Fig 1.
Expression of small membrane proteins in E. coli and membrane localization of YohP.
(A) In vivo expression of plasmid-encoded YohP, AzuC, YshB, and YkgR. Expression was induced by 0.2% arabinose (“Ara”) when indicated and whole cells were TCA precipitated, before SDS-PAGE and western blotting using α-His antibodies. The predicted amino acid sequence is shown on the right and the putative transmembrane domains are indicated in bold. Positively charged residue are shown in red. (B) YohP-GFP, SecY-YFP, and the cytosolic control protein YchF-GFP were in vivo expressed and exponentially grown cells were analyzed with a DeltaVision Ultra High Resolution Widefield Microscope (GE Healthcare, Munich, Germany) at 100× magnification. Recording, using camera sCMOS pro edge (PCO, Kelheim, Germany), was performed using a 3-μm Z-scan with 0.1-μm sectioning, and the different scans of both the fluorescence channel and the merged fluorescence/bright-field picture is shown. (C) WT E. coli cells and cells expressing YohPHis from the IPTG-inducible pET19b-YohPHis were analyzed by transmission electron microscopy. (D) E. coli cells expressing YohPHis were induced with 1 mM IPTG when indicated and subsequently fractionated by differential centrifugation. S30/P30 refer to the supernatant/pellet after the first centrifugation (30,000g) following cell breakage. The S30 fraction was then further centrifuged (150,000g), and the observed pellet (P150, containing crude membranes) was subjected to sucrose gradient centrifugation, separating the INV and the OM. Of each fraction, an aliquot (40 μg protein) was separated by SDS-PAGE and, after western transfer, decorated with α-His antibodies (YohPHis), α-SecY, α-SecG, and α-YchF antibodies. A representative image of two independent biological replicates is shown. GFP, green fluorescent protein; INV, inner membrane vesicle; IPTG, isopropyl 1-thio-β-D-galactopyranoside; OM, outer membrane fraction; TCA, trichloroacetic acid; WT, wild type; YFP, yellow fluorescent protein; YohPHis, His-tagged YohP.
Fig 2.
Membrane localization of YohP is not influenced by the His-Tag.
(A) YohP and YohPHis were in vivo expressed and pulse-labeled with 35S methionine/cysteine for 5 minutes. Whole cells were then TCA precipitated after the indicated time points, separated by SDS-PAGE, and YohPHis/YohP was detected by autoradiography. Indicated are the monomeric and dimeric versions. ± refers to a nonspecifically labeled band that was also detected in the control sample, containing cells without plasmid. (B) YohP was in vivo expressed and pulse-labeled as above. Cells were disrupted by ultrasonic treatment and subjected to cell fractionation into an S30, S150, and P150 extract, as described in the legend to Fig 1. Quantification was performed on three independent experiments, and the relative mean values of monomeric and dimeric YohP found in the supernatant (S150) or the membrane fraction (P150), respectively, are shown. Underlying data for this figure can be found in S1 Data. (C) INVs of WT cells and cells expressing YohPHis were treated directly with proteinase (“Prot.”) K or only after carbonate extraction and separation into carbonate-resistant integral membrane proteins (“P”) and carbonate-sensitive membrane-associated proteins (“S”). In addition, carbonate extraction was performed without subsequent proteinase K treatment. YohP was detected by its C-terminal His-tag, whereas the control membrane protein YfgM was detected by polyclonal antibodies raised against the native protein. A representative gel of at least two independent biological replicates with at least two technical replicates is shown. INV, inner membrane vesicle; TCA, trichloroacetic acid; WT, wild type; YohPHis, His-tagged YohP.
Fig 3.
YohP does not spontaneously insert into the membrane but requires the SRP pathway.
