Volume 26, Issue 4, Pages 511-521 (May 2007) The Plug Domain of the SecY Protein Stabilizes the Closed State of the Translocation Channel and Maintains a Membrane Seal  Weikai Li, Sol Schulman, Dana Boyd, Karl Erlandson, Jon Beckwith, Tom A. Rapoport  Molecular Cell  Volume 26, Issue 4, Pages 511-521 (May 2007) DOI: 10.1016/j.molcel.2007.05.002 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Plug Deletion Mutants of SecY Support Translocation In Vivo and In Vitro (A) Wild-type SecY or plug deletion mutants were expressed from a plasmid in an E. coli strain harboring a temperature-sensitive (ts) SecY allele. The chromosomal SecY allele is indicated on the upper line; the plasmid-encoded copy of SecY is denoted on the lower line. The bacteria were grown at the nonpermissive temperature of 42°C. (B) To test whether SecY alleles can translocate the lacZ portion of a MalF-lacZ fusion protein, plasmids encoding wild-type SecY or plug deletion mutants were introduced into a ts SecY strain expressing the fusion protein. The signal-sequence suppressor mutant prlA4 was tested in parallel. The cells were grown at 37°C to log phase, and β-galactosidase activity was measured (expressed in Miller units). Error bars represent the standard deviation of measurements with two unique colonies each in duplicate. (C) Purified plug deletion mutants of SecY were reconstituted into proteoliposomes and tested for translocation of 35S-labeled proOmpA in the presence of SecA and ATP. Translocated material was determined after treatment with proteinase K (HK) and autoradiography. Controls were performed by depleting ATP with glucose/hexokinase and by solubilizing the proteoliposomes with Triton X-100 prior to proteolysis. Lane 1 shows 10% of the input material. Molecular Cell 2007 26, 511-521DOI: (10.1016/j.molcel.2007.05.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Plug Deletion Mutants of SecY Suppress Defective or Missing Signal Sequences (A) Plasmids encoding either wild-type SecY, the prlA4 mutant, or the plug deletion mutants were transformed into cells expressing alkaline phosphatase with a weakly impaired signal sequence (L8Q mutation) from the chromosome. A control was performed with an identical plasmid lacking the SecY coding sequence. Cells were grown to midlog phase, and the alkaline phosphatase (AP) activity was measured. Activity units in the figure are defined as (OD420 nm − 1.75 OD550 nm) × 1000/(OD600 nm × volume of cell suspension × min). Error bars represent the standard deviation from measurements of two unique colonies each in duplicate. (B) As in (A), but with an alkaline phosphatase that carries a more severe mutation in its signal sequence (L14R). (C) As in (A), but with an alkaline phosphatase that lacks a signal sequence altogether (Δ2–22). In this case, the alkaline phosphatase gene was induced from a plasmid. Molecular Cell 2007 26, 511-521DOI: (10.1016/j.molcel.2007.05.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 New Plugs Are Formed in the Plug Deletion Mutants (A) Stereo view of the plug region in the half-plug deletion mutant (Δ60–65). A solvent-flattened map was calculated by using starting phases from a rigid body refinement of the 1RH5 model lacking residues 53–67. The helix in the built model is shown as a ribbon with residues in stick-and-ball representation. (B) As in (A), but for the full-plug deletion mutant (Δ57–67). A prime-and-switch map was calculated with starting phases as in (A). Molecular Cell 2007 26, 511-521DOI: (10.1016/j.molcel.2007.05.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Comparison of the Structures of Wild-Type SecY with Those of the Plug Deletion Mutants (A) Top views of the channels. The original and new plugs are shown in red, the β-hairpin connecting TM2a and TM2b in brown, TM2b in gold, and TM5 and TM10 in green. (B) Side views of the channels. (C) An enlarged view of the plug region, with the wild-type plug in red, the new plug of the half-plug deletion mutant in dark pink, and the new plug of the full-plug deletion mutant in purple, all superimposed onto the rest of the structures. The helices surrounding the plug that contain pore ring residues are shown in the same colors as in (A). The pore residues are shown in ball-and-stick representation. Molecular Cell 2007 26, 511-521DOI: (10.1016/j.molcel.2007.05.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Interactions of the Plugs with the Lateral Gate (A) The helices TM2b and TM7 that form the lateral gate interact only weakly. (B and C) In the wild-type, the interaction between TM2b and TM7 is stabilized by multiple interactions with the plug (TM2a) in red and the following β-hairpin in brown. (D and E) The new plugs of the half- and full-plug deletion mutants interact only weakly with TM2b and TM7. Molecular Cell 2007 26, 511-521DOI: (10.1016/j.molcel.2007.05.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Space-Filling Models of the Wild-Type Channel and Plug Deletion Mutants (A) The wild-type channel with the plug domain removed. Pore ring residues are shown in ball-and-stick representation. (B–D) The wild-type channel, the half-plug mutant, and the full-plug mutant, all with the plug domain in red. Molecular Cell 2007 26, 511-521DOI: (10.1016/j.molcel.2007.05.002) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Proposed Model for Channel Gating (A) Resting channel with the plug locking its closed state. (B) Channel after loop insertion of a translocation substrate. The plug is unlocked. Arrows indicate domain movements and plug widening. Molecular Cell 2007 26, 511-521DOI: (10.1016/j.molcel.2007.05.002) Copyright © 2007 Elsevier Inc. Terms and Conditions