Bacterial protein translocation requires only one copy of the SecY complex in vivo - PubMed (original) (raw)
Bacterial protein translocation requires only one copy of the SecY complex in vivo
Eunyong Park et al. J Cell Biol. 2012.
Abstract
The transport of proteins across the plasma membrane in bacteria requires a channel formed from the SecY complex, which cooperates with either a translating ribosome in cotranslational translocation or the SecA ATPase in post-translational translocation. Whether translocation requires oligomers of the SecY complex is an important but controversial issue: it determines channel size, how the permeation of small molecules is prevented, and how the channel interacts with the ribosome and SecA. Here, we probe in vivo the oligomeric state of SecY by cross-linking, using defined co- and post-translational translocation intermediates in intact Escherichia coli cells. We show that nontranslocating SecY associated transiently through different interaction surfaces with other SecY molecules inside the membrane. These interactions were significantly reduced when a translocating polypeptide inserted into the SecY channel co- or post-translationally. Mutations that abolish the interaction between SecY molecules still supported viability of E. coli. These results show that a single SecY molecule is sufficient for protein translocation.
Figures
Figure 1.
SecY complexes interact in vivo through different surfaces. (A) View of Thermotoga maritima SecY complex from the periplasm. The N- and C-terminal halves of SecY are colored blue and red, respectively, SecG in green, and SecE in yellow. Balls in magenta indicate positions that were mutated in the E. coli protein to cysteines. Note that two cysteines are part of an inserted segment. (B) The accessibility of cysteine residues introduced into SecY was tested in intact E. coli by modification with biotin-PEG2-maleimide, followed by incubation with streptavidin (SA). The samples were analyzed by SDS-PAGE and immunoblotting with SecY antibodies (anti-SecY). The two bands correspond to one or two SecY molecules bound to tetrameric streptavidin. (C) The interaction of SecYs with the indicated cysteines was tested by addition of bismaleimide-PEG3 (BM-PEG3) to resuspended intact E. coli cells, pretreated with rifampicin for 30 min. The samples were analyzed by SDS-PAGE and immunoblotting for SecY. SecY2, cross-linked SecY dimers. (D) The front-to-front interaction of SecYs with the indicated cysteines was tested by spontaneous disulfide bridge formation in vivo. Where indicated, the samples were treated with β-mercaptoethanol (β-ME). (E) Scheme of a SecY complex containing SecE, SecG, and SecY in a single polypeptide chain. The gray dotted segments are added linkers. Residue L106 at the back of SecE (see A) is indicated. (F) The back-to-back interaction of single-chain SecY complexes with a cysteine at position 106 of SecE was tested by disulfide bridge formation after addition of the oxidant CuPh3 to cell lysates. Controls were performed with protein lacking a cysteine and with β-ME addition after cross-linking.
Figure 2.
Saturation of SecY channels with a cotranslational translocation intermediate. (A) Scheme of a cotranslational translocation intermediate generated with the substrate DsbA-SecM. The nascent chain contains the signal sequence of DsbA at the N terminus, a Myc-tag for detection, and a C-terminal SecM-stalling sequence. Its insertion into the SecY channel is monitored by disulfide bridge formation between cysteines in the nascent chain and SecY. (B) DsbA-SecM was expressed from the arabinose (Ara)-inducible promoter in cells producing SecY at approximately endogenous level using a GUG translational start codon (plasmid pACYC-SecYEG). After addition of the oxidant CuPh3 to the cell culture to induce disulfide bridge formation, the lysate was analyzed by SDS-PAGE and blotting with SecY antibodies. Where indicated, rifampicin (Rif) was added for different time periods. Red arrows and black asterisks indicate cross-links between SecY and substrate or endogenous proteins, respectively. For quantification of three independent experiments, see Fig. 3 C, right panel.
Figure 3.
Saturation of SecY channels with a post-translational translocation intermediate. (A) Scheme of a post-translational translocation intermediate generated with SecA and the substrate OmpA-GFP. The translocating chain contains the signal sequence of OmpA at the N terminus and the “superfolder” GFP at the C terminus. Its insertion into the SecY channel is monitored by disulfide bridge formation between cysteines in the substrate and SecY. (B) The insertion of OmpA-GFP, containing a cysteine at position 21 (21C), into SecY containing a cysteine at position 68 (68C) was tested by disulfide bridge formation after the addition of the oxidant CuPh3 to intact E. coli cells. Where indicated, the substrate or SecY lacked a cysteine or the cysteines were blocked with _N_-ethylmaleimide (NEM) before addition of CuPh3. As a control, a substrate was used with a defective signal sequence (RR-21C). Where indicated, disulfide bridges were reduced by β-mercaptoethanol (β-ME). All samples were analyzed by SDS-PAGE followed by blotting with SecY or GFP antibodies. (C) OmpA-GFP was expressed from the arabinose (Ara)-inducible promoter in cells producing SecY at approximately endogenous level using a GUG translational start codon (plasmid pACYC-SecYEG). After addition of CuPh3, the lysate was analyzed by SDS-PAGE and blotting with SecY antibodies. Where indicated, rifampicin (Rif) was added for different time periods. Red arrows and black asterisks indicate cross-links between SecY and substrate or endogenous proteins, respectively. The right panel shows quantification of the cross-linking efficiency between SecY and translocation substrates, based on the decrease of noncross-linked SecY in experiments such as shown in the left panel and Fig. 2 B. Three different experiments were analyzed (mean and SD). (D) OmpA-GFP with either a wild-type (WT) or defective (RR) signal sequence was expressed under the arabinose (Ara) promoter in cells producing wild-type SecY or SecY lacking its plug domain (ΔP). Controls were performed with an empty vector (vec) and without Ara induction. Biotin-maleimide was added to the cells, and the modification of proteins was probed by SDS-PAGE and blotting with HRP-conjugated streptavidin. The samples were also probed with SecY, GFP, and trigger factor (TF; loading control) antibodies. Where indicated, cells were pretreated with rifampicin (Rif) before addition of biotin-maleimide. p30, a prominently modified cytosolic protein. The blue arrowheads indicate biotinylation of translocation-incompetent OmpA-GFP carrying a defective signal sequence.
