The proregion of cathepsin L is required for proper folding, stability, and ER exit.
To investigate the role of the proregion in the biosynthesis and trafficking of mouse cathepsin L, cathepsin L cDNAs encoding proteins with altered proregions were constructed and their expression in COS cells was examined. As in transformed cells, normal mouse cathepsin L was secreted by COS cells. In contrast, two altered proregion cathepsin L proteins, one in which the proregion was deleted and a second in which the proregion was replaced with that of a homologous protein (aleurain), were retained within the cell and degraded over a period of 2-6 h. Immunofluorescence localization and the lack of effect of NH4Cl and brefeldin A on the turnover of the altered cathepsin L proteins indicated that their degradation occurred in the endoplasmic reticulum (ER). By using brefeldin A to induce colocalization of the UDPGlcNAc: lysosomal enzyme N-acetylglucosamine-1-phosphotransferase with the cathepsin L proteins in the ER, it was shown that the altered proteins were not susceptible to mannose phosphorylation as they exist in the ER. Trypsin sensitivity assays indicated that altered proregion proteins synthesized in COS cells or in vitro are misfolded. Taken together, these results indicate that the proregion plays an essential role in proper folding of cathepsin L. ER retention, decreased stability, and lack of mannose phosphorylation of the altered proteins are most likely secondary effects resulting from improper folding.
Tao
K
K
Department of Physiology, School of Medicine, Tufts University, Boston, Massachusetts 02111.
Stearns
N A
NA
Dong
J
J
Wu
Q L
QL
Sahagian
G G
GG
D016475:3T3 Cells; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D020126:Brefeldin A; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003517:Cyclopentanes / Q000494:pharmacology; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary / Q000737:chemistry; D010450:Endopeptidases; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004795:Enzyme Stability; D005455:Fluorescent Antibody Technique; D008358:Mannose / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D003517:Cyclopentanes; D018076:DNA, Complementary; D011498:Protein Precursors; D000643:Ammonium Chloride; D020126:Brefeldin A; D002403:Cathepsins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; D008358:Mannose