Glycolipid MPIase is essential for the TAT (twin-arginine translocation) pathway
- Department of Biological Chemistry and Food Sciences, Faculty of Agriculture, Iwate University, Japan
Figures
Figure 1 with 2 supplements
MPIase depletion causes the accumulation of TAT substrate precursors.
(A) Detection of SufI and TorA precursors upon MPIase depletion. SufI (top) and TorA (bottom) were expressed in the indicated strains. IPTG (1 mM) was added at early log phase, and cultivation was continued for 2 hr. The cells were subjected to TCA precipitation, followed by SDS-PAGE/immunoblotting. The precursor and mature forms were then detected as indicated. MPIase depletion was confirmed in KS23 (middle left). Defects in the processing of SufI (middle right) and TorA (bottom right) with the KK signal were also confirmed. The symbol '' denotes degradation products of the SufI precursor. (B) Pulse-chase experiments of SufI processing after MPIase depletion. EK413/pTac-TatABC harboring pTet-SufI(RR) (top left) or pTet-SufI(KK) (top right), and KS23/pAra-CdsA/pTac-TatABC harboring pTet-SufI(RR) (+MPIase; bottom left, ΔMPIase; bottom right) were pulse-labeled for 30 s and then chased for the indicated periods. SufI(RR) and SufI(KK) were isolated using the attached His tags, followed by SDS-PAGE/autoradiography. (C) Subcellular localization of SufI and TorA-GFP after MPIase depletion. KS23/pTet-TatABC/pTac-SufI(RR) (left) and KS23/pTet-TatABC/pTac-TorA-GFP(RR) (right) were cultivated in the presence (+MPIase) or absence (ΔMPIase) of arabinose. The TAT substrates were leaky expressed from the tac promoter. The cells were then fractionated into periplasmic (peri) and spheroplast (sph) fractions. The indicated proteins were detected by immunoblotting. (D) Subcellular localization of TorA-GFP after MPIase depletion as revealed by fluorescence microscopy. The indicated strains were cultivated, TorA-GFP was induced, and then it was observed by fluorescence microscopy. The scale bars represent 10 μm. A magnified view of part of the '+MPIase' sample is shown on the left.
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Figure 1—source data 1
PDF file containing original immunoblots and autoradiographs for Figure 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for immunoblot and pulse-chase analysis displayed in Figure 1.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig1-data2-v1.zip
Figure 1—figure supplement 1
Structure of MPIase.
The repeating unit of the glycan chain consists of a trisaccharide unit of 4-acetamido-4-deoxyfucose (Fuc4NAc), 2-acetamido-2-deoxymannuronic acid (ManNAcA), and N-acetylglucosamine (GlcNAc). R1: acyl chain of C16 or C18, R2: Ac or H. n=9–11.
Figure 1—figure supplement 2
The level of inclusion bodies of TAT substrates.
EK413 cells that expressed the specified proteins were disrupted using a French press (whole cells; wc), followed by centrifugation at 15,000 × g for 10 min at 4 °C, yielding a pellet (ppt 1). The resulting supernatant was centrifuged again under the same conditions to obtain the supernatant (sup) and a second pellet (ppt2). Samples equivalent to 10 μg of whole-cell proteins were analyzed by SDS-PAGE/immunoblotting. The positions of the specified proteins, including the precursor and mature forms, are shown. Stars denote degradation products.
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Figure 1—figure supplement 2—source data 1
PDF file containing original immunoblots for Figure 1—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig1-figsupp2-data1-v1.zip
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Figure 1—figure supplement 2—source data 2
Original files for immunoblot analysis displayed in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig1-figsupp2-data2-v1.zip
Figure 2 with 5 supplements
TAT substrates are translocated into INV in vitro when TatABC and MPIase are both overproduced.
(A) Translocation of SufI and TorA-GFP into TatABC-overproducing INV. SufI (top) and TorA-GFP (bottom) were synthesized in vitro in the presence of INV prepared from the indicated strains. INV was omitted in the '-' samples. The signal sequences on the left are wild-type (RR), while the sequences on the right are mutants (KK). Translocated materials were generated after PK digestion (+). One-fifth of the reaction mixture was analyzed as the translation standard (-). Translocation activities, expressed as a percentage of the amount of the synthesized proteins, are shown at the bottom of each autoradiogram. (B) Depletion of MPIase resulted in the failure of TAT translocation. INV were prepared from KS46/pAra-CdsA harboring pT7-TatABC, which was cultivated in the presence (+MPIase) or absence (ΔMPIase) of arabinose. After washing arabinose, IPTG was added at 1 mM to induce TatABC, and cultivation was continued for 2 hr prior to INV preparation. SufI translocation was then performed as described in (A). (C) The level of TatC in INV used in (A) and (B). Each INV (10 μg) was analyzed by SDS-PAGE, followed by immunoblotting using anti-RpmJ-His antibodies. The position of TatC-His was shown. (D) MPIase is upregulated upon TatABC overproduction. The expression levels of MPIase in the strains used in (B) were determined by immunoblotting. CdsA and TatABC were induced as indicated. SecB levels were determined as a loading control. (E) MPIase is co-purified with TatABC. INV prepared from BL21/pT7-TatABC-His induced with 1 mM IPTG (+++) or not induced (-), and BL21/pT7-Pm-F0b-His induced with 1 mM IPTG, solubilized with DDM, were applied to a TALON column, then eluted. The eluates were analyzed by TLC, followed by immunodetection of MPIase. The positions of the origin/front and MPIase are shown. Purified MPIase was also analyzed at right.
