Department of Neurochemistry, Stockholm University, Svante Arrhenius väg 16B, SE-106 91 Stockholm, Sweden.
NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich, Germany.
Biochim Biophys Acta Biomembr. 2018 Jun;1860(6):1326-1334. doi: 10.1016/j.bbamem.2018.03.001. Epub 2018 Mar 3.
Samp1, spindle associated membrane protein 1, is a type II integral membrane protein localized in the inner nuclear membrane. Recent studies have shown that the inner nuclear membrane protein, Emerin and the small monomeric GTPase, Ran are direct binding partners of Samp1. Here we addressed the question whether Ran could regulate the interaction between Samp1 and Emerin in the inner nuclear membrane. To investigate the interaction between Samp1 and Emerin in live cells, we performed FRAP experiments in cells overexpressing YFP-Emerin. We compared the mobility of YFP-Emerin in Samp1 knock out cells and cells overexpressing Samp1. The results showed that the mobility of YFP-Emerin was higher in Samp1 knock out cells and lower in cells overexpressing Samp1, suggesting that Samp1 significantly attenuates the mobility of Emerin in the nuclear envelope. FRAP experiments using tsBN2 cells showed that the mobility of Emerin depends on RanGTP. Consistently, in vitro binding experiments showed that the affinity between Samp1 and Emerin is decreased in the presence of Ran, suggesting that Ran attenuates the interaction between Samp1 and Emerin. This is the first demonstration that Ran can regulate the interaction between two proteins in the nuclear envelope.
Samp1，纺锤体相关膜蛋白 1，是一种定位于核内膜的 II 型整合膜蛋白。最近的研究表明，核内膜蛋白埃美琳和小分子单体 GTP 酶 Ran 是 Samp1 的直接结合伴侣。在这里，我们探讨了 Ran 是否可以调节内核膜中 Samp1 和 Emerin 之间的相互作用。为了在活细胞中研究 Samp1 和 Emerin 之间的相互作用，我们在过表达 YFP-Emerin 的细胞中进行了 FRAP 实验。我们比较了 Samp1 敲除细胞和过表达 Samp1 的细胞中 YFP-Emerin 的流动性。结果表明，Samp1 敲除细胞中 YFP-Emerin 的流动性较高，而过表达 Samp1 的细胞中流动性较低，表明 Samp1 显著降低了核膜中 Emerin 的流动性。使用 tsBN2 细胞进行的 FRAP 实验表明，Emerin 的流动性取决于 RanGTP。一致地，体外结合实验表明，Ran 的存在降低了 Samp1 和 Emerin 之间的亲和力，表明 Ran 减弱了 Samp1 和 Emerin 之间的相互作用。这是首次证明 Ran 可以调节核膜中两种蛋白质之间的相互作用。
