Kakhlon Or, Sakya Prabhat, Larijani Banafshe, Watson Rose, Tooze Sharon A
Secretory Pathway Laboratories, Cancer Research UK, London Research Institute, London, UK.
EMBO J. 2006 Apr 19;25(8):1590-602. doi: 10.1038/sj.emboj.7601067. Epub 2006 Apr 6.
Secretory granule (SG) maturation has been proposed to involve formation of clathrin-coated vesicles (CCVs) from immature SGs (ISGs). We tested the effect of inhibiting CCV budding by using the clathrin adaptor GGA (Golgi-associated, gamma-ear-containing, ADP-ribosylation factor-binding protein) on SG maturation in neuroendocrine cells. Overexpression of a truncated, GFP-tagged GGA, VHS (Vps27, Hrs, Stam)-GAT (GGA and target of myb (TOM))-GFP led to retention of MPR, VAMP4, and syntaxin 6 in mature SGs (MSGs), suggesting that CCV budding from ISGs is inhibited by the SG-localizing VHS-GAT-GFP. Furthermore, VHS-GAT-GFP-overexpression disrupts prohormone convertase 2 (PC2) autocatalytic cleavage, processing of secretogranin II to its product p18, and the correlation between PC2 and p18 levels. All these effects were not observed if full-length GGA1-GFP was overexpressed. Neither GGA1-GFP nor VHS-GAT-GFP perturbed SG protein budding from the TGN, or homotypic fusion of ISGs. Reducing GGA3 levels by using short interfering (si)RNA also led to VAMP4 retention in SGs, and inhibition of PC2 activity. Our results suggest that inhibition of CCV budding from ISGs downregulates the sorting from the ISGs and perturbs the intragranular activity of PC2.
分泌颗粒（SG）成熟过程被认为涉及从不成熟SG（ISG）形成网格蛋白包被囊泡（CCV）。我们通过使用网格蛋白衔接蛋白GGA（高尔基体相关、含γ耳、ADP核糖基化因子结合蛋白）抑制CCV出芽，来测试其对神经内分泌细胞中SG成熟的影响。截短的、绿色荧光蛋白（GFP）标记的GGA、VHS（Vps27、Hrs、Stam）-GAT（GGA和myb靶标（TOM））-GFP的过表达导致成熟SG（MSG）中MPR、VAMP4和Syntaxin 6的滞留，这表明SG定位的VHS-GAT-GFP抑制了从ISG出芽的CCV。此外，VHS-GAT-GFP的过表达破坏了激素原转化酶2（PC2）的自催化切割、分泌粒蛋白II向其产物p18的加工以及PC2和p18水平之间的相关性。如果过表达全长GGA1-GFP，则未观察到所有这些效应。GGA1-GFP和VHS-GAT-GFP均未干扰从反式高尔基体网络出芽的SG蛋白，或ISG的同型融合。使用小干扰（si）RNA降低GGA3水平也导致VAMP4在SG中的滞留，并抑制PC2活性。我们的结果表明，抑制从ISG出芽的CCV会下调从ISG的分选，并扰乱PC2的颗粒内活性。
