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Vol. 20, Issue 6, 1804-1815, March 15, 2009

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Visualization of Distinct Entities of the SecYEG Translocon during Translocation and Integration of Bacterial Proteins

Diana Boy^*,, and Hans-Georg Koch^*

*Institut für Biochemie und Molekularbiologie, ZBMZ, and Fakultät für Biologie, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany

Submitted August 28, 2008; Revised December 19, 2008; Accepted January 13, 2009
Monitoring Editor: Reid Gilmore

The universally conserved SecYEG/Sec61 translocon constitutes the major protein-conducting channel in the cytoplasmic membrane of bacteria and the endoplasmic reticulum membrane of eukaryotes. It is engaged in both translocating secretory proteins across the membrane as well as in integrating membrane proteins into the lipid phase of the membrane. In the current study we have detected distinct SecYEG translocon complexes in native Escherichia coli membranes. Blue-Native-PAGE revealed the presence of a 200-kDa SecYEG complex in resting membranes. When the SecA-dependent secretory protein pOmpA was trapped inside the SecYEG channel, a smaller SecY-containing complex of 140-kDa was observed, which probably corresponds to a monomeric SecYEG–substrate complex. Trapping the SRP-dependent polytopic membrane protein mannitol permease in the SecYEG translocon, resulted in two complexes of 250 and 600 kDa, each containing both SecY and the translocon-associated membrane protein YidC. The appearance of both complexes was correlated with the number of transmembrane domains that were exposed during targeting of mannitol permease to the membrane. These results suggest that the assembly or the stability of the bacterial SecYEG translocon is influenced by the substrate that needs to be transported.

Address correspondence to: Hans-Georg Koch (Hans-Georg.Koch{at}biochemie.uni-freiburg.de)

Abbreviations used: BN-PAGE, Blue-Native-PAGE; DDM, dodecyl-maltoside; INV, inner membrane vesicles; MtlA, mannitol permease; OmpA, outer membrane protein A; RNC, ribosome-associated nascent chain; TM, transmembrane domain; wt, wild type.

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