Yamaguchi Toshio, Apse Maris P, Shi Huazhong, Blumwald Eduardo
Department of Pomology, University of California, Davis, CA 95616.
Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12510-5. doi: 10.1073/pnas.2034966100. Epub 2003 Oct 6.
We conducted an analysis of the topology of AtNHX1, an Arabidopsis thaliana vacuolar Na+/H+ antiporter. Several hydrophilic regions of the antiporter were tagged with a hemagglutinin epitope, and protease protection assays were conducted to determine the membrane topology of the antiporter by using yeast as a heterologous expression system. The overall structure of AtNHX1 is distinct from the human Na+/H+ antiporter NHE1 or any known Na+/H+ antiporter. It is comprised of nine transmembrane domains and a hydrophilic C-terminal domain. Three hydrophobic regions do not appear to span the tonoplast membrane, yet appear to be membrane associated. Our results also indicate that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region resides in the vacuolar lumen. Deletion of the hydrophilic C terminus resulted in a dramatic increase in the relative rate of Na+/H+ transport. The ratio of Na+/K+ transport was twice that of the unmodified AtNHX1. This altered ratio resulted from a relatively small decrease in K+/H+ transport with a large increase in Na+/H+ transport. The vacuolar localization of the C terminus of the AtNHX1, taken together with the regulation of the antiporter selectivity by its C terminus, demonstrates the existence of luminal vacuolar regulatory mechanisms of the antiporter activity.
我们对拟南芥液泡Na⁺/H⁺逆向转运蛋白AtNHX1的拓扑结构进行了分析。该逆向转运蛋白的几个亲水区用血凝素表位进行了标记，并通过蛋白酶保护试验，以酵母作为异源表达系统来确定该逆向转运蛋白的膜拓扑结构。AtNHX1的整体结构不同于人类Na⁺/H⁺逆向转运蛋白NHE1或任何已知的Na⁺/H⁺逆向转运蛋白。它由九个跨膜结构域和一个亲水性C端结构域组成。三个疏水区似乎并未跨越液泡膜，但似乎与膜相关。我们的结果还表明，虽然AtNHX1的N端面向细胞质，但几乎整个C端亲水区都位于液泡腔中。亲水性C端的缺失导致Na⁺/H⁺转运的相对速率急剧增加。Na⁺/K⁺转运比率是未修饰的AtNHX1的两倍。这种改变的比率是由于K⁺/H⁺转运相对小幅下降以及Na⁺/H⁺转运大幅增加所致。AtNHX1的C端在液泡中的定位，连同其C端对逆向转运蛋白选择性的调节，证明了存在液泡腔对逆向转运蛋白活性的调节机制。
