The interaction network of the YidC insertase with the SecYEG translocon, SRP and the SRP receptor FtsY - PubMed (original) (raw)
The interaction network of the YidC insertase with the SecYEG translocon, SRP and the SRP receptor FtsY
Narcis-Adrian Petriman et al. Sci Rep. 2018.
Abstract
YidC/Oxa1/Alb3 are essential proteins that operate independently or cooperatively with the Sec machinery during membrane protein insertion in bacteria, archaea and eukaryotic organelles. Although the interaction between the bacterial SecYEG translocon and YidC has been observed in multiple studies, it is still unknown which domains of YidC are in contact with the SecYEG translocon. By in vivo and in vitro site-directed and para-formaldehyde cross-linking we identified the auxiliary transmembrane domain 1 of E. coli YidC as a major contact site for SecY and SecG. Additional SecY contacts were observed for the tightly packed globular domain and the C1 loop of YidC, which reveals that the hydrophilic cavity of YidC faces the lateral gate of SecY. Surprisingly, YidC-SecYEG contacts were only observed when YidC and SecYEG were present at about stoichiometric concentrations, suggesting that the YidC-SecYEG contact in vivo is either very transient or only observed for a very small SecYEG sub-population. This is different for the YidC-SRP and YidC-FtsY interaction, which involves the C1 loop of YidC and is efficiently observed even at sub-stoichiometric concentrations of SRP/FtsY. In summary, our data provide a first detailed view on how YidC interacts with the SecYEG translocon and the SRP-targeting machinery.
Conflict of interest statement
The authors declare that they have no competing interests.
Figures
Figure 1
YidC contacts the SecYEG translocon via TM1 and the C1-loop in vivo. (a) The crystal structure of E. coli YidC (PDB accession no.: 3WFV) visualised from the membrane (left) or from the periplasmic side (right). The red spheres indicate the positions for pBpa insertion. Residues which show the strongest contacts to SecY are displayed in bold and underlined. Residues with weaker contacts are shown in black and residues that did not show significant cross-links are shown in magenta. The dashed green lines indicate TM1 and the C-terminus of YidC, which have not been crystallized so far. (b) The co-expression system shows balanced expression of YidC and SecYEG as revealed by western blotting. 1 × 108 E. coli BL21 cells expressing yidC under the arabinose promoter from plasmid pBad24 or co-expressing P_lac_ secYEG-yidC from plasmid pTrc99a were TCA precipitated, separated by SDS-PAGE and after western transfer decorated with the indicated antibodies. WT refers to wild type YidC and D399pBpa to a YidC variant with pBpa inserted at position 399, when pBpa was added to the growth medium. (c) In vivo photo-cross-linking performed with BL21 E coli cells expressing either yidC alone (-SecYEG) or co-expressing yidC and secYEG (+SecYEG). Either wild type YidC (WT) or YidC variants containing pBpa at position V15 or D399 were analysed. After UV-exposure, samples were purified via metal affinity chromatography using an N-terminal His-tag on YidC. A sample without UV-exposure served as a control. Samples were decorated with antibodies against SecY or YidC as indicated. The 95 kDa YidC-SecY cross-link is indicated. (d) In vivo para-formaldehyde (PFA) cross-linking with BL21 cells expressing either only YidC from plasmid pBad24 or YidC together with SecYEG from plasmid pTrc99a. All samples were treated as described in experimental procedures and YidC was further purified from the membrane fraction via its N-terminal His-tag and analyzed by western blotting using α-SecY antibodies for detecting SecY-YidC cross-links. α-YidC antibodies determined comparable amounts of YidC in all samples. Uncropped images are displayed in Supplementary Figure S4.
Figure 2
YidC makes multiple contacts to SecY through its transmembrane and periplasmic domains. In vivo photo cross-linking was performed as described in Fig. 1 using the co-expression system with YidC variants containing pBpa at the indicated positions. After UV-exposure, samples were purified via the C-terminal His-tag on SecY and analysed on western blot with α-YidC antibodies (a–c). The 95 kDa YidC-SecY cross-links are indicated. For position D236, an additional strong band below the 130 kDa was recognized by α-YidC antibodies (*). In addition, α-YidC antibodies recognized a UV-dependent band at approx. 65 kDa (#). (d) In vitro photo cross-linking was performed using inner membrane vesicles carrying 10 μg of wild type YidC or YidC-pBpa variants co-expressed with SecYHisEG. Samples were purified by metal affinity chromatography using the His-tag on SecY and subsequently analysed on western blot with α-YidC antibodies. Uncropped images are displayed in Supplementary Figure S4.
Figure 3
YidC contacts the SecY associated proteins SecE and SecG. The cross-linked and purified material prepared for Figs 1c and 2a,c was decorated with antibodies against SecE (a,b) or SecG (c–e). (c) The samples were analysed with antibodies against SecG both at the endogenous SecYEG levels (-SecYEG) and in the co-expression system (+SecYEG). The cross-link products are indicated. The UV dependent 45 kDa bands (*) in Fig. 2d–e likely represent a proteolytic cleavage product of the YidC-SecG cross-links. Uncropped images are displayed in Supplementary Figure S4.
