Erythropoietin (EPO) stimulates erythroid differentiation and maturation. Though the transcriptional regulation of EPO has been well studied, the molecular determinants of EPO secretion remain unknown. Here, we generated a HEK293T reporter cell line that provides a quantifiable and selectable readout of intracellular EPO levels and performed a genome-scale CRISPR screen that identified SURF4 as an important mediator of EPO secretion. Targeting SURF4 with multiple independent single guide RNAs (sgRNAs) resulted in intracellular accumulation and extracellular depletion of EPO. Both of these phenotypes were rescued by expression of SURF4 cDNA. Additionally, we found that disruption of SURF4 resulted in accumulation of EPO in the endoplasmic reticulum (ER) compartment and that SURF4 and EPO physically interact. Furthermore, SURF4 disruption in Hep3B cells also caused a defect in the secretion of endogenous EPO under conditions mimicking hypoxia, ruling out an artifact of heterologous overexpression. This work demonstrates that SURF4 functions as an ER cargo receptor that mediates the efficient secretion of EPO. Our findings also suggest that modulating SURF4 may be an effective treatment for disorders of erythropoiesis that are driven by aberrant EPO levels. Finally, we show that SURF4 overexpression results in increased secretion of EPO, suggesting a new strategy for more efficient production of recombinant EPO.

中文翻译：

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内质网货物受体 SURF4 促进高效促红细胞生成素分泌


促红细胞生成素 (EPO) 刺激红细胞分化和成熟。尽管 EPO 的转录调控已得到充分研究，但 EPO 分泌的分子决定因素仍然未知。在这里，我们生成了 HEK293T 报告细胞系，该细胞系提供了细胞内 EPO 水平的可量化和可选择的读数，并进行了基因组规模的 CRISPR 筛选，确定 SURF4 是 EPO 分泌的重要介质。用多个独立的单向导RNA (sgRNA) 靶向SURF4会导致EPO 细胞内积累和细胞外耗尽。这两种表型均通过SURF4 cDNA 的表达得到挽救。此外，我们发现 SURF4 的破坏导致 EPO 在内质网 (ER) 区室中积累，并且 SURF4 和 EPO 发生物理相互作用。此外，Hep3B 细胞中 SURF4 的破坏也会导致在模拟缺氧的条件下内源性 EPO 的分泌缺陷，从而排除了异源过度表达的假象。这项工作表明 SURF4 作为 ER 货物受体发挥作用，介导 EPO 的有效分泌。我们的研究结果还表明，调节 SURF4 可能是治疗由 EPO 水平异常引起的红细胞生成障碍的有效方法。最后，我们发现 SURF4 过表达会导致 EPO 分泌增加，这提出了一种更有效生产重组 EPO 的新策略。