Preserving the membrane barrier for small molecules during bacterial protein translocation

Abstract

Many proteins are translocated through the SecY channel in bacteria and archaea and through the related Sec61 channel in eukaryotes. The channel has an hourglass shape with a narrow constriction approximately halfway across the membrane, formed by a pore ring of amino acids. While the cytoplasmic cavity of the channel is empty, the extracellular cavity is filled with a short helix called the plug, which moves out of the way during protein translocation. The mechanism by which the channel transports large polypeptides and yet prevents the passage of small molecules, such as ions or metabolites, has been controversial. Here, we have addressed this issue in intact Escherichia coli cells by testing the permeation of small molecules through wild-type and mutant SecY channels, which are either in the resting state or contain a defined translocating polypeptide chain. We show that in the resting state, the channel is sealed by both the pore ring and the plug domain. During translocation, the pore ring forms a 'gasket-like' seal around the polypeptide chain, preventing the permeation of small molecules. The structural conservation of the channel in all organisms indicates that this may be a universal mechanism by which the membrane barrier is maintained during protein translocation.

Figure 1. Testing the permeability of the resting SecY channel

a, Structure of the modification reagent BM. b, Wild-type(WT) SecY or the ΔP mutant were expressed under the inducible Tet promoter. Cells were incubated with BM, and the proteins separated by SDS-PAGE, followed by blotting with streptavidin-HRP conjugate, with SecY-antibodies, or with trigger factor (TF)-antibodies (loading control). Where indicated, rifampicin (Rif) was added before BM. Endogenous SecY was tagged at its C-terminus, abolishing recognition by SecY antibodies (Fig. S2). p30, a prominent biotinylated protein. c, As in b, but after incubation with BM, cells(T)were fractionated into periplasm (P), membranes (M), and cytosol (C). Fractionation was controlled by immunoblotting for the indicated marker proteins. MBP, maltose-binding protein, TetR, tetracycline repressor. d, Spheroplasts were diluted into an iso-osmotic solution of xylitol and the change in turbidity followed over time. f, As in e, but with dilution into iso-osmotic KCl containing valinomycin.

Figure 2. Testing the permeability of a translocating SecY channel

a, Schematics of the model nascent chain (NC100) (upper panel)and its insertion into the SecY channel (lower panel). The indicated cysteines in NC100 and SecY form a disulfide bridge. Residues 39–60 are inside the channel. b, NC100 with Cys19 was expressed under an arabinose-inducible promoter together with SecY_68C under the endogenous promoter. The start-codon of SecY_68C was changed from AUG to GUG. Where indicated, cells were treated with the oxidant copper phenanthroline (CuPh3). Rifampicin(Rif) was added prior to oxidation for 15 min or 1 hr, as indicated. %SecY, percentage of non-crosslinked SecY. c, NC100 with either a wild-type(WT) or defective (RR) signal sequence was expressed together with SecY_68C or the ΔP mutant. Where indicated, cells were pretreated with Rif before addition of BM. The samples were analyzed by SDS-PAGE, followed by blotting with streptavidin-HRP conjugate or myc-(detects NC100), SecY-, and TF-antibodies. d, Nascent chains (NC) of different lengths, all with Cys19, were expressed together with SecY_68C, and crosslinked with CuPh3. The samples were analyzed by SDS-PAGE with or without prior reduction with β-mercaptoethanol (β-ME), followed by immunoblotting. Red arrowhead, crosslinked product between SecY and NC-tRNA. e, Nascent chains (NC) of different lengths with either wild-type(WT-NC) or defective (RR-NC) signal sequence were expressed together with ΔP channel. As a control, NC100 synthesis was not induced (−NC). BM was added to the cells and biotinylated proteins detected by SDS-PAGE, followed by blotting. The modification of p30 was quantified (error bars, s.d.; n=3). f, As in e, but spheroplasts were diluted into an iso-osmotic solution of xylitol and the initial, linear rate of turbidity decrease was determined (error bars, s.d.; n=3). Spheroplasts were also analyzed after treatment with Rif. The ΔP-SecY/+Rif sample showed an initial lag phase, which was ignored. g, As in f, but with dilution into iso-osmotic KCl containing valinomycin.

Figure 3. Permeability in pore ring mutants

a, Wild-type SecY or pore mutants in which Ile86, Ile191, Ile278, or Ile408(IIII) were replaced by Gly or Ala were expressed under the Tet promoter. Cells containing the ΔP mutant oran empty vector were also analyzed. After treatment of cells with BM, the samples were analyzed by SDS-PAGE and blotting with streptavidin-HRP conjugate or SecY-and TF-antibodies. b, NC100 with wild-type (WT) or defective (RR) signal sequence was expressed from the inducible arabinose promoter together with SecY pore mutants under the Tet promoter. Where indicated, rifampicin (Rif) was added before BM. The samples were analyzed by SDS-PAGE, followed by blotting with streptavidin-HRP conjugate or myc-(detects NC100), SecY-, and TF-antibodies. Addition of Rif does not clear the mutant channels of nascent chains (lanes 5 and 11), likely because peptidyltransferase activity is compromised in these dying cells. c, As in b, but spheroplasts containing the GGGI pore mutant were diluted into an iso-osmotic xylitol solution, and the turbidity change followed over time. d, As in c, but spheroplasts were diluted into iso-osmotic KCl containing valinomycin. e, As in c, but with the IIIG pore mutant under the constitutive promoter with a GUG start-codon. The cells were treated with Rifor induced for NC100 expression. f, As in e, but spheroplasts were diluted into iso-osmotic KCl containing valinomycin.

Figure 4. Model for the maintenance of the membrane barrier by the SecY channel

For details, see text.