Cysteine residues in the transmembrane regions of M13 procoat suggest that oligomeric coat proteins assemble onto phage progeny

1 2 The M13 phage assembles in the inner membrane of Escherichia coli. During 3 maturation, about 2700 copies of the major coat protein move from the membrane 4 onto a single-stranded phage DNA molecule that extrudes out of the cell. The major 5 coat protein is synthesized as a precursor, termed procoat protein and inserts into 6 the membrane via a Sec-independent pathway. It is processed by leader peptidase 7 from its leader (signal) peptide before it is assembled onto the phage DNA. The 8 transmembrane regions of the procoat protein play an important role in all these 9 processes. With cysteine mutants in the transmembrane regions of the procoat and 10 coat protein we investigated which of the residues are involved in multimer formation, 11 interaction with leader peptidase and formation of M13 progeny particles. We found 12 that most single cysteine residues do not interfere with the membrane insertion, 13 processing and assembly of the phage. Treatment of the cells with copper 14 phenanthroline showed that the cysteines were readily engaged in dimer and 15 multimer formation. This suggests that the coat proteins assemble into multimers 16 before they proceed onto the nascent phage particles. 17 In addition, we found that when a cysteine is located in the leader peptide at the –6 18 position, processing of the mutant procoat and of other exported proteins is affected. 19 This inhibition of leader peptidase results in a lethality of the cell and shows that 20 there are distinct amino acid residues in the M13 procoat protein involved at specific 21 steps of the phage assembly process. 22 AC CE PT ED on S etem er 3, 2017 by gest http/jb.asm .rg/ D ow nladed fom