Fine interaction profiling of VemP and mechanisms responsible for its translocation-coupled arrest-cancelation
- Institute for Frontier Life and Medical Sciences, Kyoto University, Japan
Figures
Figure 1 with 5 supplements
In vivo crosslinking reveals interaction of a VemP nascent polypeptide with Ffh and PpiD as well as uL22 and translocon.
(A) Summary of proteins crosslinked with VemP in the arrested state. Colored circles represent the positions at which crosslinking with the indicated proteins was observed in B–F. Dashed red line …
Figure 1—figure supplement 1
Systematic PiXie analysis of a nascent VemP.
(A) Systematic PiXie analysis of VemP. Cells were grown as described in Materials and methods, induced with 1 mM IPTG for 15 min to express VemP-F3M derivatives with pBPA at the indicated position, …
Figure 1—figure supplement 2
Identification and characterization of VemPxSecA crosslinked products.
(A) VemPxSecA XLs. Celle were grown, induced to express a VemP-F3M derivative with pBPA at the indicated position (WT indicates the protein with no pBPA), pulse-chased, and UV-irradiated as in Figure…
Figure 1—figure supplement 3
Identification of VemP crosslinked products.
(A) VemPxSecG XLs. (B) VemPxPpiD XLs. The cells of the indicated strains were grown, induced to express a VemP-F3M derivative with pBPA at the indicated position (WT indicates the protein with no pBP…
Figure 1—figure supplement 4
Identification of VemP crosslinked products using His-tagged derivatives of candidate proteins.
(A–D) VemP PiXie analysis in cells expressing His10-tagged proteins. E. coli strains, RM2834 (expressing SecY-His10 from the chromosome, A), RM2831 (Ffh-His10, B), and RM2935 (YidC-His10,C) were …
Figure 1—figure supplement 5
Immunoprecipitation of VemP-crosslinked products using anti-FLAG antibodies.
The cells of the indicated strains were grown, induced to express a VemP-F3M derivative with pBPA at the indicated position (WT indicates the protein with no pBPA), and analyzed by PiXie as in Figure…
Figure 2 with 4 supplements
The VemP nascent polypeptide interacts sequentially with uL22/Ffh, the translocon, and PpiD.
(A) PiXie analysis of VemP. Cells were grown, induced to express a VemP derivative with pBPA, pulse-labeled and chased as in Figure 1. At the indicated time points in the chase period, cells were …
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Figure 2—source data 1
Zip file containing gel images and quantified band intensity data for the PiXie experiments.
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig2-data1-v3.zip
Figure 2—figure supplement 1
PiXie analysis of VemP interactions.
(A) Stability of the arrested form of the VemPpBPA derivatives. Cells were grown, induced to express a VemP-F3M derivative with pBPA at the indicated position (WT indicates the protein with no pBPA),…
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Figure 2—figure supplement 1—source data 1
Zip file containing gel images and quantified band intensity data for the pulse-chase experiments.
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig2-figsupp1-data1-v3.zip
Figure 2—figure supplement 2
The procedure of the quantification of immunoprecipitated bands.
(A) A schematic picture of VemP-F3M. The regions corresponding to three VemP-F3M-derived species (FL-m, AP-un and AP-pro) observed during PiXie analysis are indicated below by gray bars. The …
Figure 2—figure supplement 3
Effects of the secA51 mutation on the stability of the arrested VemP.
(A) secA+ cells or secA51 cells carrying the vemP-3xflag-myc plasmid were grown at 30 °C until early log phase. At 2 hr after the temperature shift to 42°C, cells were induced, pulse-labeled and …
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Figure 2—figure supplement 3—source data 1
Zip file containing gel images and quantified band intensity data for the pulse-chase experiments.
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig2-figsupp3-data1-v3.zip
Figure 2—figure supplement 4
Effects of NaN3 treatment on VemP crosslinking.
(A) Wild type cells (upper gel) and mutant cells expressing Ffh-His10 (middle gel), and SecY-His10 (lower gel) from the chromosome were used. The cells were grown, induced to express a VemP-F3M …
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Figure 2—figure supplement 4—source data 1
Zip file containing gel images and quantified band intensity data for the PiXie experiments.
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig2-figsupp4-data1-v3.zip
Figure 3 with 1 supplement
Ffh (SRP) functions in the targeting of VemP.
(A) Effects of Ffh-depletion on stability of the arrested-VemP. (left) Ffh-depletable cells carrying a vemP-f3m plasmid were grown in the M9 medium with (+) or without (–) 0.05% arabinose, induced, …
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Figure 3—source data 1
Zip file containing gel images.
(A–C) and quantified band intensity data for the pulse-chase experiments using the anti-VemP antibody (A), pulse-labeling experiments using the anti-SecD2 antibody (B) and immunoblotting with the anti-SecD2 antibody (C).
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig3-data1-v3.zip
Figure 3—figure supplement 1
Effects of a secB-deletion on the stability of the arrested VemP.
