Reconstitution of rapid and asymmetric assembly of M13 procoat protein into liposomes which have bacterial leader peptidase.
The leader peptidase of Escherichia coli cleaves a 23-residue leader sequence from M13 procoat to yield mature coat protein in virus-infected cells. We have reconstituted pure leader peptidase into vesicles of E. coli lipids and found that these liposomes are active in the conversion of procoat to coat. Trypsin removes all but 10% of the leader peptidase, yet the vesicles retain nearly full capacity to convert procoat to coat, suggesting that only procoat which inserts across the liposomal membrane is a substrate for leader peptidase. This is confirmed by the finding that over 70% of the coat protein produced by these liposomes spans the membrane. The rate at which leader peptidase inside protease-treated liposomes cleaves externally added procoat is comparable to the rate of procoat cleavage by the same amount of leader peptidase in detergent micelles. Thus, procoat can rapidly integrate across a liposomal membrane and be cleaved to coat protein. These findings confirm the central part of the membrane trigger hypothesis that certain proteins (such as procoat) can cross a bilayer without the aid of a proteinaceous pore or transport system.
D000936:Antigen-Antibody Complex; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins; D003090:Coliphages / Q000201:enzymology* / Q000235:genetics; D010450:Endopeptidases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007106:Immune Sera; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins; D008823:Micelles; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012697:Serine Endopeptidases; D014357:Trypsin; D014764:Viral Proteins / Q000378:metabolism*
D000936:Antigen-Antibody Complex; D036022:Capsid Proteins; D007106:Immune Sera; D008081:Liposomes; D008565:Membrane Proteins; D008823:Micelles; D011498:Protein Precursors; D012333:RNA, Messenger; D014764:Viral Proteins; C025668:coat protein, Bacteriophage M13; D010450:Endopeptidases; D012697:Serine Endopeptidases; D014357:Trypsin; C026515:type I signal peptidase