Intracellular targeting of lysosomal cathepsin D in COS cells.
Cathepsin D carries a mannose 6-phosphate sorting signal which is recognized by a specific mannose 6-phosphate receptor, presumably at the site of the trans Golgi network, which segregates cathepsin D from the secretory proteins, and results in targeting of the enzyme to the acidic prelysosomal compartments and lysosomes in mammalian cells. Recent evidence implies that another sorting signal resides within the polypeptide backbone of the precursor cathepsin D. To evaluate the role of the propeptide region of cathepsin D in mannose 6-phosphate receptor-independent targeting to lysosomes, we prepared a deletion mutant of rat cathepsin D lacking the propeptide portion and analyzed its intracellular targeting mechanism after transfection of the mutant cDNA as well as the wild-type cDNA into COS cells. The glycosylated mutant protein was retained intracellularly, and extracellular release of mutant protein was not observed after a 48 h chase. A cell fractionation experiment demonstrated that in the cells expressing the wild-type cathepsin D, the processed form of 44 kDa cathepsin D was recovered in the dense lysosomal fraction. In contrast, in the cells expressing the mutant protein, virtually all of the cell-associated cathepsin D was present in the light fraction which was enriched in the marker enzyme NADPH cytochrome c reductase, and this molecular form of cathepsin D was not observed in the dense lysosomal fraction. An immunofluorescence study revealed that the deletion mutant protein was accumulated within the endoplasmic reticulum, unlike the wild-type protein. These results suggest that the mutant cathepsin D is not correctly recognized by the intracellular sorting system in the endoplasmic reticulum, implying that the propeptide region of cathepsin D is essential for the export of cathepsin D from the endoplasmic reticulum.
Takeshima
H
H
Division of Physiological Chemistry, Kyushu University, Fukuoka.
Sakaguchi
M
M
Mihara
K
K
Murakami
K
K
Omura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002402:Cathepsin D / Q000032:analysis* / Q000235:genetics; D002458:Cell Fractionation; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics*; D005069:Evaluation Studies as Topic; D019084:Fluorescent Antibody Technique, Indirect; D017353:Gene Deletion; D006031:Glycosylation; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis; D013347:Subcellular Fractions
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D002402:Cathepsin D