Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>)
The post-translational import of nuclear-encoded chloroplast preproteins is critical for chloroplast biogenesis, and the Toc159 family of proteins is the receptor for this process. Our previous work identified and analyzed the Toc GTPase in tomato; however, the tomato-specific transport substrate fo...
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MDPI AG
2022-10-01
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| Online Access: | https://www.mdpi.com/2223-7747/11/21/2923 |
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| author | Qi Wang Jiang Yue Chaozhong Zhang Jianmin Yan |
| author_facet | Qi Wang Jiang Yue Chaozhong Zhang Jianmin Yan |
| author_sort | Qi Wang |
| collection | DOAJ |
| description | The post-translational import of nuclear-encoded chloroplast preproteins is critical for chloroplast biogenesis, and the Toc159 family of proteins is the receptor for this process. Our previous work identified and analyzed the Toc GTPase in tomato; however, the tomato-specific transport substrate for Toc159 is still unknown, which limits the study of the function of the TOC receptor in tomato. In this study, we expand the number of preprotein substrates of slToc159 receptor family members using slToc159-1 and slToc159-2 as bait via a split-ubiquitin yeast two-hybrid membrane system. Forty-one specific substrates were identified in tomato for the first time. Using slToc159-1GM and slToc159-2GM as bait, we compared the affinity of the two bait proteins, with and without the A domain, to the precursor protein, which suggested that the A domain endowed the proproteins with subclass specificity. The presence of the A domain enhanced the interaction intensity of slToc159-1 with the photosynthetic preprotein but decreased the interaction intensity of slToc159-2 with the photosynthetic preprotein. Similarly, the presence of the A domain also altered the affinity of slToc159 to non-photosynthetic preproteins. Bimolecular fluorescence complementation (BiFC) analysis showed that A domain had the ability to recognize the preprotein, and the interaction occurred in the chloroplast. Further, the localization of the A domain in Arabidopsis protoplasts showed that the A domain did not contain chloroplast membrane targeting signals. Our data demonstrate the importance of a highly non-conserved A domain, which endows the slToc159 receptor with specificity for different protein types. However, the domain containing the information on targeting the chloroplast needs further study. |
| first_indexed | 2024-03-09T18:44:19Z |
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| id | doaj.art-ae022143405346a5821b3bbb6309a645 |
| institution | Directory Open Access Journal |
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| language | English |
| last_indexed | 2024-03-09T18:44:19Z |
| publishDate | 2022-10-01 |
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| series | Plants |
| spelling | doaj.art-ae022143405346a5821b3bbb6309a6452023-11-24T06:25:16ZengMDPI AGPlants2223-77472022-10-011121292310.3390/plants11212923Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>)Qi Wang0Jiang Yue1Chaozhong Zhang2Jianmin Yan3College of Agriculture, Guizhou University, Guiyang 550025, ChinaCollege of Agriculture, Guizhou University, Guiyang 550025, ChinaCollege of Agriculture, Guizhou University, Guiyang 550025, ChinaCollege of Agriculture, Guizhou University, Guiyang 550025, ChinaThe post-translational import of nuclear-encoded chloroplast preproteins is critical for chloroplast biogenesis, and the Toc159 family of proteins is the receptor for this process. Our previous work identified and analyzed the Toc GTPase in tomato; however, the tomato-specific transport substrate for Toc159 is still unknown, which limits the study of the function of the TOC receptor in tomato. In this study, we expand the number of preprotein substrates of slToc159 receptor family members using slToc159-1 and slToc159-2 as bait via a split-ubiquitin yeast two-hybrid membrane system. Forty-one specific substrates were identified in tomato for the first time. Using slToc159-1GM and slToc159-2GM as bait, we compared the affinity of the two bait proteins, with and without the A domain, to the precursor protein, which suggested that the A domain endowed the proproteins with subclass specificity. The presence of the A domain enhanced the interaction intensity of slToc159-1 with the photosynthetic preprotein but decreased the interaction intensity of slToc159-2 with the photosynthetic preprotein. Similarly, the presence of the A domain also altered the affinity of slToc159 to non-photosynthetic preproteins. Bimolecular fluorescence complementation (BiFC) analysis showed that A domain had the ability to recognize the preprotein, and the interaction occurred in the chloroplast. Further, the localization of the A domain in Arabidopsis protoplasts showed that the A domain did not contain chloroplast membrane targeting signals. Our data demonstrate the importance of a highly non-conserved A domain, which endows the slToc159 receptor with specificity for different protein types. However, the domain containing the information on targeting the chloroplast needs further study.https://www.mdpi.com/2223-7747/11/21/2923tomatotranslocon of chloroplast outer membraneToc159split-ubiquitin two-hybrid screeninteracting protein |
| spellingShingle | Qi Wang Jiang Yue Chaozhong Zhang Jianmin Yan Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>) Plants tomato translocon of chloroplast outer membrane Toc159 split-ubiquitin two-hybrid screen interacting protein |
| title | Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>) |
| title_full | Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>) |
| title_fullStr | Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>) |
| title_full_unstemmed | Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>) |
| title_short | Split-Ubiquitin Two-Hybrid Screen for Proteins Interacting with slToc159-1 and slToc159-2, Two Chloroplast Preprotein Import Receptors in Tomato (<i>Solanum lycopersicum</i>) |
| title_sort | split ubiquitin two hybrid screen for proteins interacting with sltoc159 1 and sltoc159 2 two chloroplast preprotein import receptors in tomato i solanum lycopersicum i |
| topic | tomato translocon of chloroplast outer membrane Toc159 split-ubiquitin two-hybrid screen interacting protein |
| url | https://www.mdpi.com/2223-7747/11/21/2923 |
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