The lysosomal proenzyme receptor that binds procathepsin L to microsomal membranes at pH 5 is a 43-kDa integral membrane protein.

Abstract

Two lysosomal proenzymes, procathepsins L and D, bind to mouse fibroblast microsomal membranes at acidic pH. This membrane association is independent of the mannose-6-phosphate receptors and requires the presence of the N-terminal propeptides of the enzymes. We have identified the protein that specifically binds procathepsin L at pH 5. A 43-kDa membrane protein coimmunoprecipitated with procathepsin L at pH 5 but not at pH 7 when cells were denatured with detergents. Similarly, a 43-kDa integral membrane protein bound procathepsin L in three kinds of ligand blots at pH 5 but not at pH 7. A synthetic peptide containing the 24 N-terminal residues of mouse procathepsin L blocked the binding of procathepsin L to this integral membrane protein on ligand blots. These results indicate that the 43-kDa integral membrane protein is a lysosomal proenzyme receptor that specdflcally binds the procathepsin L activation peptide at acidic pH.Phosphomannosyl residues added post-translationally mediate binding of lysosomal proenzymes to mannose-6-phosphate (M6P) receptors (MPRs) within the Golgi (1, 2). The MPRs deliver the ligands either to early endosomes (3) or directly to late endosomal compartments or prelysosomes, where the low pH causes the proenzymes to dissociate from the MPRs (4, 5). It is not clear how enzymes destined for lysosomes are sorted within prelysosomes from the MPRs, which are recycled back to the Golgi.While MPRs play a major role in the intracellular transport of newly synthesized lysosomal enzymes in fibroblasts, additional proteins are probably involved in transporting the enzymes to lysosomes. For example, the transport of lysosomal enzymes in hepatocytes, Kupffer cells, and leukocytes is unaffected by a deficiency in the phosphotransferase enzyme, the protein that places phosphate residues on the high-mannose carbohydrate chains of lysosomal enzymes (6, 7). This suggests that there is an alternative M6P-independent intracellular pathway for lysosomal proteins in these cells.MPR-independent membrane association has been reported for an increasing number' of lysosomal proteins. Procathepsin D is membrane-associated in macrophages (8) and HepG2 cells (9, 10) in the presence of 10 mM M6P. Glucocerebrosidase and prosaposin are similarly bound to membranes in HepG2 cells (9), while procathepsin C is transiently membrane bound in Morris hepatoma 7777 cells (11), which lack the cation-independent MPR. It has not been established whether these proenzymes are interacting directly with membranes or with yet-unidentified receptor proteins embedded in the membranes. The role the membrane association plays in the intracellular transport of these proenzymes is also not known.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.We have demonstrated that at least two lysosomal proenzymes, procathepsins L and D, specifically bind to microsomal membranes at pH...

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