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 cathepsin D in yeast cells, we produced cathepsin D in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin D coding sequence produced an intermediate species which had a slightly higher molecular weight than that of the mature cathepsin D. Cell fractionation experiments demonstrated that the cathepsin D polypeptide was colocalized to the yeast vacuoles with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. A biosynthesis study with pulse-chase kinetic analysis revealed that the precursor polypeptide was accurately sorted to the yeast vacuoles as determined by cell fractionation, and that N-linked carbohydrate modifications were not required for vacuolar sorting of this protein. To elucidate the role of the propeptide region of cathepsin D, which might function in the intracellular targeting to the vacuole, a deletion mutant of cathepsin D lacking the propeptide was prepared and its intracellular targeting was examined after transfection into yeast cells. Immunoblotting analysis demonstrated that the propeptide-deleted mutant protein was recovered in a low quantity as compared with that in the case of yeast cells expressing the wild-type protein in the isolated vacuolar fraction. Immunofluorescence 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 mechanisms similar to those involved in the intracellular sorting of vacuolar proteins through the ER/Golgi/vacuolar sorting pathway in yeast cells, and that the propeptide has an important function in translocation of the cathepsin D polypeptide to the vacuole.
Division of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka.
Y
Y
Nishimura
H
H
Takeshima
M
M
Sakaguchi
K
K
Mihara
T
T
Omura
K
K
Kato
M
M
Himeno
D000818:Animals; D001692:Biological Transport; D002402:Cathepsin D / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000096:biosynthesis*; D019084:Fluorescent Antibody Technique, Indirect; D017353:Gene Deletion; D006031:Glycosylation; D009154:Mutation; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae; D014415:Tunicamycin / Q000494:pharmacology; D014617:Vacuoles / Q000187:drug effects* / Q000235:genetics
D018076:DNA, Complementary; D011994:Recombinant Proteins; D014415:Tunicamycin; D002402:Cathepsin D