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Figure 2—source data 1
PDF file containing original immunoblots and autoradiographs for Figure 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for immunoblot and translocation analysis displayed in Figure 2.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-data2-v1.zip
Figure 2—figure supplement 1
Schematic diagram of the in vitro protein translocation assay system using INV or proteoliposomes.
Proteins are synthesized in vitro in the presence of INV or proteoliposomes. After the translocation reaction, the portion of the protein that has been translocated into the membrane vesicles is protected from cleavage by externally added proteinase K (PK).
Figure 2—figure supplement 2
Expression levels of TatA in the INV used in Figure 2A.
Expression levels of TatA in the BL21(DE3)/pT7-TatABC and BL21(DE3)/pT7 (empty) cells (10 μg of protein each) were detected by SDS-PAGE and immunoblotting with an anti-TatA antibody. As a loading control, SecB was detected in the same cells using an anti-SecB antibody.
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Figure 2—figure supplement 2—source data 1
PDF file containing original immunoblots for Figure 2—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp2-data1-v1.zip
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Figure 2—figure supplement 2—source data 2
Original files for immunoblot analysis displayed in Figure 2—figure supplement 2.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp2-data2-v1.zip
Figure 2—figure supplement 3
The anti-MPIase antibody inhibits SufI translocation into TatABC-overexpressed INV.
INV prepared from BL21/pT7-TatABC (4 μg) and anti-MPIase IgG (αMPIase IgG) (4 μg/μL) were mixed and incubated on ice for 30 min. The translocation assay was performed, as described in the legend to Figure 2A. Samples were analyzed by SDS-PAGE/autoradiography. One-fifth of the reaction mixture was used as the translation control (-PK). Translocation activity is shown at the bottom of each gel.
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Figure 2—figure supplement 3—source data 1
PDF file containing original autoradiograms for Figure 2—figure supplement 3, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp3-data1-v1.zip
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Figure 2—figure supplement 3—source data 2
Original files for translocation analysis displayed in Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp3-data2-v1.zip
Figure 2—figure supplement 4
SecYEG overproduction does not induce MPIase upregulation.
BL21/pAra-SecYEG was cultivated with or without 0.2% arabinose and then incubated for an additional 1 hr. An aliquot (500 μL) of each sample was treated with TCA to a final concentration of 5%. The TCA precipitate was recovered by centrifugation (10,000 × g, 4°C, 5 min) followed by washing with acetone. The samples were then analyzed by SDS-PAGE/immunoblotting using an anti-MPIase antibody (left) or Coomassie Brilliant Blue staining (right).
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Figure 2—figure supplement 4—source data 1
PDF file containing original immunoblots for Figure 2—figure supplement 4, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp4-data1-v1.zip
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Figure 2—figure supplement 4—source data 2
Original files for immunoblot analysis displayed in Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp4-data2-v1.zip
Figure 2—figure supplement 5
Purification of Pm-F0b and TatABC.
BL21(DE3)/pT7-Pm-F0b-His (left) and BL21(DE3)/pT7-TatABC-His (right) were grown in LB medium to OD600 nm ~0.5 at 37 °C, followed by induction with IPTG (1 mM). INV, then prepared, was solubilized, and then subjected to TALON column chromatography. The eluted fraction (~5 μg) was analyzed by SDS-PAGE/Coomassie Brilliant Blue staining. The positions of the specified proteins are indicated.
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Figure 2—figure supplement 5—source data 1
PDF file containing original Coomassie Brilliant Blue-stained gels for Figure 2—figure supplement 5, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp5-data1-v1.zip
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Figure 2—figure supplement 5—source data 2
Original files for gels displayed in Figure 2—figure supplement 5.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig2-figsupp5-data2-v1.zip
Figure 3 with 1 supplement
Reconstitution of the TAT system.
