Abstract
PCSK9 is a secreted protein that regulates plasma cholesterol levels and cardiovascular disease risk. Prior studies suggested the presence of an ER cargo receptor that recruits PCSK9 into the secretory pathway, but its identity has remained elusive. Here, we apply a novel approach that combines proximity-dependent biotinylation and proteomics together with genome-scale CRISPR screening to identify SURF4, a homologue of the yeast cargo receptor Erv29p, as a primary mediator of PCSK9 secretion in HEK293T cells. 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. SURF4 was found to localize to the early secretory pathway where it physically interacts with PCSK9. Deletion of SURF4 resulted in ER accumulation and decreased extracellular secretion of PCSK9. These findings support a model in which SURF4 functions as an ER cargo receptor mediating the efficient cellular secretion of PCSK9.
Highlights
PCSK9 is a proprotein convertase that acts as a negative regulator of the LDL receptor (Seidah et al, 2014)
The alpha-1 antitrypsin (A1AT) cargo receptor LMAN1 was labeled by both PCSK9-BirA* and A1AT-BirA*, suggesting that the restricted environment of the complex II (COPII) vesicle may lead to nonspecific labeling of adjacent cargo receptors
We compared the interactome of PCSK9 to that of SAR1A and SAR1B (Figure 1D), COPII proteins that localize to the cytoplasmic surface of budding COPII vesicles, identifying a total of 35 candidate ER cargo receptors interacting with both PCSK9 and either SAR1A or SAR1B (Figure 1E–F, Supplementary file 1)
Summary
PCSK9 is a proprotein convertase that acts as a negative regulator of the LDL receptor (Seidah et al, 2014). PCSK9 is synthesized primarily in hepatocytes and secreted into the bloodstream. Circulating PCSK9 binds to the LDL receptor and diverts it to lysosomes for degradation, thereby leading to decreased LDL receptor abundance at the hepatocyte cell surface, decreased LDL clearance, and hypercholesterolemia. PCSK9 was originally implicated in cardiovascular disease when human genetic studies identified gain-of-function PCSK9 mutations as a cause of familial hypercholesterolemia (Abifadel et al, 2003). Loss-of-function PCSK9 variants were associated with decreased plasma cholesterol and lowered lifetime incidence of cardiovascular disease (Cohen et al, 2006; Benn et al, 2010). Therapeutic inhibitors of PCSK9 have been recently developed that exhibit potent lipid-lowering effects and are associated with a reduction in cardiovascular events (Open-Label Study of Long-Term Evaluation against LDL Cholesterol (OSLER) Investigators et al, 2015; ODYSSEY LONG TERM Investigators et al, 2015)
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