| Abstract: | 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.The eukaryotic secretory pathway plays important roles in delivering a variety of newly synthesized proteins to their specific resident compartments. The fidelity of protein transport in the secretory pathway depends on accurate sorting of specific cargo proteins into transport vesicles. Defects in cargo sorting cause protein mistargeting and induce defects in establishing cell polarity, immunity, as well as other physiological processes (1).A variety of cytosolic proteins are recruited to the membrane and play important roles in the protein sorting process. These cytosolic proteins include small GTPases of the Arf family and cargo adaptors (1, 2). The Arf family GTPases cycle between a GDP-bound cytosolic state and a GTP-bound state. Upon GTP binding, Arf proteins undergo conformational changes in which the N-terminal amphipathic helix is exposed to bind membranes and the switch domains change their conformation to recruit various cytosolic cargo adaptors. Once recruited onto the membranes, these cargo adaptors recognize sorting motifs on the cargo proteins. This recognition step is important for efficiently capturing cargo proteins into vesicles.The Arf family protein, Sar1, regulates packaging of cargo proteins into vesicles at the endoplasmic reticulum (ER). GTP-bound Sar1 mediates membrane recruitment of the coat protein complex II (COPII) to capture cargo proteins (2). Soluble cargo proteins in the lumen of the ER cannot be directly recognized by COPII coat and such proteins are thought to be linked to the cargo sorting machinery on the cytosolic side by transmembrane cargo receptors. One cargo receptor in mammalian cells, ERGIC53, is a mannose lectin and functions in capturing specific N-linked glycoproteins in the lumen of the ER (3). ERGIC53 regulates ER export of blood coagulation factors V and VIII, a cathepsin-Z–related protein, and alpha1-antittrypsin (4–7). Another cargo receptor, SURF4, binds amino-terminal tripeptide motifs of soluble cargo proteins and regulates ER export of soluble cargo proteins, including the yolk protein VIT-2 in Caenorhabditis elegans (8), and PCSK9 and apolipoprotein B in mammalian cells (9–11).Although significant progress has been made in understanding the general steps of cargo sorting, the spectrum of cargo clients of a specific Arf family member, cargo adaptor, or cargo receptor remains largely underinvestigated. To deepen our understanding of protein sorting in the secretory pathway, it is important to develop a robust approach to systematically reveal cargo proteins that depend on a specific factor to be efficiently packaged into vesicles. Revealing this will provide significant insight into the functions and the specificity of cargo sorting. Since distinct cytosolic proteins are recruited to membranes by different GTP-bound Arf family proteins, systematic approaches are needed to characterize budding events associated with a specific GTP-bound Arf family protein.A cellular imaging approach, pairing analysis of cargo receptors (PAIRS), has been utilized to identify the spectrum of cargo proteins that depend on a specific cargo receptor for ER export in yeast. This analysis focused on around 150 cargo molecules labeled with fluorescent tags (12). An in vitro assay that reconstitutes packaging of cargo proteins into vesicles has been used to reveal protein profiles of vesicles budded with purified COPII or COPI proteins (13). However, this analysis did not identify any non-ER resident transmembrane proteins or secretory proteins (13). This is possibly due to an unappreciated requirement for other cytosolic factors in addition to the COP coats. Affinity chromatography has been utilized to reveal cytosolic proteins that specifically interact with GTP-bound Arf or Rab proteins (14–16). In this approach, the membranes are disrupted, which might preclude identification of membrane-associated effectors. Thus, it is important to develop additional approaches to reveal novel cytosolic proteins that associate with GTP-bound Arf proteins on membranes.Here, we used an in vitro assay to reconstitute packaging of cargo proteins into transport vesicles utilizing rat liver cytosol (RLC) as a source of cytosolic proteins. Analysis of vesicle fractions by quantitative mass spectrometry (MS) revealed cytosolic proteins that are associated with vesicles dependent on GTP or GTP-bound Sar1A, and that regulate protein trafficking. One of the identified proteins, PRRC1, regulates membrane association of the COPII coat and facilitates ER-to-Golgi trafficking. We also revealed cargo proteins that depend on specific cargo receptors, ERGIC53 or SURF4, to be efficiently packaged into vesicles. Our study indicates that the vesicle formation assay is a robust tool to reveal functional roles of specific factors in protein sorting, and to uncover novel factors that regulate vesicular trafficking in the secretory pathway. |
