An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking Yan Huanga,1, Haidi Yinb,c,1, Baiying Lid,1, Qian Wub,c, Yang Liua, Kristina Poljake, Julija Maldutytee, Xiao Tanga, Mo Wanga, Zhixiao Wua, Elizabeth A. Millere, Liwen Jiangd, Zhong-Ping Yaob,c,2, and Yusong Guoa,f,g,2 aDivision of Life Science and State Key Laboratory of Molecular Neuroscience, The Hong Kong University of Science and Technology, Hong Kong, China; bState Key Laboratory of Chemical Biology and Drug Discovery, Research Institute for Future Food, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong Special Administrative Region, China; cState Key Laboratory of Chinese Medicine and Molecular Pharmacology (Incubation), Shenzhen Key Laboratory of Food Biological Safety Control, Shenzhen Research Instituteof Hong Kong Polytechnic University, Shenzhen 518057, China; dSchool of Life Sciences, Centre for Cell and Developmental Biology, State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China; eCell Biology Division, Medical Research Council Laboratory of Molecular Biology, CB2 0QH, Cambridge, United Kingdom; fShenzhen Research Institute, The Hong Kong University of Science and Technology, 518057 Shenzhen, China; and gHong Kong Branch of Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China Edited by Peter J. Novick, University of California San Diego, La Jolla, CA, and approved July 13, 2021 (received for review January 21, 2021) The fidelity of protein transport in the secretory pathway relies on II (COPII) to capture cargo proteins (2). Soluble cargo proteins in the accurate sorting of proteins to their correct destinations. To the lumen of the ER cannot be directly recognized by COPII coat deepen our understanding of the underlying molecular mechanisms, and such proteins are thought to be linked to the cargo sorting it is important to develop a robust approach to systematically machinery on the cytosolic side by transmembrane cargo receptors. reveal cargo proteins that depend on specific sorting machinery to One cargo receptor in mammalian cells, ERGIC53, is a mannose be enriched into transport vesicles. Here, we used an in vitro assay lectin and functions in capturing specific N-linked glycoproteins in that reconstitutes packaging of human cargo proteins into vesicles the lumen of the ER (3). ERGIC53 regulates ER export of blood to quantify cargo capture. Quantitative mass spectrometry (MS) coagulation factors V and VIII, a cathepsin-Z–related protein, and analyses of the isolated vesicles revealed cytosolic proteins that alpha1-antittrypsin (4–7). Another cargo receptor, SURF4, binds are associated with vesicle membranes in a GTP-dependent man- amino-terminal tripeptide motifs of soluble cargo proteins and CELL BIOLOGY ner. We found that two of them, FAM84B (also known as LRAT regulates ER export of soluble cargo proteins, including the yolk domain containing 2 or LRATD2) and PRRC1, contain proline-rich protein VIT-2 in Caenorhabditis elegans (8), and PCSK9 and domains and regulate anterograde trafficking. Further analyses apolipoprotein B in mammalian cells (9–11). revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit Although significant progress has been made in understanding sites, interacts with the inner COPII coat, and its absence increases the general steps of cargo sorting, the spectrum of cargo clients membrane association of COPII. In addition, we uncovered cargo of a specific Arf family member, cargo adaptor, or cargo receptor proteins that depend on GTP hydrolysis to be captured into vesicles. remains largely underinvestigated. To deepen our understand- Comparing control cells with cells depleted of the cargo receptors, ing of protein sorting in the secretory pathway, it is important to SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation as- Significance say in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular traf- ficking and to uncover cargo clients of specific cellular factors. Protein sorting in the secretory pathway is a fundamentally im- portant cellular process, but the clients of a specific cargo sorting machinery remains largely underinvestigated. Here, utilizing a cargo sorting | secretory pathway | intracellular protein transport | vesicle formation assay to profile proteins associated with vesi- COPII | cargo receptor cles, we identified cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner or that interact he eukaryotic secretory pathway plays important roles in de- with GTP-bound Sar1A. We found that two of them, FAM84B and Tlivering a variety