(A) YohP and YohPHis were in vitro synthesized using a purified coupled transcription/translation system (CTF system). In vitro synthesis was performed in the presence of inverted INVs or liposomes (“Lipos”) or buffer, as a control. Liposomes were generated from E. coli phospholipids and contained 70% PE, 25% PG, and 5% CL. After 20 minutes of in vitro synthesis, half of the sample was directly TCA precipitated, whereas the other half was first treated with PK before TCA precipitation. Samples were then separated by SDS-PAGE and analyzed by autoradiography. Indicated are the in vitro–synthesized YohP and YohPHis. The quantification is based on three independent experiments and the mean values (±SD) are shown. Underlying data for this figure can be found in S1 Data. (B) YohPHis was in vitro synthesized as above, in the presence of either wild-type INV, INV(OE), or U-INV(OE). When indicated, purified SecA (80 ng/μl final concentration) was added. The PK-protected fragments of YohPHis are indicated (#). Please note that in these experiments, the amount of in vitro–synthesized YohP was reduced for preventing any saturation effects. (C) As in (B), but purified SRP, FtsY, or both (20 ng/μl each final concentration) were added when indicated. (D) Quantification of YohP insertion using the different conditions described in (B) and (C). The values are means of three independent experiments and the SD is indicated by error bars. Underlying data for this figure can be found in S1 Data. CL, cardiolipin; CTF, cytosolic translation factor; INV, inner membrane vesicle; INV(OE), INV from a SecYEG-overproducing E. coli strain; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PK, proteinase K; SD, standard deviation; SRP, signal recognition particle; TCA, trichloroacetic acid; U-INV, urea-treated INV; YohPHis, His-tagged YohP.
Fig 4.
Posttranslational recognition and insertion of YohP by SRP.
(A) YohP was in vitro synthesized in the absence of INV and translation was terminated by the addition of chloramphenicol (35 mg/ml). Samples were centrifuged for removing ribosomes and aggregates, and the supernatant was incubated with INV or U-INV for 10 minutes. When indicated, purified SRP and FtsY were added together with the U-INV. Samples were then processed as described in the legend to Fig 3. Quantification is based on three independent experiment, and the SD is shown in brackets. Underlying data for this figure can be found in S1 Data. (B) YohP(I4pBpa)His was in vitro synthesized and radioactively labeled. When indicated, YohP(I4pBpa)His was incubated with purified SRP (100 ng/μl) and UV-exposed. Samples were then TCA precipitated and separated by SDS-PAGE and analyzed by autoradiography. When indicated, a 10-fold scaled-up reaction was subjected to cross-linking and subsequently immune-precipitated by α-Ffh antibodies, covalently coupled to sepharose beads. (C) Cross-linking was performed as in (B), but when indicated, puromycin (1 mM) (“Puro”) was added prior to UV exposure for dissociating the ribosome. INV, inner membrane vesicle; IP, immune precipitation; PK, proteinase K; SD, standard deviation; SRP, signal recognition particle; TCA, trichloroacetic acid; U-INV, urea-treated INV; YohP(I4pBpa)His, YohP containing the UV-reactive cross-linker para-benzoyl-L-phenylalanine at position 4.
Fig 5.
SRP binds to YohP in vitro and in vivo.
(A) Either YohP(I4pBpa)His or YohP(F27pBpa)His were in vitro synthesized in the presence or absence of purified SRP (100 ng/μl) and UV-exposed. When indicated, UV exposure was performed in the presence of INV or after puromycin treatment (“Puro”). Lane 5 contains a sample in which puromycin was added at the start of the in vitro synthesis (+*) for controlling its ribosome dissociating activity. (B) YohPHis and YohP(I4pBpa)His were in vivo expressed in wt E. coli cells and when indicated, in vivo cross-linking in whole cells was induced by UV exposure. YohP and its cross-linked partner proteins were subsequently enriched by a single-step metal-affinity purification, TCA precipitated, and analyzed by western blotting using α-Ffh antibodies. Indicated are the copurifying Ffh and the Ffh-YohP cross-linking product. (C) The same material as in (B) was decorated with antibodies against the cytosolic chaperone Trigger factor. Indicated is the copurifying Trigger factor, but no specific cross-linking product was observed. INV, inner membrane vesicle; SRP, signal recognition particle; TCA, trichloroacetic acid; wt, wild type; YohP(I4pBpa)His, YohP containing the UV-reactive cross-linker para-benzoyl-L-phenylalanine at position 4; YohP(F27pBpa)His, YohP containing the UV-reactive cross-linker para-benzoyl-L-phenylalanine at position 27; YohPHis, His-tagged YohP.
