The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9
The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9
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DOI：
10.1101/336016
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发表时间：
2018-05
期刊：
影响因子：
7.7
通讯作者：
Brian T. Emmer;G. Hesketh;E. Kotnik;Vi T. Tang;Paul J. Lascuna;Jie Xiang;A. Gingras;Xiao-Wei Chen-Xiao-Wei-C
中科院分区：
文献类型：
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作者：
Brian T. Emmer;G. Hesketh;E. Kotnik;Vi T. Tang;Paul J. Lascuna;Jie Xiang;A. Gingras;Xiao-Wei Chen-Xiao-Wei-C
前蛋白转化酶枯草杆菌蛋白酶/kexin 9型（PCSK 9）是一种分泌性蛋白，在调节血浆胆固醇和心血管疾病风险中起重要作用。PCSK 9分泌独特地依赖于细胞质COPII蛋白SEC 24 A，表明存在介导这种相互作用的跨膜ER货物受体。在这里，我们报告了一种新的方法，该方法将邻近依赖性生物素化和蛋白质组学与基因组规模的CRISPR筛选相结合，以鉴定促进HEK 293 T细胞中异源表达的PCSK 9有效分泌的蛋白质。我们首先鉴定了35种候选蛋白，这些蛋白通过BirA* 与PCSK 9和COPII组分SAR 1A或SAR 1B的融合物标记。然后，我们进行了基因组规模的合并CRISPR诱变以鉴定其扰动导致PCSK 9-eGFP而不是对照A1 AT-mCherry的细胞内积累的基因。该筛选中4种最富集的sgRNA都靶向SURF 4，SURF 4是酵母内质网（ER）货物受体Erv 29 p的同源物，也是通过邻近依赖性生物素化鉴定的唯一候选物。SURF 4对PCSK 9分泌的功能贡献用多种独立的SURF 4靶向sgRNA、克隆SURF 4缺陷细胞系和SURF 4 cDNA的功能拯救来证实。与SURF 4作为PCSK 9的货物受体的功能兼容，荧光显微镜将SURF 4定位于早期分泌途径，免疫共沉淀揭示了SURF 4和PCSK 9之间的物理相互作用，SURF 4缺失导致PCSK 9的细胞外分泌减少和PCSK 9在ER中的积累。总之，这些发现支持了一种模型，其中SURF 4作为ER货物受体用于PCSK 9的有效细胞分泌。
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.