUOLES

To investigate the intracellular transport mechanisms of lysosomal cat hepsin D in yeast cells, we produced cathepsin D in Saccharomyces cere visiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, Im munoblotting analysis by the use of an antibody specific for rat cathe psin D coding sequence produced an intermediate species which had a sl ightly higher molecular weight than that of the mature cathepsin D, Ce ll fractionation experiments demonstrated that the cathepsin D polypep tide was colocalized to the yeast vacuole with the marker enzyme carbo xypeptidase Y in a Ficoll step gradient, A biosynthesis study with pul se-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fraction ation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein, To elucidate the role of the pro peptide region of cathepsin D, which might function in the intracellul ar targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examin ed after transfection into yeast cells, Immunoblotting analysis demons trated that the propeptide-deleted mutant protein was recovered in a l ow quantity as compared with that in the case of yeast cells expressin g the wild-type protein in the isolated vacuolar fraction, Immunofluor escence analysis revealed that the deletion mutant protein appeared to be accumulated within the intracellular small vesicles but not in the carboxypeptidase Y-positive vacuoles, Overall, these results indicate that the rat cathepsin D precursor polypeptide is recognized by mecha nisms similar to those involved in the intracellular sorting of vacuol ar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cel ls, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.