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The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

By Brian T Emmer, Geoffrey G Hesketh, Emilee Kotnik, Vi T Tang, Paul J Lascuna, Jie Xiang, Anne-Claude Gingras, Xiao-Wei Chen, David Ginsburg

Posted 31 May 2018
bioRxiv DOI: 10.1101/336016 (published DOI: 10.7554/elife.38839)

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that plays an important role in regulating plasma cholesterol and cardiovascular disease risk. PCSK9 secretion uniquely depends on the cytoplasmic COPII protein SEC24A, suggesting the presence of a transmembrane ER cargo receptor mediating this interaction. Here, we report a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify proteins that facilitate the efficient secretion of PCSK9 heterologously expressed in HEK293T cells. We first identified 35 candidate proteins that were labeled by BirA* fusions to PCSK9 and either COPII component SAR1A or SAR1B. We then performed genome-scale pooled CRISPR mutagenesis to identify genes whose perturbation resulted in intracellular accumulation of PCSK9-eGFP but not the control A1AT-mCherry. The 4 most enriched sgRNAs in this screen all targeted SURF4, a homologue of the yeast endoplasmic reticulum (ER) cargo receptor Erv29p and the only candidate also identified by proximity-dependent biotinylation. The functional contribution of SURF4 to PCSK9 secretion was confirmed with multiple independent SURF4-targeting sgRNAs, clonal SURF4-deficient cell lines, and functional rescue with SURF4 cDNA. Compatible with a function of SURF4 as a cargo receptor for PCSK9, fluorescence microscopy localized SURF4 to the early secretory pathway, coimmunoprecipitation revealed a physical interaction between SURF4 and PCSK9, and SURF4 deletion resulted in decreased extracellular secretion of PCSK9 and PCSK9 accumulation in the ER. Taken together, these findings support a model in which SURF4 functions as an ER cargo receptor for the efficient cellular secretion of PCSK9.

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