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

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.</p

Molecular Mechanisms of SURF4- mediated Protein Secretion

Protein secretion is an essential process that drives organelle biogenesis, cell growth and communication. Once synthesised and processed in the Endoplasmic Reticulum (ER), secretory proteins are incorporated into transport carriers that are generated by the COPII coat. Efficient and accurate cargo incorporation into ER-derived carriers is driven by transmembrane cargo receptors. ER export receptors are especially important for secretion of soluble cargo as they provide a transmembrane bridge to the cytosolic COPII coat. This study focused on SURF4, a cargo receptor for soluble cargo. I characterised the spectrum of SURF4 clients in HEK-293TREx and Huh7 cells using mass spectrometry. Amongst the top hits, I identified many oligomeric Ca2+-binding proteins, including Cab45 and NUCB1. Using *in vitro* translation and sitespecific photo-crosslinking, I showed direct co-translational SURF4 engagement with the cargo via an N-terminal ER-ESCAPE motif, exposed after signal peptide cleavage. This result supports a fast-export mechanism for preventing improper cargo oligomerization in an early organelle, and for the first time shows a cargo receptor can interact with an unfolded and incompletely translated client. Furthermore, with the aid of structural prediction-guided mutagenesis and site-specific cross-linking, I mapped a putative ER-ESCAPE interaction surface on SURF4 to an ER lumen-facing pocket. Additionally, my work examined SURF4 interactions with the COPII cargo adaptor, SEC24. Whereas Cab45 and NUCB1 use SEC24C/D isoforms, a previously described SURF4 cargo, PCSK9, which exposes ER-ESCAPE after propeptide autocleavage, exclusively employs SEC24A. I show that this discrepancy is due to a PCSK9 requirement for a co-receptor, TMED10. Using a protein-protein interaction assay and various SEC24 and SURF4 binding mutants, I showed that SURF4 uses C-terminal hydrophobic and acidic amino acids to bind the B-site on SEC24C. Conversely, SEC24A recognises a cytosolic loop on SURF4 via the D-site, while the B-site is likely occupied by TMED10 cytosolic tail. Finally, knock-down of TMED10 both reduced the SURF4-SEC24A interaction and abrogated PCSK9 secretion. Altogether, my PhD work defined the SURF4 cargo repertoire and described the biochemical basis for differential cargo recruitment into COPII vesicles.Medical Research Counci

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

Protein secretion is an essential process that drives cell growth, movement, and communication. Protein traffic within the secretory pathway occurs via transport intermediates that bud from one compartment and fuse with a downstream compartment to deliver their contents. Here, we explore the possibility that protein secretion can be selectively inhibited by perturbing protein-protein interactions that drive capture into transport vesicles. Human proprotein convertase subtilisin/kexin type 9 (PCSK9) is a determinant of cholesterol metabolism whose secretion is mediated by a specific cargo adaptor protein, SEC24A. We map a series of protein-protein interactions between PCSK9, its endoplasmic reticulum (ER) export receptor SURF4, and SEC24A that mediate secretion of PCSK9. We show that the interaction between SURF4 and SEC24A can be inhibited by 4-phenylbutyrate (4-PBA), a small molecule that occludes a cargo-binding domain of SEC24. This inhibition reduces secretion of PCSK9 and additional SURF4 clients that we identify by mass spectrometry, leaving other secreted cargoes unaffected. We propose that selective small-molecule inhibition of cargo recognition by SEC24 is a potential therapeutic intervention for atherosclerosis and other diseases that are modulated by secreted proteins