Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport

Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES). The penta-EF-hand (PEF) protein apoptosis-linked gene 2 (ALG-2) stabilizes sec31A at ER exit sites (ERES) and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK) cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport—peflin is a negative regulator of transport.

内质网腔钙离子（Luminal calcium）可调控分泌通路早期阶段的囊泡运输。在从内质网到高尔基体的转运过程中，腔内钙离子的耗竭会显著抑制转运过程，并导致处于出芽阶段及新形成的包被蛋白II囊泡（COPII vesicles）与囊泡蛋白的积累。腔内钙离子对转运的调控作用，可能通过内质网出口位点（ER exit sites, ERES）附近的胞质钙离子传感器介导。五EF手结构域蛋白（penta-EF-hand, PEF）家族的凋亡相关基因2（apoptosis-linked gene 2, ALG-2）可在内质网出口位点（ERES）稳定Sec31A蛋白，并促进COPII囊泡内外壳组分的组装。然而，在基础生理条件下，ALG-2究竟是正向、负向调控因子，还是完全不具备调控作用，目前尚未通过体外实验与完整细胞实验得到明确结论。ALG-2可与另一PEF蛋白peflin结合形成胞质异二聚体，该复合物可响应钙离子信号发生解离。但目前尚未证实peflin的生物学功能，也未探究peflin及ALG-2与peflin的相互作用是否会调控囊泡转运。本研究利用完整单细胞形态学实验体系，检测正常大鼠肾细胞（normal rat kidney, NRK cells）中的内质网到高尔基体转运过程。我们发现，通过小干扰RNA（siRNA）敲低peflin后，膜运输货物水泡性口炎病毒G蛋白（VSV-G）的转运速率显著加快。同时敲低peflin与ALG-2则会阻断peflin敲低所引发的转运速率提升，这表明ALG-2在该加速转运过程中发挥了关键作用。进一步实验显示，peflin敲低会导致ALG-2向内质网出口位点的靶向募集增多，同时增强ALG-2与Sec31A的相互作用，这提示peflin通常可通过抑制ALG-2与Sec31A的结合，负向调控囊泡转运。本研究首次明确了PEF蛋白在内质网到高尔基体转运中的稳态调控功能——peflin是囊泡转运的负向调控因子。