Flegelova Hana, Haguenauer-Tsapis Rosine, Sychrova Hana
Department of Membrane Transport, Institute of Physiology AS CR, Videnska 1083, 142 20, Prague 4, Czech Republic.
Biochim Biophys Acta. 2006 Mar;1760(3):504-16. doi: 10.1016/j.bbagen.2006.01.014. Epub 2006 Feb 17.
Na+/H+ antiporters, integral membrane proteins that exchange protons for alkali metal cations, play multiple roles in probably all living organisms (preventing cells from excessive amounts of alkali metal cations, regulating intracellular pH and cell volume). In this work, we studied the functionality of rat plasma membrane NHE1-3 exchangers upon their heterologous expression in alkali-metal-cation sensitive Saccharomyces cerevisiae, and searched for conditions that would increase their level in the plasma membrane and improve their functionality. Though three tested exchangers were partially localized to the plasma membrane (and two of them (NHE2 and NHE3) in an active form), the bulk of the synthesized proteins were arrested along the secretory pathway, mainly in the ER. To increase the level of exchangers in the yeast plasma membrane several approaches (truncation of C-terminal regulatory sequences, expression in mutant yeast strains, construction of rat/yeast protein chimeras, various growth conditions and chemical chaperones) were tested. The only increase in the amount of NHE exchangers in the plasma membrane was obtained upon expression in a strain with the npi1 mutation, which significantly lowers the level of Rsp5 ubiquitin ligase in cells. This mutation helped to stabilize proteins in the plasma membrane.
钠/氢逆向转运蛋白是一种将质子与碱金属阳离子进行交换的整合膜蛋白，可能在所有生物体中都发挥着多种作用（防止细胞内碱金属阳离子过量、调节细胞内pH值和细胞体积）。在这项研究中，我们研究了大鼠质膜NHE1 - 3交换蛋白在碱金属阳离子敏感的酿酒酵母中异源表达后的功能，并寻找能够提高其在质膜中的水平并改善其功能的条件。尽管三种测试的交换蛋白部分定位于质膜（其中两种（NHE2和NHE3）以活性形式存在），但大部分合成蛋白在分泌途径中受阻，主要滞留在内质网中。为了提高酵母质膜中交换蛋白的水平，我们测试了几种方法（截断C末端调节序列、在突变酵母菌株中表达、构建大鼠/酵母蛋白嵌合体、各种生长条件和化学伴侣）。在npi1突变的菌株中表达时，质膜中NHE交换蛋白的量唯一增加，该突变显著降低了细胞中Rsp5泛素连接酶的水平。这种突变有助于稳定质膜中的蛋白质。