Figure 4
The periplasmic loop of YidC contacts SecE and SecG, while TM1 is only in contact with SecG. In vivo photo-cross-linking was performed with a SecYHisEG-YidC or SecYHisE-YidC co-expression system with pBpa incorporated either in TM1 (position 15) or within the P1 loop (position 236) of YidC. After purification via the C-terminal His-tag on SecY, the samples were analysed using antibodies against YidC (a), SecG (b) or SecE (c). The 95 kDa YidC-SecY cross-link and the 65 kDa YidC-SecE/G cross-links are indicated (#). For pBpa-insertion at position 236 of YidC, in addition to the 95 kDa YidC-SecY cross-link, a second cross-link product below the 130 kDa marker was observed (*), as already shown in Fig. 2a. Uncropped images are displayed in Supplementary Figure S4.
Figure 5
The C1-loop and TM1 form a composite binding site for SecY. (a) The positions of the partial YidC deletions used in (b) (Δ3 + Δ4 and Δ2-23) and (d) (Δ3, Δ4, Δ3 + Δ4) as well as the position of proline 388 which was mutated to alanine (P388A) (b,d) are indicated on the E. coli YidC crystal structure (PDB accession no. 3WFV). In addition, the amphipathic helix EH1 (orange) and the periplasmic loop 1 (magenta) are displayed. The dashed lines indicate TM1 and the C-terminus of YidC for which no structural information is available. (b) The conditional YidC depletion strain JS7131 was used to monitor the functionality of different pTrc99a-YidC-SecYEG co-expression systems. The presence of arabinose in the media results in expression of the endogenous YidC via the araBAD promoter while the absence of arabinose (replaced by fructose) induces YidC depletion. SecYHisEG-YidC corresponds to a co-expression system with a C-terminal His-tag on SecY, SecYEG-YidCHis to the co-expression system with a N-terminal His-tag on YidC; SecYEG-YidCHis-(Δ2-23)YidCHis to a co-expression system in which YidC lacked TM1 (residues 2–23), SecYEG-(Δ3 + Δ4)YidCHis to a co-expression system in which most of the C1 loop was deleted, SecYEG-(P388A)YidCHis to a co-expression system in which proline 388 of YidC was replaced by alanine. The empty plasmid (pTrc99a) or pTrc99a expressing just SecYHisEG served as control. Cells were sequentially diluted in LB medium and spotted onto LB plates with or without arabinose; cell growth was monitored after overnight incubation at 37 °C (upper panel). (c) In vivo photo-cross-linking was performed in the co-expression system with a YidC variant in which TM1 was replaced by the PelB signal sequence (PelB-YidC). When indicated pBpa was inserted at position 399 of either PelB-YidC (PelB-D399pBpa) or wild type YidC (D399pBpa-YidC). After UV-exposure of 1 × 108 cells, the material was not further purified but directly TCA precipitated and analysed by western blotting using α-YidC antibodies. Indicated are the cross-links (SecY-YidC), the endogenous YidC (YidCendogenous), PelB-YidC and the truncated YidC variants produced by incomplete suppression of the amber stop codon at position 399. In addition, a YidC dimer is indicated (2 × YidC). The N-terminal sequence of the PelB-YidC variant is displayed in the lower panel. The 65 kDa cross-link products between YidC and SecE/SecG are indicated (#) (d) In vivo photo-cross-linking was performed in the co-expression system with the indicated YidC variants. YidC was purified by metal affinity chromatography via an N-terminal His-tag on YidC and further analysed by western blotting with antibodies against SecY and YidC. The sequence information of the deletion mutants used in this experiment is displayed in the lower panel. CH1 and CH2 correspond to the two α-helices of the C1-loop. Uncropped images are displayed in Supplementary Figure S4.
Figure 6
The cytosolic loop 2 of YidC constitutes a binding site for SRP and its receptor FtsY. (a) In vivo photo-cross-linking was performed in the single-expression system using BL21 cells expressing yidC with pBpa at position 399. Subsequently YidC was purified via metal affinity chromatography and analysed on western blot with antibodies directed against Ffh, the protein subunit of the E. coli SRP. The 110 kDa YidC-Ffh cross-link is indicated. Note, a very weak, apparently also UV-dependent band was recognized by α-Ffh antibodies in the wild type. (b) BL21 cells expressing YidC were treated in vivo with p-formaldehyde (PFA) or buffer as a control. Subsequently, YidC was purified together with its cross-linking partners by metal affinity chromatography and analysed on western blot with α-Ffh antibodies. The 1YidC-Ffh species likely corresponds to a YidC-Ffh-4.5SRNA cross-link product and the 2YidC-Ffh species to the YidC-Ffh cross-link. (c) As in (b), but with cells expressing either wild type YidC or the YidC(Δ3 + Δ4) variant, which lacks most of the C1-loop. (d) The same material shown in (a) was decorated with α-FtsY antibodies, which revealed two cross-link products migrating at about 150 kDa and 180 kDa. FtsY-14 corresponds to the proteolytic cleavage product of FtsY, which is frequently observed. (e,f) The same material as in (b,c) was decorated with α-FtsY antibodies. Uncropped images are displayed in Supplementary Figure S4.
Figure 7
The presence of SecYEG prevents binding of FtsY to the C1 loop of YidC. In vivo photo cross-linking was performed with BL21 cells expressing either just YidC or together with SecYEG. In both cases pBpa was present at position 399 of YidC. YidC was purified via its His-tag and analysed using antibodies against Ffh (a) or FtsY (b). Note that the YidC amounts which are present in (a) and (b) are displayed in Fig. 1c (α-YidC -bottom). (c) In vivo p-formaldehyde cross-linking was performed with BL21 cells expressing YidC only or together with SecYEG. The cross-linked material was analysed with α-FtsY (top) and α-YidC (bottom) antibodies. Uncropped images are displayed in Supplementary Figure S4.
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