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PLoS One. 2017 Aug 18;12(8):e0183434. doi: 10.1371/journal.pone.0183434. eCollection 2017.

SecY-SecA fusion protein retains the ability to mediate protein transport.

Author information

1
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan.
2
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

Abstract

In bacteria, the membrane protein complex SecY/E/G and SecA ATPase are essential for protein translocation. About 30% of newly synthesized proteins in the cytosol are targeted to and translocated across the cytoplasmic membrane by the Sec factors. Although a number of single-molecule analyses and structural studies, including the crystal structure of SecYEG complexed with SecA, have been published, the underlying molecular mechanisms and the functional oligomer states remain elusive. In this study, we constructed a fusion protein SecY-SecA, which induces the formation of the SecY-A/SecE/SecG complex (SecYAEG), to enable investigation of the molecular mechanisms by advanced single-molecule analyses. SecYAEG-reconstituted liposomes were found to possess protein translocation activity in vitro and form stable intermediates capable of the translocation using a mutant substrate protein. We additionally found that one unit of SecYAEG complex embedded into a nanodisc, using membrane scaffold proteins, interacts strongly with the substrate. The isolated SecYAEG-reconstituted nanodisc is a promising tool for investigation of the molecular mechanisms by which a single unit of Sec machinery mediates protein translocation.

PMID:
28820900
PMCID:
PMC5562318
DOI:
10.1371/journal.pone.0183434
[Indexed for MEDLINE]
Free PMC Article

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