ARF鸟苷酸交换因子BIGs对高尔基体相关的囊泡转运的调控作用  

ADP-Ribosylation Factor-Guanine Nucleotide Exchange Factors,BIGs,Regulates Golgi-Related Vesicular Transport

作　　者：林思思[1] 林巍[1] 王莹[1] 周春[1] 李翠限[1] 沈晓燕[1] 

机构地区：[1]中山大学药学院药理毒理实验室,广东广州510006

出　　处：《中山大学学报（医学科学版）》2012年第3期311-315,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：国家自然科学基金(31070924;81173056);教育部高等学校博士学科点科研基金(20100171110052)

摘　　要：【目的】探讨ARF鸟苷酸交换因子BIG1和BIG2在高尔基体相关囊泡转运方面的功能。【方法】利用脂质体将siRNA干扰序列转入细胞中,western blotting方法检测转染效率;利用细胞免疫荧光染色方法检测Hela细胞中BIGs蛋白水平的表达分布;采用Alexa568标记的转铁蛋白孵育Hela细胞,检测转铁蛋白相关的内吞体循环;利用脂质体转染VSVG-YFP病毒质粒,检测新生蛋白经从内质网经高尔基体转运至胞膜的途径。【结果】BIG1和BIG2的siRNA干扰效率均高于70%,且特异性良好;干扰掉BIGs后,细胞内TGN230结构变松散,呈现短片状或点状;干扰BIG2可导致细胞内转铁蛋白的积聚,而同时干扰BIG1则可进-步加剧转铁蛋白的积聚;BIG1或/和BIG2干扰均抑制了新生蛋白的从内质网向高尔基及细胞表面的转运过程。【结论】BIGs蛋白主要位于反面高尔基体网络,对维持其结构完整性非常重要;它们均参与调控高尔基体相关的囊泡转运,两者具有协同作用。[Objective] To investigate the roles of Brefeldin A-inhibited GEPs （BIG1 and BIG2） in Golgi-related vesicular transport. [Methods] Small interfering RNAs targeting BIG1 and BIG2 were transfected by liposome and the efficiency was verified by Western blotting. Immunostaining of BIG1 or BIG2 combined with TGN230 was used to determine the expression and localization of BIGs. The release of uptaked Alexa568-Transferrin was used to detect the transferrin recycling. Hela cells transfected with VSVG-YFP were used to analyze the progression of VSVG along the secretory pathway. [Results] BIGland BIG2 protein expression were suppressed at least 70% with high specificity. The staining of trans-Golgi network （TGN） marker TGN230 was fragmented after BIGs depletion. BIG2 siRNA caused transferrin accumulation, and eotransfection withBIG1 siRNA could enhance this effect. BIG1 or/and BIG2 siRNA inhibited the secretory pathway of VSVG. [Conclusion] BIG1 and BIG2 both localize at the TGN, and are required for the integrality of TGN. BIGs co-operate in regulating of Golgi-related vesicular transport.

关 键 词：RNAI BIGs 反面高尔基体网络 囊泡转运 VSVG—YFP 

分 类 号：Q2[生物学—细胞生物学]