An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking
| العنوان: | An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking |
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| المؤلفون: | Yusong Guo, Haidi Yin, Qian Wu, Xiao Tang, Baiying Li, Liwen Jiang, Yang Liu, Elizabeth A. Miller, Kristina Poljak, Mo Wang, Julija Maldutyte, Zhongping Yao, Yan Huang, Zhixiao Wu |
| المصدر: | Proceedings of the National Academy of Sciences of the United States of America |
| بيانات النشر: | National Academy of Sciences, 2021. |
| سنة النشر: | 2021 |
| مصطلحات موضوعية: | intracellular protein transport, Golgi Apparatus, GTPase, In Vitro Techniques, Endoplasmic Reticulum, Mass Spectrometry, Cytosol, COPII, Humans, Receptor, Transport Vesicles, Secretory pathway, Monomeric GTP-Binding Proteins, Multidisciplinary, Secretory Pathway, Chemistry, Endoplasmic reticulum, Vesicle, Membrane Proteins, Cell Biology, Intracellular Membranes, Biological Sciences, Transport protein, Cell biology, Neoplasm Proteins, Protein Transport, HEK293 Cells, cargo sorting, cargo receptor, Guanosine Triphosphate, COP-Coated Vesicles, Carrier Proteins |
| الوصف: | Significance Protein sorting in the secretory pathway is a fundamentally important cellular process, but the clients of a specific cargo sorting machinery remains largely underinvestigated. Here, utilizing a vesicle formation assay to profile proteins associated with vesicles, we identified cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner or that interact with GTP-bound Sar1A. We found that two of them, FAM84B and PRRC1, regulate anterograde trafficking. Moreover, we revealed specific clients of two export adaptors, SURF4 and ERGIC53. These analyses demonstrate that our approach is powerful to identify factors that regulate vesicular trafficking and to uncover clients of specific cargo receptors, providing a robust method to reveal insights into the secretory pathway. The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors. |
| اللغة: | English |
| تدمد: | 1091-6490 0027-8424 |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::a0baeb53ed642a34ffcd2371a176a7f2 http://europepmc.org/articles/PMC8536394 |
| حقوق: | OPEN |
| رقم الأكسشن: | edsair.doi.dedup.....a0baeb53ed642a34ffcd2371a176a7f2 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 10916490 00278424 |
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