TY - JOUR
T1 - An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking
AU - Huang, Yan
AU - Yin, Haidi
AU - Li, Baiying
AU - Wu, Qian
AU - Liu, Yang
AU - Poljak, Kristina
AU - Maldutyte, Julija
AU - Tang, Xiao
AU - Wang, Mo
AU - Wu, Zhixiao
AU - Miller, Elizabeth A.
AU - Jiang, Liwen
AU - Yao, Zhong Ping
AU - Guo, Yusong
N1 - Funding Information:
ACKNOWLEDGMENTS. We thank Dr. Randy Schekman (University of California, Berkeley) for providing purified COPII components and antibodies against COPII components. We thank Dr. Xiao-wei Chen (Peking University) for antibodies against SURF4. This work was supported by Hong Kong Research Grants Council (RGC) grants 16102921, 26100315, 16101116, 16102218, 16103319, AoE/M-05/12, C4002-17G, and C4012-16E (to Y.G.). This work was also supported by grants from the National Natural Science Foundation of China (NSFC31871421 and NSFC3207050042 to Y.G. and NSFC 81601828 to H.Y.) and a Free Explore Project from the Shenzhen Science and Technology Innovation Committee (JCYJ20180306174847511 to Y.G.). This study was supported in part by the Innovation and Technology Commission (ITCPD/17-9 to Y.G.). This study was also partially supported by the Hong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou) (SMSEGL20SC01 to Y.G.). In addition, this work was supported by the Medical Research Council (MC_UP_1201/10) to E.A.M., and Z.-P.Y. is supported by Hong Kong RGC grants R5013-19, R4005-18, C5031-14E, 15304117, 15304020, and the University Research Facility in Chemical and Environmental Analysis and University Research Facility in Life Sciences of PolyU. L.J. is supported by grants from Hong Kong RGC (C4012-16E, C4002-17G, C4033-19E, and AoE/M-05/12).
Publisher Copyright:
© 2021 National Academy of Sciences. All rights reserved.
PY - 2021/8/31
Y1 - 2021/8/31
N2 - 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.
AB - 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.
KW - Cargo receptor
KW - Cargo sorting
KW - COPII
KW - Intracellular protein transport
KW - Secretory pathway
UR - http://www.scopus.com/inward/record.url?scp=85113481544&partnerID=8YFLogxK
U2 - 10.1073/pnas.2101287118
DO - 10.1073/pnas.2101287118
M3 - Journal article
C2 - 34433667
AN - SCOPUS:85113481544
SN - 0027-8424
VL - 118
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 35
M1 - e2101287118
ER -