Inner membrane YfgM–PpiD heterodimer acts as a functional unit that associates with the SecY/E/G translocon and promotes protein translocation

PpiD and YfgM are inner membrane proteins that are both composed of an N-terminal transmembrane segment and a C-terminal periplasmic domain. Escherichia coli YfgM and PpiD form a stable complex that interacts with the SecY/E/G (Sec) translocon, a channel that allows protein translocation across the cytoplasmic membrane. Although PpiD is known to function in protein translocation, the functional significance of PpiD-YfgM complex formation as well as the molecular mechanisms of PpiD-YfgM and PpiD/YfgM- Sec translocon interactions remain unclear. Here, we conducted genetic and biochemical studies using yfgM and ppiD mutants and demonstrated that a lack of YfgM caused partial PpiD degradation at its C-terminal region and hindered the membrane translocation of VemP, a Vibrio secretory protein in both Escherichia coli and Vibrio alginolyticus. While ppiD disruption also impaired VemP translocation, we found that the yfgM and ppiD double deletion exhibited no additive or synergistic effects. Together, these results strongly suggest that both PpiD and YfgM are required for efficient VemP translocation. Furthermore, our site-directed in vivo photo-crosslinking analysis revealed that the tetratricopeptide repeat domain of YfgM and a conserved structural domain (NC domain) in PpiD interact with each other and that YfgM, like PpiD, directly interacts with the SecG translocon subunit. Crosslinking analysis also suggested that PpiD/YfgM complex formation is required for these proteins to interact with SecG. In summary, we propose that PpiD and YfgM form a functional unit that stimulates protein translocation by facilitating proper interactions with the Sec translocon

Structural basis of Sec-independent membrane protein insertion by YidC

[プレスリリース]バイオサイエンス研究科膜分子複合機能学研究室の塚崎智也准教授らの研究グループが、タンパク質を細胞膜に組み込むメカニズムを解明しました（2014/04/17）Newly synthesized membrane proteins must be accurately inserted into the membrane, folded and assembled for proper functioning. The protein YidC inserts its substrates into the membrane, thereby facilitating membrane protein assembly in bacteria; the homologous proteins Oxa1 and Alb3 have the same function in mitochondria and chloroplasts, respectively1, 2. In the bacterial cytoplasmic membrane, YidC functions as an independent insertase and a membrane chaperone in cooperation with the translocon SecYEG3, 4, 5. Here we present the crystal structure of YidC from Bacillus halodurans, at 2.4 Å resolution. The structure reveals a novel fold, in which five conserved transmembrane helices form a positively charged hydrophilic groove that is open towards both the lipid bilayer and the cytoplasm but closed on the extracellular side. Structure-based in vivo analyses reveal that a conserved arginine residue in the groove is important for the insertion of membrane proteins by YidC. We propose an insertion mechanism for single-spanning membrane proteins, in which the hydrophilic environment generated by the groove recruits the extracellular regions of substrates into the low-dielectric environment of the membrane

Structural Basis of the Sec Translocon and YidC Revealed Through X-ray Crystallography

出版社許諾条件により、本文は2020年4月10日以降に公

Structure-based working model of SecDF, a proton-driven bacterial protein translocation factor

The bacterial membrane protein SecDF enhances protein translocation across the membrane driven by the complex of SecA ATPase and SecYEG. Many newly synthesized proteins in the cytoplasm are programmed to be translocated to the periplasm via the narrow channel that is formed in the center of SecYEG. During the protein-translocation process, SecDF is proposed to undergo repeated conformational transitions to pull out the precursor protein from the SecYEG channel into the periplasm. Once SecDF captures the precursor protein on the periplasmic surface, SecDF can complete protein translocation even if SecA function is inactivated by ATP depletion, implying that SecDF is a protein-translocation motor that works independent of SecA. Structural and functional analyses of SecDF in 2011 suggested that SecDF utilizes the proton gradient and interacts with precursor protein in the flexible periplasmic region. The crystal structures of SecDF in different states at more than 3Å resolution were reported in 2017 and 2018, which further improved our understanding of the dynamic molecular mechanisms of SecDF. This review summarizes recent structural studies of SecDF