Efficient translocation of positively charged residues of M13 procoat protein across the membrane excludes electrophoresis as the primary force for membrane insertion.
The coat protein of bacteriophage M13 is inserted into the Escherichia coli plasma membrane as a precursor protein, termed procoat, with a typical leader peptide of 23 amino acid residues. Its membrane insertion requires the electrochemical potential but not the cellular components SecA and SecY. Since the electrochemical gradients result in the periplasmic side of the membrane being positively charged, the membrane potential could contribute to the transfer of the negatively charged central region of procoat across the membrane. Here we demonstrate that the central domain following the leader peptide can be translocated across the membrane even when the net charge of the region is changed from -3 to +3. This rules out an electrophoresis-like insertion mechanism for procoat. We also show that the sec independence of procoat insertion is linked to the presence of the second apolar domain. The deletion of most of the second apolar domain from a procoat fusion protein results in sec dependent membrane insertion of the hybrid protein. Moreover, like other proteins that require the sec genes, translocation of this sec dependent procoat protein is inhibited when positively charged residues are introduced after the leader peptide. Loop models involving one or two hydrophobic regions are presented that account for the differences in tolerance of positively charged residues.
Kuhn
A
A
Department of Microbiology, University of Basel, Switzerland.
Zhu
H Y
HY
Dalbey
R E
RE
D000595:Amino Acid Sequence; D002213:Capsid; D036022:Capsid Proteins; D002462:Cell Membrane / Q000378:metabolism; D003090:Coliphages / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational
D036022:Capsid Proteins; D011498:Protein Precursors; C025668:coat protein, Bacteriophage M13