A heretofore unidentified receptor which may be critical for protease targeting to lysosomes has been identified in this laboratory. Two lysosomal proproteases, procathepsins L and D, are membrane associated at acidic pH in microsomal vesicles derived from mouse fibroblasts. The binding, which is specific for the pro but not the mature enzymes, is saturable and is mediated by a factor which can be removed from the membranes by protease treatment, suggesting that this is a specific interaction with a receptor protein. As the association occurs at the pH at which lysosomal enzymes are released from mannose-6-phosphate receptors, the membrane association could be involved in protein sorting in prelysosomes. Synthetic peptides based on the amino-terminal sequence of procathepsin L will be used in binding assays to establish which region of the capthepsin L propeptide associates with membranes. Specific amino acids in the receptor binding regions will be altered by site-directed mutagenesis. These analyses will identify specific amino acids responsible for correct subcellular targeting. Altered recombinant protein will be expressed from an SV40 promoter in COS cells, isolated from cell culture medium, and assayed for membrane binding ability. Following this initial screen in which an altered protein is identified with has lost its ability to associate with membranes in a pH-dependent manner, an in vivo functional assay will be performed. The cDNA plasmid will be expressed in HeLa cells which have been demonstrated to target wild-type recombinant lysosomal enzymes correctly. These studies will establish whether the stretch of amino acids responsible for the pH-dependent membrane binding contains information essential for lysosomal targeting. %%% The interior of a eukaryotic cell is highly compartmentalized, in order for the cell to carry out its necessary functions. One set of critical functions, involving the hydrolytic degradation of endocytosed or autophagocytosed macromolecules, is restricted to a membrane-enclosed acidic compartment called the lysosome. These degradations are catalyzed by proteolytic, glycosidic, and other acid hydrolytic enzymes, which are contained within the lysosomes. This project addresses the question of how the hydrolytic enzymes get into the lysosomes. Some years ago, it was found that many lysosomal enzymes shared a common oligosaccharide structure, specifically a mannosyl-6-phosphate residue, which is recognized and bound by two membrane bound man-6-P receptors. These receptors traffic through the endosome-lysosome system and deliver bound lysosomal enzymes to the endosomal-lysosomal compartment. However, evidence has been accumulating in the literature that the man-6-P receptors cannot account for all instances of lysosomal enzyme targeting in cells. The discovery of a new lysosomal enzyme binding membrane protein, which does not act via the man-6-P residues on lysosomal enzymes, is therefore an important finding which will undoubtedly broaden our view of how lysosomal enzymes get into lysosomes.
