SUBCELLULAR-LOCALIZATION AND TARGETING OF CATHEPSIN-E

The subcellular distribution and targeting of the nonlysosomal asparti c proteinase cathepsin E have been studied using mouse L cells and mon key 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 sign ificant degradation and its single N-linked oligosaccharide remained s ensitive to endo-beta-N-acetyl-glucosaminidase H. When cathepsin E was overexpressed by transient transfection in Cos 1 cells, it was very s lowly secreted into the media. The intracellular form of the enzyme co ntained a high mannose oligosaccharide which was processed to a comple x type species upon secretion. In double label immunofluorescence stud ies, cathepsin E co-localized with cathepsin D myc-KDEL, an endoplasmi c reticulum (ER) marker. Subcellular fractionation on a Percoll densit y gradient showed that the cathepsin E co-migrated with membranous ves icles that were distinct from dense lysosomes. Only a trace amount of the enzyme was recovered in the soluble fraction. These findings indic ate that in L cells and Cos 1 cells, the intracellular location of cat hepsin E is the endoplasmic reticulum. To identify the protein sequenc es required for ER retention, we made chimeric proteins between cathep sin 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 impo rtant for its retention in the ER. Within this region, Cys(7), which i s involved in covalent dimer formation, plays a significant role in th e retention.