Related Experiment Videos
Access of proteinase K to partially translocated nascent polypeptides in intact and detergent-solubilized membranes
T Connolly1, P Collins, R Gilmore
1Department of Biochemistry, University of Massachusetts Medical School, Worcester, Massachusetts 01655.
Abstract:
We have used proteinase K as a probe to detect cytoplasmically and luminally exposed segments of nascent polypeptides undergoing transport across mammalian microsomal membranes. A series of translocation intermediates consisting of discrete-sized nascent chains was prepared by including microsomal membranes in cell-free translations of mRNAs lacking termination codons. The truncated mRNAs were derived from preprolactin and the G protein of vesicular stomatitis virus and encoded nascent chains ranging between 64 and 200 amino acid residues long. Partially translocated nascent chains of 100 amino acid residues or less were insensitive to protease digestion from the external surface of the membrane while longer nascent chains were susceptible to digestion by externally added protease. We conclude that the increased protease sensitivity of larger nascent chains is due to the exposure of a segment of the nascent polypeptide on the cytoplasmic face of the membrane. In contrast, low molecular weight nascent chains were remarkably resistant to protease digestion even after detergent solubilization of the membrane. The protease resistant behaviour of detergent solubilized nascent chains could be abolished by release of the polypeptide from the ribosome or by the addition of protein denaturants. We propose that the protease resistance of partially translocated nascent chains can be ascribed to components of the translocation apparatus that remain bound to the nascent chain after detergent solubilization of the membrane.
Insights
Researchers used proteinase K to study nascent polypeptide transport across membranes. Longer chains exposed more protein to protease, indicating cytoplasmic presence, while shorter chains showed membrane-bound resistance.
Area of Science:
- Molecular Biology
- Cell Biology
- Protein Trafficking
Background:
- Nascent polypeptide chains are synthesized on ribosomes and can be translocated across or inserted into cellular membranes.
- Understanding the mechanism of protein translocation across the endoplasmic reticulum membrane is crucial for protein folding and function.
Purpose of the Study:
- To investigate the topological orientation and membrane association of nascent polypeptide chains during translocation.
- To identify distinct translocation intermediates and their protease sensitivity.
Main Methods:
- Utilized cell-free translation systems with truncated mRNAs encoding preprolactin and vesicular stomatitis virus G protein nascent chains.
- Incorporated mammalian microsomal membranes to generate translocation intermediates.
- Employed proteinase K digestion to probe exposed segments of nascent polypeptides on the cytoplasmic or luminal side of the membrane.
- Investigated protease resistance of detergent-solubilized nascent chains.
Main Results:
- Nascent chains of 100 amino acid residues or less were protease-resistant, while longer chains (>100 amino acids) showed increased sensitivity to externally added proteinase K.
- This protease sensitivity correlated with the exposure of polypeptide segments to the cytoplasmic face of the membrane.
- Low molecular weight nascent chains remained protease-resistant even after detergent solubilization, suggesting association with translocation machinery.
Conclusions:
- The length of the nascent polypeptide chain dictates its exposure and protease sensitivity during membrane translocation.
- Protease resistance of short, detergent-solubilized nascent chains suggests stable interactions with components of the protein translocation apparatus.