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The pH-dependent membrane association of procathepsin L is mediated by a 9-residue sequence within the propeptide
G F McIntyre1, G D Godbold, A H Erickson
1Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Abstract:
The lysosomal proprotease procathepsin L binds to mouse fibroblast microsomal membranes at pH 5, but mature active cathepsin L does not (McIntyre, G.F., and Erickson, A. H. (1991) J. Biol. Chem. 266, 15438-15445). This binding is not dependent on N-linked carbohydrate as procathepsin L synthesized in cells treated with tunicamycin still shows pH-dependent membrane association. These results suggest that the propeptide (Thr18-Lys113) of the cysteine protease mediates its pH-dependent membrane association. Synthetic peptides containing either 24 or 9 residues from the N-terminal portion of the mouse procathepsin L propeptide inhibited the binding of mouse procathepsin L to microsomal membranes 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-terminal residues from mature mouse cathepsin L. The 24-residue peptide chemically coupled to horseradish peroxidase bound to microsomes at pH 5, but not at pH 7. On ligand blots, the same conjugate bound specifically to a 43-kDa integral membrane protein, identifying the microsomal protein that mediates the proenzyme binding. The 9-residue propeptide sequence 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 association of procathepsin L may play a role in the transport of the proenzyme to lysosomes, the vacuolar equivalent in mammalian cells.
Insights
The propeptide of procathepsin L mediates its pH-dependent binding to fibroblast microsomal membranes, identifying a specific 43-kDa protein involved in lysosomal transport.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Lysosomal proproteases require specific trafficking pathways.
- Procathepsin L exhibits pH-dependent binding to microsomal membranes, unlike its mature form.
- This binding is independent of N-linked glycosylation.
Purpose of the Study:
- To investigate the mechanism of pH-dependent microsomal membrane association of procathepsin L.
- To identify the specific region of procathepsin L responsible for membrane binding.
- To identify the microsomal membrane protein that interacts with procathepsin L.
Main Methods:
- Utilizing synthetic peptides from the procathepsin L propeptide to inhibit membrane binding.
- Employing chemical conjugation of peptides to horseradish peroxidase for binding assays.
- Performing ligand blot analysis to identify the interacting membrane protein.
Main Results:
- The N-terminal propeptide of procathepsin L mediates pH-dependent membrane binding.
- A 24-residue peptide from the propeptide inhibited binding, while scrambled or mature cathepsin L peptides did not.
- A 43-kDa integral membrane protein was identified as the specific binding partner for procathepsin L at acidic pH.
- The inhibitory 9-residue propeptide sequence shares similarities with yeast vacuolar sorting signals.
Conclusions:
- The propeptide of procathepsin L is crucial for its pH-dependent association with microsomal membranes.
- A specific 43-kDa integral membrane protein facilitates this interaction.
- Procathepsin L membrane binding may be a mechanism for its transport to lysosomes.