Fig 6.
SRP-dependent insertion of YohP via the SecYEG translocon or YidC.
(A) The SecYEG-dependent secretory protein OmpA was in vitro synthesized in the absence or presence of INV that were pretreated with the inhibitor IpomF or with DMSO as control (0 mM IpomF). Samples were then subjected to PK treatment. Indicated are pOmpA and mature OmpA, which occurs after signal sequence cleavage. (B) Quantification of OmpA, MtlA, and YohP transport in IpomF-treated INV. Transport in the absence of IpomF was set to 100%. Indicated are the mean values of at least three (MtlA, OmpA) or five (YohP) independent experiments, and the SD is shown by error bars. Underlying data for this figure can be found in S1 Data. (C) YohP was in vitro synthesized in the absence of membranes, translation was stopped by the addition of chloramphenicol, and the sample was centrifuged for removing aggregates. The supernatant was subsequently incubated with INV, liposomes (“Lipos”), or proteoliposomes containing the SecYEG complex (100 ng/μl) or the YidC insertase (100 ng/μl) and in the presence of absence of purified SRP/FtsY (20 ng/μl, each). Samples were then subjected to PK treatment as before. (D) Quantification of at least three independent experiments performed as in (C). The SD is indicated by error bars. INV, inner membrane vesicle; IpomF, Ipomoeassin F; MtlA, mannitol permease; PK, proteinase K; pOmpA, pro-OmpA; SD, standard deviation; SRP, signal recognition particle.
Fig 7.
Translation-independent membrane enrichment of the YohP mRNA.
(A) E. coli wild-type cells carrying pBad24-MS2-GFP were induced with 0.1 mM arabinose and 5 μL culture solution were placed on a sterile glass-bottom dish (35-mm dish with 20-mm bottom well, glass thickness 0.16–0.19 mm). Imaging was performed with the DeltaVision Ultra microscope (exposure 0.2 seconds for GFP at 35% laser intensity and 0.075 seconds for bright field at 5% laser intensity), and 3-μm Z-scans were recorded with an interval of 1 μm. The displayed images were taken at the focal point. The image was developed with the ImageJ Fiji software. (B) As in (A) but cells were coexpressing the pSC-bglB-MS2.6x plasmid or the pSC-yohP-MS2.6x plasmid, encoding the bglB or yohP mRNA, respectively, each with a deleted ribosome binding site and a hexa-repeat MS2 stem-loop recognition sequence at the 3′ UTR. Expression of the respective mRNAs was induced at an OD600 = 0.5 with 4 mM IPTG, and MS2-GFP was induced 40 minutes after IPTG addition with 0.1 mM arabinose. Samples were analyzed as above and Nile red was used to stain the membrane. Nile red staining was monitored with an exposure time of 0.2 seconds at a laser intensity of 35%. GFP, green fluorescent protein; IPTG, isopropyl 1-thio-β-D-galactopyranoside.
Fig 8.
Model for the posttranslational SRP-dependent insertion of the small membrane protein YohP.
(A) yohP mRNA is recognized by cytosolic ribosomes and translated. YohP is released from the ribosome and bound by SRP, which targets YohP to its receptor FtsY that is bound to either the SecYEG translocon or YidC. SRP dissociates from YohP upon contacting FtsY, and YohP inserts into SecY or YidC and subsequently into the lipid phase of the membrane. (B) In addition, yohP mRNA that is not bound by cytosolic ribosomes is targeted by a so far unknown mechanism to a membrane-bound receptor. The membrane-bound mRNA is then translated and released from the ribosome before it is bound by SRP, which then delivers YohP to SecYEG or YidC as in (A). SRP, signal recognition particle.