Nagler Christof, Nagler Gisela, Kuhn Andreas
Institute of Microbiology and Molecular Biology, University of Hohenheim, 70599 Stuttgart, Germany.
J Bacteriol. 2007 Apr;189(7):2897-905. doi: 10.1128/JB.01551-06. Epub 2007 Jan 19.
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.
M13噬菌体在大肠杆菌的内膜中组装。在成熟过程中，约2700个主要衣壳蛋白拷贝从膜上转移到一个挤出细胞的单链噬菌体DNA分子上。主要衣壳蛋白以前体形式合成，称为前衣壳蛋白，并通过不依赖Sec的途径插入膜中。在组装到噬菌体DNA上之前，它会被一种前导肽酶从前导（信号）肽上加工下来。前衣壳蛋白的跨膜区域在所有这些过程中都起着重要作用。我们利用在前衣壳蛋白和衣壳蛋白跨膜区域有突变的半胱氨酸突变体，研究了哪些残基参与多聚体形成、与前导肽酶的相互作用以及M13子代颗粒的形成。我们发现大多数单个半胱氨酸残基不会干扰噬菌体的膜插入、加工和组装。用菲咯啉铜处理细胞表明，半胱氨酸残基很容易参与二聚体和多聚体的形成。这表明衣壳蛋白在转移到新生噬菌体颗粒之前先组装成多聚体。此外，我们发现当一个半胱氨酸位于前导肽的-6位置时，突变前衣壳蛋白和其他输出蛋白的加工会受到影响。前导肽酶的这种抑制作用导致细胞死亡，表明M13前衣壳蛋白中存在特定的氨基酸残基参与噬菌体组装过程的特定步骤。