(A) Membrane fusion of TatABC proteoliposomes with MPIase liposomes enabled the detection of the SufI translocation. INV, prepared from BL21/pT7-TatABC, were solubilized with OG, and then mixed with purified F0F1-ATPase. The proteoliposomes were then formed by removing the detergent. PL/DAG liposomes or PL/DAG/MPIase liposomes were fused to the reconstituted proteoliposomes. SufI translocation was performed co-translationally (left) or post-translationally (right) in the presence of these proteoliposomes. SufI(RR) (top left) or SufI(KK) (bottom left) was used as the substrate. One-ninth of the reaction mixture was analyzed as the translation standard (-) for the co-translational reactions. For the post-translational reactions, ATP was added as indicated. Translocation activities are shown at the bottom of the left panels. (B) Purification of F0F1-ATPase and the TAT components. F0F1-ATPase from a thermophilic Bacillus PS3 (left), and E. coli TatA-His (lane 1), TatB-His (lane 2), and TatC-His (lane 3) (right) were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining. (C, D) Complete reconstitution of the TAT system using the purified components. (C) Purified TatA, TatB, and TatC (in a 1:1:1 ratio) and F0F1-ATPase were mixed with phospholipids. Then, detergent was removed to form proteoliposomes. The reconstituted proteoliposomes were then fused with PL/DAG liposomes or PL/DAG/MPIase liposomes. Translocation activity was assayed with SufI(RR) (left) or SufI(KK) (right). (D) Proteoliposomes were reconstituted with the indicated components, followed by TorA-GFP translocation. The TatA:TatB:TatC ratio was 10:1:1. CCCP (20 μM), a protonophore, was added to the sample on the left. TorA-GFP(RR) (top) or TorA-GFP(KK) (bottom) was used as the substrate. Translocation activities are shown at the bottom of each gel. Errors denote ± SD (N=3).
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Figure 3—source data 1
PDF file containing original autoradiographs for Figure 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for translocation analysis displayed in Figure 3.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig3-data2-v1.zip
Figure 3—figure supplement 1
In vivo complementation of the SDS-sensitive growth by His-tagged Tat components.
The ΔTatE mutation was transferred to the ΔTatA strain via P1 transduction. Overnight cultures of the ΔTatA/ΔTatE, ΔTatB, and ΔTatC strains harboring the indicated plasmid were adjusted to an OD600 nm of ~0.5, serially diluted by a factor of 10, and then spotted on LB agar plates supplemented with or without 2% SDS. All plates were incubated at 37°C for 1 day.
Figure 4 with 2 supplements
MPIase is a membrane receptor for TAT substrates.
(A,B) TorA-GFP is targeted to the membrane in an MPIase-dependent manner. TorA-GFP(RR) (A; left half) or TorA-GFP(KK) (A; right half) was expressed in the specified strains. The cells were then disrupted to obtain the supernatant (sup; cytosol and periplasm) and the precipitate (ppt; membranes) fractions. The fluorescence intensity of each fraction was measured. The ratio to the whole cell was determined and shown as percentages. The means and standard deviations were calculated from at least three independent experiments. **, p<0.01. In (B), the indicated strains were cultivated, TorA-GFP was induced, and then it was observed by fluorescence microscopy. The scale bars represent 10 μm. Part of each picture was magnified on each side. Scale bars represent 10 μm. (C) TorA-GFP interacts with INV expressing MPIase. Purified TorA-GFP was mixed with the indicated INV, followed by fluorescence detection (top). As a control, each INV was mixed with NBD-PE, a fluorescent phospholipid, and observed (bottom). Scale bars represent 10 μm.
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Figure 4—source data 1
Excel files including the raw data for Figure 4A, Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
GFP without the TAT signal is not targeted to the membrane.
KS23/pTac-GFP cells were cultivated in the presence (+MPIase) or absence (ΔMPIase) of arabinose. EK413/pTac-GFP and JW3815 (ΔTatC)/pTac-GFP were cultivated as well. After inducing GFP with 1 mM IPTG, the cells were disrupted and fractionated to obtain the supernatant (sup; cytosol) and the pellet (ppt; membranes) fractions. The fluorescence intensity of each fraction was measured. The ratio to the whole cell was determined and shown as percentages. The average of three independent experiments, along with the standard deviation, is shown with each value.
Figure 4—figure supplement 2
Purification of TorA-GFP.
BL21(DE3)/pT7-TorA-GFP was grown in LB medium to OD600 nm ~0.5. Then, 1 mM IPTG was added to induce TorA-GFP, and INV was prepared. The solubilized membrane was prepared and then applied to a TALON column. An eluted fraction (5 μL) was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue.
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Figure 4—figure supplement 2—source data 1
PDF file containing original Coomassie Brilliant Blue-stained gel for Figure 4—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Original files for the gel displayed in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/112170/elife-112170-fig4-figsupp2-data2-v1.zip
Figure 5
Working model of mechanisms underlying MPIase-dependent TAT translocation.
MPIase recognizes the signal sequence of the TAT substrates and inserts them into the membrane. The TatBC complex on the membrane then determines whether the signal sequence of the membrane-inserted TAT substrate is valid. The TatABC translocon then forms a pore the same size as the substrate. This allows the substrate protein to be translocated across the membrane. Finally, Lep cleaves the signal sequence, and the mature protein is released into the periplasm. See the text for details.
Additional files
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Supplementary file 1
Bacterial strains (Table A), plasmids (Table B), and oligonucleotides (Table C) used in this study.
- https://cdn.elifesciences.org/articles/112170/elife-112170-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/112170/elife-112170-mdarchecklist1-v1.docx
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Glycolipid MPIase is essential for the TAT (twin-arginine translocation) pathway
eLife 15:RP112170.
https://doi.org/10.7554/eLife.112170.4
