SecAAA trimer is fully functional as SecAA dimer in the membrane: Existence of higher oligomers?
Adenosine Triphosphatases
0301 basic medicine
0303 health sciences
SecA Proteins
Xenopus
Membrane Transport Proteins
Microscopy, Atomic Force
03 medical and health sciences
Cross-Linking Reagents
Bacterial Proteins
Oocytes
Animals
Protein Multimerization
SEC Translocation Channels
DOI:
10.1016/j.bbrc.2014.03.116
Publication Date:
2014-04-01T22:03:56Z
AUTHORS (7)
ABSTRACT
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.
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