Sato M, Hresko R, Mueckler M
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1998 Sep 25;273(39):25203-8. doi: 10.1074/jbc.273.39.25203.
The Glut1 glucose transporter is a glycoprotein whose membrane topology has been verified by a number of experimental observations, all of which are consistent with a 12-transmembrane helix model originally based on hydrophobicity analysis. We used Glut1 as a model multispanning membrane protein to test the Charge Difference Hypothesis (Hartmann, E., Rapoport, T. A., and Lodish, H. F. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5786-5790), which asserts that the topology of a eucaryotic multispanning membrane protein is determined solely by the amino acid charge difference across the first transmembrane segment. The charge difference across the first transmembrane segment of Glut1 was progressively inverted in two independent series of mutants, one series in which only the number of positively charged amino acid residues in the two flanking domains was altered and the other in which only the number of negatively charged residues in the two flanking domains was changed. The results indicate that the charge difference across the first transmembrane segment does affect the topology of the protein, but that contrary to the hypothesis, it only dictates the orientation of the first transmembrane segment and the disposition of the amino terminus and the first linker domain. Charge inversion resulted in the formation of aberrant molecules in which either the first or second transmembrane segment failed to insert into the membrane. The topology of downstream regions of Glut1 was unaffected by charge inversion across the first transmembrane segment, indicating that downstream sequences are important in determining the local topological disposition of the molecule.
葡萄糖转运蛋白1（Glut1）是一种糖蛋白，其膜拓扑结构已通过多项实验观察得到验证，所有这些观察结果均与最初基于疏水性分析的12跨膜螺旋模型一致。我们以Glut1作为多跨膜蛋白模型来检验电荷差异假说（哈特曼，E.，拉波波特，T. A.，和洛迪什，H. F.（1989年）《美国国家科学院院刊》86，5786 - 5790），该假说认为真核多跨膜蛋白的拓扑结构仅由第一个跨膜片段上的氨基酸电荷差异决定。在两个独立的突变体系列中，Glut1第一个跨膜片段上的电荷差异被逐步反转，一个系列中仅改变两个侧翼结构域中带正电荷氨基酸残基的数量，另一个系列中仅改变两个侧翼结构域中带负电荷残基的数量。结果表明，第一个跨膜片段上的电荷差异确实会影响蛋白质的拓扑结构，但与该假说相反的是，它仅决定第一个跨膜片段的方向以及氨基末端和第一个连接结构域的布局。电荷反转导致形成异常分子，其中第一个或第二个跨膜片段未能插入膜中。Glut1下游区域的拓扑结构不受第一个跨膜片段电荷反转的影响，这表明下游序列在决定分子的局部拓扑布局方面很重要。
