Valentin M, Kühlkamp T, Wagner K, Krohne G, Arndt P, Baumgarten K, Weber W, Segal A, Veyhl M, Koepsell H
Anatomical Institute, University of Wurzburg, Koellikerstrasse 6, 97070 Wurzburg, Germany.
Biochim Biophys Acta. 2000 Sep 29;1468(1-2):367-80. doi: 10.1016/s0005-2736(00)00277-7.
Previously we cloned membrane associated (M(r) 62000-67000) polypeptides from pig (pRS1), rabbit (rbRS1) and man (hRS1) which modified transport activities that were expressed in Xenopus laevis oocytes by the Na(+)-D-glucose cotransporter SGLT1 and/or the organic cation transporter OCT2. These effects were dependent on the species of RS1 and on the target transporters. hRS1 and rbRS1 were shown to be intronless single copy genes which are expressed in various tissues and cell types. Earlier immunohistochemical data with a monoclonal IgM antibody suggested an extracellular membrane association of RS1. In the present paper antibodies against recombinant pRS1 were raised and the distribution and membrane localization of RS1 reevaluated. After subcellular fractionation of renal cortex RS1 was found associated with brush border membranes and an about 1:200 relation between RS1 and SGLT1 protein was estimated. Also after overexpression in X. laevis oocytes RS1 was associated with the plasma membrane, however, at variance to the kidney it was also observed in the cytosol. Labeling experiments with covalently binding lipid-permeable and lipid-impermeable biotin analogues showed that RS1 is localized at the inner side of the plasma membrane. Western blots with plasma membranes from Xenopus oocytes revealed that SGLT1 protein in the plasma membrane was reduced when hRS1 was coexpressed with human SGLT1 which leads to a reduction in V(max) of expressed glucose transport. Measurements of membrane capacitance and electron microscopic inspection showed that the expression of hRS1 leads to a reduction of the oocyte plasma membrane surface. The data suggest that RS1 is an intracellular regulatory protein that associates with the plasma membrane. Overexpression of RS1 may effect the incorporation and/or retrieval of transporters into the plasma membrane.
此前，我们从猪（pRS1）、兔（rbRS1）和人（hRS1）中克隆出了膜相关多肽（分子量62000 - 67000），这些多肽可调节由钠 - D - 葡萄糖共转运蛋白SGLT1和/或有机阳离子转运蛋白OCT2在非洲爪蟾卵母细胞中表达的转运活性。这些效应取决于RS1的种类和靶转运蛋白。hRS1和rbRS1被证明是无内含子的单拷贝基因，在各种组织和细胞类型中表达。早期用单克隆IgM抗体进行的免疫组织化学数据表明RS1与细胞外膜相关。在本文中，制备了针对重组pRS1的抗体，并重新评估了RS1的分布和膜定位。肾皮质亚细胞分级分离后，发现RS1与刷状缘膜相关，估计RS1与SGLT1蛋白的比例约为1:200。在非洲爪蟾卵母细胞中过表达后，RS1也与质膜相关，然而，与肾脏不同的是，在细胞质中也观察到了RS1。用共价结合的脂质可渗透和脂质不可渗透的生物素类似物进行的标记实验表明，RS1定位于质膜内侧。用非洲爪蟾卵母细胞质膜进行的蛋白质印迹显示，当hRS1与人SGLT1共表达时，质膜中的SGLT1蛋白减少，这导致表达的葡萄糖转运的Vmax降低。膜电容测量和电子显微镜检查表明，hRS1的表达导致卵母细胞质膜表面积减少。数据表明RS1是一种与质膜相关的细胞内调节蛋白。RS1的过表达可能影响转运蛋白并入和/或从质膜回收。
