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A trap mutant reveals the physiological client spectrum of TRC40
The transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Company of Biologists Ltd
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633398/ https://www.ncbi.nlm.nih.gov/pubmed/31182645 http://dx.doi.org/10.1242/jcs.230094 |
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author | Coy-Vergara, Javier Rivera-Monroy, Jhon Urlaub, Henning Lenz, Christof Schwappach, Blanche |
author_facet | Coy-Vergara, Javier Rivera-Monroy, Jhon Urlaub, Henning Lenz, Christof Schwappach, Blanche |
author_sort | Coy-Vergara, Javier |
collection | PubMed |
description | The transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins that engage with TRC40 when other targeting machineries are fully operational. We use a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduces interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. We confirm known TRC40 substrates and identify many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, we show that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40. Thus, our results provide new avenues to assess the essential role of TRC40 in metazoan organisms. This article has an associated First Person interview with the first author of the paper. |
format | Online Article Text |
id | pubmed-6633398 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | The Company of Biologists Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-66333982019-08-01 A trap mutant reveals the physiological client spectrum of TRC40 Coy-Vergara, Javier Rivera-Monroy, Jhon Urlaub, Henning Lenz, Christof Schwappach, Blanche J Cell Sci Research Article The transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins that engage with TRC40 when other targeting machineries are fully operational. We use a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduces interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. We confirm known TRC40 substrates and identify many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, we show that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40. Thus, our results provide new avenues to assess the essential role of TRC40 in metazoan organisms. This article has an associated First Person interview with the first author of the paper. The Company of Biologists Ltd 2019-07-01 2019-07-01 /pmc/articles/PMC6633398/ /pubmed/31182645 http://dx.doi.org/10.1242/jcs.230094 Text en © 2019. Published by The Company of Biologists Ltd http://creativecommons.org/licenses/by/4.0This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. |
spellingShingle | Research Article Coy-Vergara, Javier Rivera-Monroy, Jhon Urlaub, Henning Lenz, Christof Schwappach, Blanche A trap mutant reveals the physiological client spectrum of TRC40 |
title | A trap mutant reveals the physiological client spectrum of TRC40 |
title_full | A trap mutant reveals the physiological client spectrum of TRC40 |
title_fullStr | A trap mutant reveals the physiological client spectrum of TRC40 |
title_full_unstemmed | A trap mutant reveals the physiological client spectrum of TRC40 |
title_short | A trap mutant reveals the physiological client spectrum of TRC40 |
title_sort | trap mutant reveals the physiological client spectrum of trc40 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6633398/ https://www.ncbi.nlm.nih.gov/pubmed/31182645 http://dx.doi.org/10.1242/jcs.230094 |
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