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
