THE PH-DEPENDENT MEMBRANE ASSOCIATION OF PROCATHEPSIN-L IS MEDIATED BY A 9-RESIDUE SEQUENCE WITHIN THE PROPEPTIDE

The lysosomal proprotease procathepsin L binds to mouse fibroblast mic rosomal membranes at pH 5, but mature active cathepsin L does not (McI ntyre, G. F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-154 45). This binding is not dependent on N-linked carbohydrate as procath epsin L synthesized in cells treated with tunicamycin still shows pH-d ependent membrane association. These results suggest that the propepti de (Thr18-Lys113) of the cysteine protease mediates its pH-dependent m embrane association. Synthetic peptides containing either 24 or 9 resi dues from the N-terminal portion of the mouse procathepsin L propeptid e inhibited the binding of mouse procathepsin L to microsomal membrane s at pH 5. In contrast, the pH-dependent membrane association was not inhibited either by a scrambled version of the 24-residue peptide, in which 3 adjacent residues likely to be positively charged at pH 5 were dispersed, or by a second control peptide containing the 11 N-termina l residues from mature mouse cathepsin L. The 24-residue peptide chemi cally coupled to horseradish peroxidase bound to microsomes at pH 5, b ut not at pH 7. On ligand blots, the same conjugate bound specifically to a 43-kDa integral membrane protein, identifying the microsomal pro tein that mediates the proenzyme binding. The 9-residue propeptide seq uence that inhibits the membrane association of procathepsin L at pH 5 resembles the vacuolar sorting sequences in the propeptides of yeast proteinase A and carboxypeptidase Y. This suggests that the membrane a ssociation of procathepsin L may play a role in the transport of the p roenzyme to lysosomes, the vacuolar equivalent in mammalian cells.