Figure 4.
Dissociation of SecY oligomers upon insertion of a translocating chain. DsbA-SecM or OmpA-GFP was expressed from the arabinose (Ara)-inducible promoter in cells producing SecY at approximately endogenous level using a GUG translational start codon. SecY carried either a cysteine at the front (SecY-151C) or at the back (SecY-212C). Cross-linking of SecYs (SecY2) was tested after addition of bismaleimide-PEG3 (BM-PEG3) to intact cells. Where indicated, cells were treated with rifampicin (Rif) before cross-linking.
Figure 5.
Preferential insertion of substrate into monomeric SecY complex. (A) A single-chain SecY complex with a cysteine at position 106 of SecE for back-to-back cross-linking was expressed under the endogenous promoter together with the cotranslational substrate DsbA-SecM under the arabinose (Ara) promoter. The level of DsbA-SecM was reduced by expression of the RNase MazF. Insertion of the nascent chain was monitored by disulfide bridge cross-linking between DsbA-SecM and SecY after addition of DTNB to intact cells (first XL). After homogenization, a cell extract was treated with the oxidant CuPh3 to induce cross-links between SecY molecules (second XL). The samples were analyzed by SDS-PAGE and immunoblotting for Myc (substrate) and His (channel) tags. The red arrow indicates the position expected for double cross-links of SecY with SecY and DsbA-SecM. (B) As in A, but with the post-translational substrate OmpA-GFP. The expression level of substrate was reduced by the use of a GUG translational start codon, and SecY channels were cleared of endogenous substrates by addition of rifampicin for 30 min. (C) SecY with a cysteine at position 154 for spontaneous front-to-front disulfide bridge formation was expressed together with DsbA-SecM. Channel insertion of DsbA-SecM was monitored by disulfide bridge formation between substrate and SecY after addition of CuPh3. Controls were performed with SecY lacking cysteines for SecY-SecY (154C) or SecY-substrate (68C) cross-linking. The red arrow indicates the position expected for double cross-links of SecY with SecY and DsbA-SecM. (D) As in C, but with OmpA-GFP. The cross-linking between substrate and position 154 of SecY (lane 6) might be explained by assuming that the proOmpA signal sequence is mobile (more so than that of DsbA), so that it comes close to position 154 at the lateral gate.
Figure 6.
Mutants disrupting the association of SecY complexes in the membrane. (A) The back-to-back association of SecY complexes was tested using a cysteine at position 212 and the bi-functional cross-linker bismaleimide-PEG3 (BM-PEG3). Mutations were introduced at position 106 of SecE to test their effect on SecY dimer (SecY2) formation. The samples were analyzed by SDS-PAGE and immunoblotting for SecY and His tag (SecE). The cross-linking efficiency was quantitated and is expressed relative to that with wild-type SecE. (B) As in A, but with the chromosomal SecE copy deleted. (C) The growth rate of the cells used in B was compared. The data shown are from a single representative experiment out of two repeats. (D) The front-to-front association of SecY complexes was tested using a cysteine at position 151 and the bi-functional cross-linker bismaleimide-PEG3 (BM-PEG3). Mutations were introduced in TM3 of SecY, as indicated (TM3RR), to disrupt SecY dimer (SecY2) formation. Where indicated, cells were treated with rifampicin (Rif) before cross-linking. (E) The growth rate of the cells used in D was compared. The data shown are from a single representative experiment out of two repeats.
Figure 7.
Oligomerization-defective SecY mutants have a negligible growth defect. (A) SecY was expressed at different levels by using plasmids with different origins of replication (approximate copy numbers: 10–12 for pACYC, 20–40 for pRN1, 5 for pSC101, and 1–2 for pBeloBAC) and different translation start codons for the secY gene (shown in brackets). Samples from equal numbers of cells were analyzed by SDS-PAGE and immunoblotting with antibodies to the C terminus of SecY. In lanes 2–9, the endogenous SecY copy was tagged with CBP at the C terminus, which makes it nondetectable by SecY antibodies. The levels of SecY are given relative to that in wild-type cells (lane 1). (B) SecY with a cysteine at position 151 (the front) or 212 (the back) was expressed at different levels from various plasmids. Cross-linking between SecYs was induced by addition of bismaleimide-PEG3 (BM-PEG3) and the samples were analyzed by SDS-PAGE and immunoblotting with SecY antibodies. (C) Wild-type (WT) SecY complex or mutant complexes defective in either front-to-front association (SecY (TM3RR)) or back-to-back association (SecE(106R)) were expressed at different levels from various plasmids in a strain lacking endogenous SecY. The growth of colonies at 37°C was analyzed at different time points after transformation. N.D., not determined.
References
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