SecAAA trimer is fully functional as SecAA dimer in the membrane: existence of higher oligomers?
SecA is an essential ATPase in bacterial Sec-dependent protein translocation pathway, and equilibrates between monomers and dimers in solution. The question of whether SecA functions as monomers or dimers in membranes during the protein translocation is controversial. We previously constructed a tail-to-head SecAA tandem dimer, and showed it is fully functional by complementation in vivo and protein translocation in vitro, indicating that SecA can function at least as a dimer in the membrane without dissociating into monomers. In this study, we further constructed genetically a tail-to-head SecAAA trimer, which is functional in complementing a temperature-sensitive secA mutant. The purified SecAAA trimer per protomer is fully active as SecAA tandem dimers in ATPase activity, in protein translocation in vitro and in ion channel activities in the oocytes. With these functional tail-to-head trimer SecAAA and tandem SecAA, we examined their surface topology in the presence of liposomes using AFM. As expected, the soluble SecAAA without lipids are larger than SecAA. However, the ring/pore structures of SecAAA trimers were, surprisingly, almost identical to the SecA 2-monomers and SecAA dimers, raising the intriguing possibility that the SecA may exist and function as hexamer ring-structures in membranes. Cross-linking with formaldehyde showed that SecA, SecAA and SecAAA could form larger oligomers, including the hexamers. The molecular modeling simulation shows that both tail-to-head and tail-to-tail hexamers in the membranes are possible.
Department of Biology, Georgia State University, Atlanta, GA 30303, United States.
Hongyun
H
Wang
Department of Biology, Georgia State University, Atlanta, GA 30303, United States.
Yamin
Y
Ma
Department of Biology, Georgia State University, Atlanta, GA 30303, United States.
Ying-Hsin
YH
Hsieh
Department of Biology, Georgia State University, Atlanta, GA 30303, United States.
Hsiuchin
H
Yang
Department of Chemistry, and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States; Key Laboratory of Chemical Biology, School of Pharmacy, Shandong University, Jinan, Shandong 250012, China.
Minyong
M
Li
Department of Chemistry, and Center for Biotechnology and Drug Design, Georgia State University, Atlanta, GA 30303, United States.
Binghe
B
Wang
Department of Biology, Georgia State University, Atlanta, GA 30303, United States. Electronic address: biopct@gsu.edu.
Phang C
PC
Tai
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D003432:Cross-Linking Reagents / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D018625:Microscopy, Atomic Force; D009865:Oocytes; D055503:Protein Multimerization; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014981:Xenopus
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins
AFM; Hexamer; SecA; Trimer