人SDCT2蛋白亚细胞定位信号分析

Subcellular Localization Signal Analysis of High-affinity Sodium-dependent Dicarboxylate Co-transporter Protein

为了确定人高亲和力钠离子依赖性二羧酸共转运蛋白 (high affinitysodium dependentdicarboxylateco transporter,SDCT2 ,NaDC3)在细胞内的定位 ,构建了SDCT2与增强型绿色荧光蛋白 (EGFP)的融合蛋白表达载体 ,并转染肾小管上皮细胞LLC PK1,激光共聚焦显微镜观察显示 ,SDCT2蛋白主要定位于细胞的基底侧膜上 .同时将SDCT2 EGFP融合基因mRNA显微注射到爪蟾卵母细胞中表达 ,可见融合蛋白的绿色荧光仅分布在细胞膜上 .为了进一步确定该蛋白质的亚细胞定位信号序列 ,将SDCT2基因的N端及C端分别缺失 ,并构建缺失突变体与EGFP的融合蛋白表达载体 ,将它们转染到LLC PK1中 ,观察SDCT2缺失体在细胞内的分布情况 .结果显示 ,N端缺失的SDCT2蛋白主要位于细胞质中 ,顶膜和基底侧膜上也有表达 ;C端缺失的SDCT2蛋白主要位于基底侧膜上 ,顶膜几乎没有表达 ,细胞质中表达很少 .免疫组化结果也显示 ,SDCT2只表达于人近端肾小管上皮细胞的基底侧膜 .这表明SDCT2蛋白的N端序列对其亚细胞定位是必需的 ,人SDCT2蛋白的基底膜定位信号位于N端序列中 .

In order to define subcellular localization of SDCT2, expression vector of SDCT2 and EGFP (enhanced green fluorescence protein) fusion protein was constructed and transfected into pig renal tubular epithelial cells LLC-PK1. Laser confocal microscopy showed that SDCT2 protein was almost exclusively located at the basal and lateral membranes of LLC-PK1 cells. When SDCT2-EGFP mRNA-was microinjected into Xenopus oocytes, green fluorescence of the fusion protein was only found at the cellular membrane. To further determine subcellular localization signal of SDCT2, its N-terminal and C-terminal sequences were deleted and fused with EGFP. These deletion mutants were transfected into LLC-PK1 and their intracellular distributions were observed. The confocal analysis indicated that the SDCT2 with N-terminal deletion was predominantly distributed in the cytoplasm and also expressed at apical and basolateral membranes. The SDCT2 with C-terminal deletion was almost entirely distributed at the basolateral membrane, nearly not expressed at the apical membrane, and seldom expressed in the cytoplasm. Immunohistochemistry staining revealed that SDCT2 only expressed at the basolateral membrane of proximal renal tubular cells. The above results suggested that the N-terminus of SDCT2 is required for its subcellular localization and the basolateral membrane localization signal of SDCT2 locates in the N-terminal sequence.