title: Subcellular localization and targeting of cathepsin E.
abstract: The subcellular distribution and targeting of the non-lysosomal aspartic proteinase cathepsin E have been studied using mouse L cells and monkey Cos 1 cells that were transfected with cDNA encoding cathepsin E. The cathepsin E was retained in L cells for at least 20 h without significant degradation and its single N-linked oligosaccharide remained sensitive to endo-beta-N-acetyl-glucosaminidase H. When cathepsin E was overexpressed by transient transfection in Cos 1 cells, it was very slowly secreted into the media. The intracellular form of the enzyme contained a high mannose oligosaccharide which was processed to a complex type species upon secretion. In double label immunofluorescence studies, cathepsin E co-localized with cathepsin D-myc-KDEL, an endoplasmic reticulum (ER) marker. Subcellular fractionation on a Percoll density gradient showed that the cathepsin E co-migrated with membranous vesicles that were distinct from dense lysosomes. Only a trace amount of the enzyme was recovered in the soluble fraction. These findings indicate that in L cells and Cos 1 cells, the intracellular location of cathepsin E is the endoplasmic reticulum. To identify the protein sequences required for ER retention, we made chimeric proteins between cathepsin E and pepsinogen, an aspartic proteinase that is rapidly secreted by Cos 1 cells. We found that amino acids 1-48 of cathepsin E are important for its retention in the ER. Within this region, Cys7, which is involved in covalent dimer formation, plays a significant role in the retention.
authors.affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
authors.forename: E M
authors.initials: EM
authors.lastname: Finley
authors.forename: S
authors.initials: S
authors.lastname: Kornfeld
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D020059:Cathepsin E; D002403:Cathepsins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002460:Cell Line; D003432:Cross-Linking Reagents; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D000882:Haplorhini; D007739:L Cells / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010436:Pepstatins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology*
chemical_list: D000906:Antibodies; D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D010436:Pepstatins; D011993:Recombinant Fusion Proteins; C014195:Streptomyces pepsin inhibitor; D012685:Sepharose; D002403:Cathepsins; D020059:Cathepsin E; C031375:pepstatin