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The lysosomal proenzyme receptor that binds procathepsin L to microsomal membranes at pH 5 is a 43-kDa integral
1Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
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 specifically binds the procathepsin L activation peptide at acidic pH.
Insights
Researchers identified a 43-kDa membrane protein that binds procathepsin L at acidic pH. This novel lysosomal proenzyme receptor binds the procathepsin L activation peptide, independent of mannose-6-phosphate receptors.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Lysosomal enzymes are synthesized as inactive proenzymes.
- Procathepsins L and D bind to fibroblast membranes at acidic pH.
- This binding is independent of mannose-6-phosphate receptors and involves propeptides.
Purpose of the Study:
- To identify the specific membrane protein responsible for binding procathepsin L at acidic pH.
- To characterize the interaction between procathepsin L and its binding protein.
Main Methods:
- Co-immunoprecipitation of procathepsin L with membrane proteins at different pH values.
- Ligand blot analysis using a 43-kDa integral membrane protein.
- Inhibition studies using a synthetic peptide mimicking the N-terminal residues of procathepsin L.
Main Results:
- A 43-kDa membrane protein was identified that specifically binds procathepsin L at acidic pH (pH 5) but not neutral pH (pH 7).
- This binding was confirmed using co-immunoprecipitation and ligand blot assays.
- A synthetic N-terminal peptide of procathepsin L blocked the binding, indicating the propeptide is crucial for interaction.
Conclusions:
- The 43-kDa integral membrane protein acts as a specific receptor for lysosomal proenzymes.
- This receptor binds the activation peptide of procathepsin L at acidic pH.
- This interaction is a novel mechanism for lysosomal proenzyme targeting or regulation.