An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking

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.

Selective inhibition of protein secretion by abrogating receptor-coat interactions during ER export

“…Deletion of SURF4 resulted in significantly reduced secretion of 10 proteins in HEK-293 cells and 18 in HuH7 cells (Figure 4C, D, Table S1). Consistent with previous observations, NUCB1 and NUCB2 were among the top hits affected in SURF4KO cells (Huang et al , 2021). Nucleobindins, together with Cab45, overlap as SURF4-dependent hits between the two cell lineages (Figure 4E).…”

“…Since multiple diverse cargo proteins interact with the Sec24 B-site (Miller et al , 2003; Mossessova et al , 2003; Mancias & Goldberg, 2008; Ma et al , 2017), perturbing SURF4-client interaction may represent a more tractable way forward to therapeutic intervention. However, the client repertoire of SURF4 seems substantial (Yin et al , 2018; Ordonez et al , 2021; Huang et al , 2021), and being able to selectively inhibit a subset of receptor-cargo interactions may be difficult. Nonetheless, like SEC24, SURF4 also appears to be a multivalent export facilitator, since it also recognises a second ER export signal, the Cardin-Weintraub (CW) motif, found on Shh (Tang et al, in press).…”

Comparative Proteomics Study of Yak Milk from Standard and Naturally Extended Lactation Using iTRAQ Technique

“…In contrast, the transporter activity was the most abundant category in the molecular function (Figure 4B). Vesicles play a key role in protein transport [29], and the difference in protein transport activity may be related to milk protein components in HL yak milk. The KEGG pathway enrichment analysis, which was based on the DEPs, revealed that the metabolic pathways with the most differences were those that involved carbohydrate metabolism, the biosynthesis of amino acids, and fat digestion and absorption.…”

ADP-Ribosylation Factor Family of Small GTP-Binding Proteins: Their Membrane Recruitment, Activation, Crosstalk and Functions

“…The vesicle formation assay is another way to identify the Arf regulators or Arf effectors (Huang et al, 2021). During this approach, the vesicle formation assay was performed in the presence or absence of the GTP-locked form of a specific ARF family protein.…”

“…This approach has an advantage to identify the Arf binding partners in conditions that lipid bilayer is not disrupted. Using this approach, a novel cytosolic factor, PRRC1, was identified to interact with Sar1A in a GTPdependent manner on vesicle membranes (Huang et al, 2021).…”