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The biosynthesis of membrane-bound M13 coat protein. Energetics and assembly intermediates
Abstract:
The major coat protein of bacteriophage M13 spans the plasma membrane of infected cells prior to its assembly into extruding virus. It is initially made as a precursor, termed procoat, with a 23-residue leader sequence at its NH2 terminus. Procoat is found bound to the inner surface of the plasma membrane. The electrical potential of the cell membrane is required for procoat insertion and conversion to coat protein, although the order of these events has been unknown. We now report studies of the conversion of a mutant procoat (procoat-R6) from the virus M13am8H1R6 to mutant coat (coat R6). The behavior of procoat-R6 differs from that of the wild type procoat in three respects. (i) Pulse-labeled procoat-R6 is largely found inserted across the cell membrane. This suggests that the active site of leader peptidase is on the periplasmic membrane face and that insertion normally precedes processing for wild type procoat as well. (ii) Despite the greater abundance of inserted procoat-R6 in M13am8H1R6-infected cells than inserted procoat in wild type infections, procoat-R6 is processed to coat-R6 more slowly than procoat is converted to coat. (iii) The membrane insertion and proteolytic processing of procoat-R6 are almost completely insensitive to uncouplers. We present a working model for the energetics and assembly intermediates of coat protein biosynthesis.
Insights
Bacteriophage M13 coat protein insertion and processing were studied using a mutant procoat. Findings suggest membrane insertion precedes processing and reveal insights into coat protein biosynthesis energetics.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Bacteriophage M13 major coat protein is synthesized as a precursor (procoat) with a leader sequence.
- Procoat is found on the inner plasma membrane surface before viral assembly.
- Membrane electrical potential is crucial for procoat insertion and conversion to coat protein.
Purpose of the Study:
- To investigate the order of procoat insertion and processing into coat protein.
- To characterize the behavior of a mutant procoat (procoat-R6) and its conversion to coat protein (coat R6).
- To elucidate the energetics and assembly intermediates of coat protein biosynthesis.
Main Methods:
- Pulse-labeling of M13-infected cells.
- Analysis of procoat-R6 and wild-type procoat insertion and processing.
- Assessment of the effect of uncouplers on membrane insertion and proteolytic processing.
Main Results:
- Pulse-labeled procoat-R6 is inserted across the cell membrane, suggesting insertion precedes processing for wild-type.
- Procoat-R6 is processed more slowly than wild-type procoat, despite higher abundance of inserted procoat-R6.
- Membrane insertion and processing of procoat-R6 are largely insensitive to uncouplers.
Conclusions:
- Leader peptidase likely acts on the periplasmic membrane face.
- A working model for coat protein biosynthesis energetics and assembly intermediates is proposed.
- The study provides new insights into the mechanism of bacteriophage M13 coat protein maturation.