Lysosomal proteases are active in cellular protein turnover and degradation of molecules which enter the cell by phagocytosis. Like secretory proteins, they are synthesized on membrane-bound ribosomes. Two pathways exist for their subsequent sorting from secretory proteins and transport to lysosomes. In fibroblasts, a phosphotransferase adds phosphate to mannose residues, forming the mannose-6-phosphate recognition marker which enables lysosomal proteins to react with specific receptors which carry them to lysosomes. In kidney and liver cells, an alternative pathway exists but the sorting sequences on the lysosomal enzymes and the cellular receptor it reacts with have not been characterized. Thus transport to the lysosome is not a default pathway but rather requires that biosynthetic forms of the lysosomal enzymes be recognized by specific cellular enzymes in a complex sequence. While the active sites of lysosomal cysteine proteases have been carefully studied, virtually nothing is known about the structural motifs which constitute processing enzyme or receptor binding sites on lysosomal enzymes. We will define these surface sequences for the lysosomal cysteine protease cathepsin L by coupling analysis of protein structure with molecular biology technology. Mutant enzymes will be constructed by site-specific and/or saturation mutagenesis techniques and introduced into eukaryotic cells by electroporation. Proper cellular segregation of the mutant enzymes will be assayed by immunofluorescence, pulse-chase analysis of the biosynthesis of the expressed protein, and cell fractionation. By this method we hope to identify the specific amino acid sequences recognized by the phosphotransferase and to identify surface sequences critical to the recognition of cathepsin L by processing proteases responsible for propeptide removal, asymmetric cleavage into light and heavy chains, and removal of carboxyl terminal amino acids. This research addresses an important and timely problem in modern cell biology, namely, what are the mechanisms responsible for the correct sorting of cellular or secretory proteins. A successful outcome of this work will increase our understanding of section protein-protein interactions and the basis of recognition specificity.