(A) WT cells or ΔsecB cells carrying a vemP-3xflag-myc plasmid were grown at 30°C until early log phase, induced, pulse-labeled, and chased as in Figure 4. At the indicated chase time points, total …
Figure 4
PpiD and SecD/F cooperate to facilitate the translocation and the arrest cancelation of VemP.
(A) Effects of the ppiD disruption on the stability of the arrested-VemP. (left) WT cells or ΔppiD cells carrying the vemP-f3m plasmid were grown, induced, pulse-labeled, and chased as in Figure 1. …
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Figure 4—source data 1
Zip file containing gel images.
(A–C) and quantified band intensity data for the pulse-chase experiments using the anti-VemP antibody (A), pulse-labeling experiments using the anti-SecD2 antibody (B) and immunoblotting with the anti-SecD2 antibody (C).
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig4-data1-v3.zip
Figure 5 with 3 supplements
Conserved Arg-85 has a role in the regulation of secretion-coupling of the VemP arrest cancelation.
(A) Effects of Arg-85 mutations on the stability of the arrested VemP in vivo. Cells were grown, induced to express the indicated VemP-F3M derivatives, and used for the pulse-chase experiments as in …
Figure 5—figure supplement 1
The conserved Arg-85 residue is important for the stability of the arrested VemP in vivo.
(A) Stability of the arrested state of the VemPpBPA variants. A schematic picture of the VemP polypeptide in the arrested state is presented with residue numbers. Arrested VemP (%) was calculated …
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Figure 5—figure supplement 1—source data 1
Zip file containing gel images and quantified band intensity data for the pulse-labeling experiments.
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig5-figsupp1-data1-v3.zip
Figure 5—figure supplement 2
Effects of a ppiD deletion on translocation and arrest cancelation of the VemP variant Cells were grown, induced to express the indicated VemP-F3M derivative, and pulse-labeled as in Figure 4.
After 30 s of chase, total cellular proteins were acid-precipitated, subjected to IP with the anti-VemP antibody and analyzed as in Figure 1. Arrested VemP (%) was calculated and the mean values are …
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Figure 5—figure supplement 2—source data 1
Zip file containing gel images and quantified band intensity data for the pulse- labeling experiments.
- https://cdn.elifesciences.org/articles/62623/elife-62623-fig5-figsupp2-data1-v3.zip
Figure 5—figure supplement 3
Sequence alignment of VemP orthologues.
Amino acids sequence of VemP orthologues were aligned using Clustal omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/); Vibrio alginolyticus (Va), Vibrio nigripulchritudo (Vn), Vibrio …
Figure 6
A model of the arrest-cancelation of a VemP nascent polypeptide.
(A) Schematic interaction maps of VemP during its translocation processes. The positions of the crosslinking with other factors and Arg-85 are mapped on the schematic picture of the arrested …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (E. coli) | MC4100 | This paper | Supplementary file 1 | |
| Strain (V. alginolyticus) | 138–2 | This paper | Supplementary file 1 | |
| Strain (P1 bacteriophage) | Laboratory stock | CGSC12133 | ||
| Recombinant DNA reagent | Plasmids | This paper | Supplementary file 2 | |
| Sequence-based reagent | This paper | PCR primers | Supplementary file 3 | |
| Antibodies | 1st antibodies | This paper | listed in the below | |
| Antibody | Goat Anti-Rabbit IgG (H + L)-HRP Conjugate | Bio-Rad Laboratories | 1706515 | |
| Chemical compound | H-p-Bz-Phe-OH | Bachem | F2800 | |
| Chemical compound | Methionine, L-[35S] Translation Grade | American Radiolabeled Chemicals | ARS 01014 | |
| Chemical compound | ANTI-FLAG M2 Affinity Gel | Sigma-Aldrich | A2220 | |
| Chemical compound | nProtein A Sepharose4 Fast Flow | GE Healthcare | 17528004 | |
| Chemical compound | Ni-NTA Agarose | QIAGEN | 30250 | |
| Commercial kit | ECL Western Blotting Detection Reagents | GE Healthcare | RPN2106 | |
| Commercial kit | ECL Prime Western Blotting Detection Reagents | GE Healthcare | RPN2232 | |
| Software, Algorithm | Microsoft Excel | Microsoft | ||
| Software, Algorithm | Bio-imaging Analyzer BAS-1800, BAS-5000 | Fujifilm/GE Healthcare | ||
| Software, Algorithm | Image Qaunt LAS 4000 mini | Fujifilm/GE Healthcare | ||
| Software, Algorithm | Multi Gauge | Fujifilm/GE Healthcare |
Additional files
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Supplementary file 1
Table S1.
Strains used in this study
- https://cdn.elifesciences.org/articles/62623/elife-62623-supp1-v3.docx
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Supplementary file 2
Table S2.
Plasmids used in this study
- https://cdn.elifesciences.org/articles/62623/elife-62623-supp2-v3.docx
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Supplementary file 3
Table S3.
Primers used in this study
- https://cdn.elifesciences.org/articles/62623/elife-62623-supp3-v3.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/62623/elife-62623-transrepform-v3.docx
