Self-Association of Purified Reconstituted ER Luminal Spacer Climp63
Membranes of the endoplasmic reticulum (ER) are shaped into cisternal sheets and cylindrical tubules. How ER sheets are generated and maintained is not clear. ER membrane protein Climp63 is enriched in sheets and routinely used as a marker of this structure. The luminal domain (LD) of Climp63 is pre...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2020-06-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fcell.2020.00500/full |
| _version_ | 1818760430602944512 |
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| author | Jinghua Zhao Junjie Hu Junjie Hu |
| author_facet | Jinghua Zhao Junjie Hu Junjie Hu |
| author_sort | Jinghua Zhao |
| collection | DOAJ |
| description | Membranes of the endoplasmic reticulum (ER) are shaped into cisternal sheets and cylindrical tubules. How ER sheets are generated and maintained is not clear. ER membrane protein Climp63 is enriched in sheets and routinely used as a marker of this structure. The luminal domain (LD) of Climp63 is predicted to be highly helical, and it may form bridges between parallel membranes, regulating the abundance and width of ER sheets. Here, we purified the LD and full-length (FL) Climp63 to analyze their homotypic interactions. The N-terminal tagged LD formed low-order oligomers in solution, but was extremely aggregation-prone when the GST tag was removed. Purified FL Climp63 formed detectable but moderate interactions with both the FL protein and the LD. When Climp63 was reconstituted into proteoliposomes with its LD facing out, the homotypic interactions were retained and could be competed by soluble LD, though vesicle clustering was not observed. These results demonstrate a direct self-association of Climp63, supporting its role as an ER luminal spacer. |
| first_indexed | 2024-12-18T06:58:30Z |
| format | Article |
| id | doaj.art-915807facb1249ff9b12099b775780d4 |
| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-12-18T06:58:30Z |
| publishDate | 2020-06-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-915807facb1249ff9b12099b775780d42022-12-21T21:17:07ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2020-06-01810.3389/fcell.2020.00500549456Self-Association of Purified Reconstituted ER Luminal Spacer Climp63Jinghua Zhao0Junjie Hu1Junjie Hu2Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, ChinaDepartment of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin, ChinaNational Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, ChinaMembranes of the endoplasmic reticulum (ER) are shaped into cisternal sheets and cylindrical tubules. How ER sheets are generated and maintained is not clear. ER membrane protein Climp63 is enriched in sheets and routinely used as a marker of this structure. The luminal domain (LD) of Climp63 is predicted to be highly helical, and it may form bridges between parallel membranes, regulating the abundance and width of ER sheets. Here, we purified the LD and full-length (FL) Climp63 to analyze their homotypic interactions. The N-terminal tagged LD formed low-order oligomers in solution, but was extremely aggregation-prone when the GST tag was removed. Purified FL Climp63 formed detectable but moderate interactions with both the FL protein and the LD. When Climp63 was reconstituted into proteoliposomes with its LD facing out, the homotypic interactions were retained and could be competed by soluble LD, though vesicle clustering was not observed. These results demonstrate a direct self-association of Climp63, supporting its role as an ER luminal spacer.https://www.frontiersin.org/article/10.3389/fcell.2020.00500/fullendoplasmic reticulumsheet biogenesismembrane tetheringhomotypic interactionsreconstitutionClimp63 |
| spellingShingle | Jinghua Zhao Junjie Hu Junjie Hu Self-Association of Purified Reconstituted ER Luminal Spacer Climp63 Frontiers in Cell and Developmental Biology endoplasmic reticulum sheet biogenesis membrane tethering homotypic interactions reconstitution Climp63 |
| title | Self-Association of Purified Reconstituted ER Luminal Spacer Climp63 |
| title_full | Self-Association of Purified Reconstituted ER Luminal Spacer Climp63 |
| title_fullStr | Self-Association of Purified Reconstituted ER Luminal Spacer Climp63 |
| title_full_unstemmed | Self-Association of Purified Reconstituted ER Luminal Spacer Climp63 |
| title_short | Self-Association of Purified Reconstituted ER Luminal Spacer Climp63 |
| title_sort | self association of purified reconstituted er luminal spacer climp63 |
| topic | endoplasmic reticulum sheet biogenesis membrane tethering homotypic interactions reconstitution Climp63 |
| url | https://www.frontiersin.org/article/10.3389/fcell.2020.00500/full |
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