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J Mol Biol. 2015 Mar 13;427(5):1023-37. doi: 10.1016/j.jmb.2014.05.005. Epub 2014 May 15.

SecA drives transmembrane insertion of RodZ, an unusual single-span membrane protein.

Author information

1
Department of Physiology and Biophysics and the Center for Biomembrane Systems, University of California at Irvine, Irvine, CA 92697-4560, USA.
2
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
3
Department of Physiology and Biophysics and the Center for Biomembrane Systems, University of California at Irvine, Irvine, CA 92697-4560, USA. Electronic address: stephen.white@uci.edu.

Abstract

The transmembrane (TM) helices of most type II single-span membrane proteins (S-SMPs) of Escherichia coli occur near the N-terminus, where the cell's targeting mechanisms can readily identify it as it emerges from the ribosome. However, the TM helices of a few S-SMPs, such as RodZ, occur a hundred or more residues downstream from the N-terminus, which raises fundamental questions about targeting and assembly. Because of RodZ's novelty and potential usefulness for understanding TM helix insertion in vivo, we examined its membrane targeting and assembly. We used RodZ constructs containing immunotags before the TM domain to assess membrane insertion using proteinase K digestion. We confirmed the N(in)-C(out) (type II) topology of RodZ and established the absence of a targeting signal other than the TM domain. RodZ was not inserted into the membrane under SecA depletion conditions or in the presence of sodium azide, which is known to inhibit SecA. Insertion failed when the TM proton gradient was abolished with Carbonyl cyanide m-chlorophenyl hydrazone. Insertion also failed when RodZ was expressed in SecE-depleted E. coli, indicating that the SecYEG translocon is required for RodZ assembly. Protease accessibility assays of RodZ in other E. coli depletion strains revealed that insertion is independent of SecB, YidC, and SecD/F. Insertion was found to be only weakly dependent on the signal recognition particle pathway: insertion was weakly dependent on the Ffh but independent of FtsY. We conclude that membrane insertion of RodZ requires only the SecYEG translocon, the SecA ATPase motor, and the TM proton motive force.

KEYWORDS:

SecA ATPase; SecYEG translocase; membrane protein assembly; single-span membrane proteins

PMID:
24846669
PMCID:
PMC4233018
DOI:
10.1016/j.jmb.2014.05.005
[Indexed for MEDLINE]
Free PMC Article

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