A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli
The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, an...
| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2019-07-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fmicb.2019.01482/full |
| _version_ | 1828228901454741504 |
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| author | Hendrik Geise Eyleen Sabine Heidrich Christoph Stefan Nikolin Denise Mehner-Breitfeld Thomas Brüser |
| author_facet | Hendrik Geise Eyleen Sabine Heidrich Christoph Stefan Nikolin Denise Mehner-Breitfeld Thomas Brüser |
| author_sort | Hendrik Geise |
| collection | DOAJ |
| description | The twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in Escherichia coli that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid p-benzoyl-l-phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatCI50Bpa resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBCI50Bpa complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatCI50Bpa. When substrate binding was abolished by point mutations, this TatBCI50Bpa complex shifted analogously to active TatABCI50Bpa complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems. |
| first_indexed | 2024-04-12T18:25:40Z |
| format | Article |
| id | doaj.art-c21b47c4c94a4659ab338f097b611beb |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-12T18:25:40Z |
| publishDate | 2019-07-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-c21b47c4c94a4659ab338f097b611beb2022-12-22T03:21:14ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2019-07-011010.3389/fmicb.2019.01482458271A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coliHendrik GeiseEyleen Sabine HeidrichChristoph Stefan NikolinDenise Mehner-BreitfeldThomas BrüserThe twin-arginine translocation (Tat) system transports folded proteins across membranes of prokaryotes, plant plastids, and some mitochondria. According to blue-native polyacrylamide gel electrophoresis after solubilization with digitonin, distinct interactions between the components TatA, TatB, and TatC result in two major TatBC-containing complexes in Escherichia coli that can bind protein substrates. We now report the first detection of a TatABC complex that likely represents the state at which transport occurs. This complex was initially found when the photo cross-linking amino acid p-benzoyl-l-phenylalanine (Bpa) was introduced at position I50 on the periplasmic side of the first trans-membrane domain of TatC. Cross-linking of TatCI50Bpa resulted in TatC-TatC-cross-links, indicating a close proximity to neighboring TatC in the complex. However, the new complex was not caused by cross-links but rather by non-covalent side chain interactions, as it was also detectable without UV-cross-linking or with an I50Y exchange. The new complex did not contain any detectable substrate. It was slightly upshifted relative to previously reported substrate-containing TatABC complexes. In the absence of TatA, an inactive TatBCI50Bpa complex was formed of the size of wild-type substrate-containing TatABC complexes, suggesting that TatB occupies TatA-binding sites at TatCI50Bpa. When substrate binding was abolished by point mutations, this TatBCI50Bpa complex shifted analogously to active TatABCI50Bpa complexes, indicating that a defect substrate-binding site further enhances TatB association to TatA-binding sites. Only TatA could shift the complex with an intact substrate-binding site, which explains the TatA requirement for substrate transport by TatABC systems.https://www.frontiersin.org/article/10.3389/fmicb.2019.01482/fulltwin-arginine translocationmembrane protein complexesprotein translocationEscherichia coliphoto cross-linking |
| spellingShingle | Hendrik Geise Eyleen Sabine Heidrich Christoph Stefan Nikolin Denise Mehner-Breitfeld Thomas Brüser A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli Frontiers in Microbiology twin-arginine translocation membrane protein complexes protein translocation Escherichia coli photo cross-linking |
| title | A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli |
| title_full | A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli |
| title_fullStr | A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli |
| title_full_unstemmed | A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli |
| title_short | A Potential Late Stage Intermediate of Twin-Arginine Dependent Protein Translocation in Escherichia coli |
| title_sort | potential late stage intermediate of twin arginine dependent protein translocation in escherichia coli |
| topic | twin-arginine translocation membrane protein complexes protein translocation Escherichia coli photo cross-linking |
| url | https://www.frontiersin.org/article/10.3389/fmicb.2019.01482/full |
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