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Procathepsins L and D are membrane-bound in acidic microsomal vesicles
1Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill 27599.
Abstract:
Procathepsins L and D, the proenzyme forms of two lysosomal proteases, are shown to bind to mouse fibroblast microsomal membranes at acidic pH. The propeptide of procathepsin L is necessary for membrane association because the mature forms of this lysosomal protein did not bind to the membranes. Both proenzymes were eluted from the membranes by increasing either the pH or the ionic strength of the buffer, so they are peripheral proteins that interact ionically with the membranes. The proenzymes were not eluted from the membranes with 50 mM mannose or 10 mM mannose 6-phosphate, which suggests that carbohydrate does not mediate membrane binding. Membrane binding is probably a specific, protein-mediated interaction since treatment of the microsomes with trypsin reduced by half the amount of procathepsin L which bound to the membranes, and binding of procathepsin L to the membranes was saturable. One or more "lysosomal proenzyme receptors" capable of binding to lysosomal proenzymes at acidic pH could complement the mannose 6-phosphate receptor system in prelysosomes and sort certain lysosomal proenzymes to lysosomes.
Insights
Procathepsins L and D bind to fibroblast membranes at acidic pH via ionic interactions, mediated by their propeptides. This suggests a novel receptor system for lysosomal enzyme sorting.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Lysosomal proteases, procathepsins L and D, are crucial for cellular degradation.
- Understanding their trafficking and sorting mechanisms is essential for lysosomal function.
Purpose of the Study:
- To investigate the mechanism of lysosomal proenzyme binding to microsomal membranes.
- To identify potential receptors involved in lysosomal sorting.
Main Methods:
- Incubation of mouse fibroblast microsomes with procathepsins L and D at acidic pH.
- Elution studies varying pH and ionic strength.
- Competition assays with mannose and mannose 6-phosphate.
- Protease treatment (trypsin) of microsomes.
Main Results:
- Procathepsins L and D bind peripherally to membranes at acidic pH via ionic interactions.
- The propeptide of procathepsin L is essential for membrane binding.
- Binding is protein-mediated, saturable, and not dependent on carbohydrate moieties.
- Trypsin treatment significantly reduces procathepsin L binding.
Conclusions:
- A novel, pH-dependent, protein-mediated binding mechanism for lysosomal proenzymes to membranes exists.
- This interaction may involve specific proenzyme receptors distinct from the mannose 6-phosphate system.
- Such receptors could play a complementary role in lysosomal proenzyme sorting.