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Tryptic dissection and reconstitution of translocation activity for nascent presecretory proteins across microsomal
Abstract:
The ability of microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer into the intravesicular space was investigated by using trypsin as a proteolytic probe. We found that under defined conditions trypsin is able to dissect the translocation activity of microsomal membranes into components that can be separated into two fractions, one soluble and the other membrane bound. The trypsinized membrane fraction has lost its translocation activity. Addition of the trypsin-generated soluble fraction, however, results in reconstitution of translocation activity. These results are compatible with the notion proposed in the signal hypothesis that the translocation activity of the microsomal membrane resides in transmembrane protein(s). We propose that trypsin effects solubilization from the membrane of cytosol-exposed domain(s) involved in recognition of the signal sequence or ribosome or both, leaving behind membrane-integrated domain(s) that provide the environment for the passage of the nascent chain across the membrane. Signal peptidase activity was unaffected by trypsinization of microsomal vesicles consistent with a localization of the active site of this enzyme on the cisternal side of the vesicles.
Insights
Investigating protein translocation across microsomal membranes using trypsin revealed that membrane proteins are essential for this process. A soluble fraction, when added back, reconstituted translocation activity, supporting the signal hypothesis.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Trafficking
Background:
- Nascent presecretory proteins must cross the microsomal membrane.
- The mechanism of protein translocation across membranes is not fully understood.
Purpose of the Study:
- To investigate the role of microsomal membranes in protein translocation.
- To identify components responsible for translocation activity.
Main Methods:
- Using trypsin as a proteolytic probe to dissect microsomal membrane activity.
- Separating membrane components into soluble and membrane-bound fractions.
- Assessing reconstitution of translocation activity.
Main Results:
- Trypsin treatment separated translocation activity into soluble and membrane-bound fractions.
- The membrane-bound fraction lost activity, but activity was restored upon addition of the soluble fraction.
- Signal peptidase activity remained unaffected, suggesting its cisternal localization.
Conclusions:
- Translocation activity resides in transmembrane proteins, as suggested by the signal hypothesis.
- Cytosol-exposed domains involved in signal sequence/ribosome recognition are solubilized by trypsin.
- Membrane-integrated domains facilitate nascent chain passage across the membrane.