SecA drives transmembrane insertion of RodZ, an unusual single-span membrane protein.
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.
Department of Physiology and Biophysics and the Center for Biomembrane Systems, University of California at Irvine, Irvine, CA 92697-4560, USA.
Swati
S
Rawat
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Lu
L
Zhu
Department of Physiology and Biophysics and the Center for Biomembrane Systems, University of California at Irvine, Irvine, CA 92697-4560, USA.
Eric
E
Lindner
Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
Ross E
RE
Dalbey
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.
Stephen H
SH
White
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D021381:Protein Transport / Q000502:physiology; D012270:Ribosomes / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D018271:Signal Recognition Particle / Q000378:metabolism
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C538795:RodZ protein, E coli; D000069816:SEC Translocation Channels; D018271:Signal Recognition Particle; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins
SecA ATPase; SecYEG translocase; membrane protein assembly; single-span membrane proteins