Cysteine residues in the transmembrane regions of M13 procoat protein suggest that oligomeric coat proteins assemble onto phage progeny.
The M13 phage assembles in the inner membrane of Escherichia coli. During maturation, about 2,700 copies of the major coat protein move from the membrane onto a single-stranded phage DNA molecule that extrudes out of the cell. The major coat protein is synthesized as a precursor, termed procoat protein, and inserts into the membrane via a Sec-independent pathway. It is processed by a leader peptidase from its leader (signal) peptide before it is assembled onto the phage DNA. The transmembrane regions of the procoat protein play an important role in all these processes. Using cysteine mutants with mutations in the transmembrane regions of the procoat and coat proteins, we investigated which of the residues are involved in multimer formation, interaction with the leader peptidase, and formation of M13 progeny particles. We found that most single cysteine residues do not interfere with the membrane insertion, processing, and assembly of the phage. Treatment of the cells with copper phenanthroline showed that the cysteine residues were readily engaged in dimer and multimer formation. This suggests that the coat proteins assemble into multimers before they proceed onto the nascent phage particles. In addition, we found that when a cysteine is located in the leader peptide at the -6 position, processing of the mutant procoat protein and of other exported proteins is affected. This inhibition of the leader peptidase results in death of the cell and shows that there are distinct amino acid residues in the M13 procoat protein involved at specific steps of the phage assembly process.
Nagler
Christof
C
Institute of Microbiology and Molecular Biology, University of Hohenheim, 70599 Stuttgart, Germany.
Nagler
Gisela
G
Kuhn
Andreas
A
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics*; D036022:Capsid Proteins / Q000737:chemistry / Q000235:genetics*; D003545:Cysteine / Q000494:pharmacology*; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000821:virology; D015967:Gene Expression Regulation, Viral; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics
D036022:Capsid Proteins; D004279:DNA, Viral; D011498:Protein Precursors; D003545:Cysteine