of newly synthesized proteins to their specific PRRC1, regulate anterograde trafficking. Moreover, we revealed resident compartments. The fidelity of protein transport in the specific clients of two export adaptors, SURF4 and ERGIC53. These secretory pathway depends on accurate sorting of specific cargo analyses demonstrate that our approach is powerful to identify proteins into transport vesicles. Defects in cargo sorting cause protein factors that regulate vesicular trafficking and to uncover clients mistargeting and induce defects in establishing cell polarity, im- of specific cargo receptors, providing a robust method to reveal munity, as well as other physiological processes (1). insights into the secretory pathway. A variety of cytosolic proteins are recruited to the membrane and play important roles in the protein sorting process. These cytosolic Author contributions: Y.H. and Y.G. designed research; Y.H., H.Y., B.L., Q.W., Y.L., K.P., proteins include small GTPases of the Arf family and cargo adaptors J.M., X.T., M.W., Z.W., and Y.G. performed research; E.A.M., L.J., Z.-P.Y., and Y.G. contrib- uted new reagents/analytic tools; Y.H., H.Y., E.A.M., L.J., Z.-P.Y., and Y.G. analyzed data; (1, 2). The Arf family GTPases cycle between a GDP-bound cytosolic and Y.H., H.Y., E.A.M., Z.-P.Y., and Y.G. wrote the paper. state and a GTP-bound state. Upon GTP binding, Arf proteins un- The authors declare no competing interest. dergo conformational changes in which the N-terminal amphipathic This article is a PNAS Direct Submission. helix is exposed to bind membranes and the switch domains change This open access article is distributed under Creative Commons Attribution License 4.0 their conformation to recruit various cytosolic cargo adaptors. Once (CC BY). recruited onto the membranes, these cargo adaptors recognize sort- 1Y.H., H.Y., and B.L. contributed equally to this work. ing motifs on the cargo proteins. This recognition step is important 2To whom correspondence may be addressed. Email: [email protected] or zhongping. for efficiently capturing cargo proteins into vesicles. [email protected]. The Arf family protein, Sar1, regulates packaging of cargo pro- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ teins into vesicles at the endoplasmic reticulum (ER). GTP-bound doi:10.1073/pnas.2101287118/-/DCSupplemental. Sar1 mediates membrane recruitment of the coat protein complex Published August 25, 2021. PNAS 2021 Vol. 118 No. 35 e2101287118 https://doi.org/10.1073/pnas.2101287118 | 1of12 Downloaded by guest on September 24, 2021 develop a robust approach to systematically reveal cargo proteins (13). This is possibly due to an unappreciated requirement for that depend on a specific factor to be efficiently packaged into other cytosolic factors in addition to the COP coats. Affinity vesicles. Revealing this will provide significant insight into the chromatography has been utilized to reveal cytosolic proteins that functions and the specificity of cargo sorting. Since distinct cyto- specifically interact with GTP-bound Arf or Rab proteins (14–16). solic proteins are recruited to membranes by different GTP-bound In this approach, the membranes are disrupted, which might Arf family proteins, systematic approaches are needed to char- preclude identification of membrane-associated effectors. Thus, it acterize budding events associated with a specific GTP-bound Arf is important to develop additional approaches to reveal novel family protein. cytosolic proteins that associate with GTP-bound Arf proteins on A cellular imaging approach, pairing analysis of cargo recep- membranes. tors (PAIRS), has been utilized to identify the spectrum of cargo Here, we used an in vitro assay to reconstitute packaging of cargo proteins that depend on a specific cargo receptor for ER export proteins into transport vesicles utilizing rat liver cytosol (RLC) as a in yeast. This analysis focused on around 150 cargo molecules source of cytosolic proteins. Analysis of vesicle fractions by quan- labeled with fluorescent tags (12). An in vitro assay that recon- titative mass spectrometry (MS) revealed cytosolic proteins that are stitutes packaging of cargo proteins into vesicles has been used to associated with vesicles dependent on GTP or GTP-bound Sar1A, reveal protein profiles of vesicles budded with purified COPII or and that regulate protein trafficking. One of the identified proteins, COPI proteins (13). However, this analysis did not identify any PRRC1, regulates membrane association of the COPII coat