Kawasaki-Nishi Shoko, Nishi Tsuyoshi, Forgac Michael
Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA.
J Biol Chem. 2003 Oct 24;278(43):41908-13. doi: 10.1074/jbc.M308026200. Epub 2003 Aug 12.
Proton translocation by the vacuolar (H+)-ATPase (or V-ATPase) has been shown by mutagenesis to be dependent upon charged residues present within transmembrane segments of subunit a as well as the three proteolipid subunits (c, c', and c"). Interaction between R735 in TM7 of subunit a and the glutamic acid residue in the middle of TM4 of subunits c and c' or TM2 of subunit c" has been proposed to be essential for proton release to the luminal compartment. In order to determine whether the helical face of TM7 of subunit a containing R735 is capable of interacting with the helical face of TM4 of subunit c' containing the essential glutamic acid residue (Glu-145), cysteine-mediated cross-linking between these subunits in yeast has been performed. Cys-less forms of subunits a and c' as well as forms containing unique cysteine residues were constructed, introduced together into a strain disrupted in both endogenous subunits, and tested for growth at neutral pH, for assembly competence and for cross-linking in the presence of cupric-phenanthroline by SDS-PAGE and Western blot analysis. Four different cysteine mutants of subunit a were each tested pairwise with ten different unique cysteine mutants of subunit c'. Strong cross-linking was observed for the pairs aS728C/c'I142C, aA731C/c'E145C, aA738C/c'F143C, aA738C/c'L147C, and aL739C/c'L147C. Partial cross-linking was observed for an additional 13 of 40 pairs analyzed. When arrayed on a helical wheel diagram, the results suggest that the helical face of TM7 of subunit a containing Arg-735 interacts with the helical face of TM4 of subunit c' centered on Val-146 and bounded by Glu-145 and Leu-147. The results are consistent with a possible rotational flexibility of one or both of these transmembrane segments as well as some flexibility of movement perpendicular to the membrane.
通过诱变已经表明，液泡（H⁺）-ATP酶（或V-ATP酶）的质子转运依赖于亚基a跨膜片段以及三个脂蛋白亚基（c、c'和c''）中存在的带电荷残基。有人提出，亚基a的TM7中的R735与亚基c和c'的TM4中间的谷氨酸残基或亚基c''的TM2之间的相互作用对于质子释放到腔室至关重要。为了确定含有R735的亚基a的TM7的螺旋面是否能够与含有必需谷氨酸残基（Glu-145）的亚基c'的TM4的螺旋面相互作用，已在酵母中对这些亚基进行了半胱氨酸介导的交联。构建了无半胱氨酸形式的亚基a和c'以及含有独特半胱氨酸残基的形式，将它们一起导入内源性两个亚基均被破坏的菌株中，并通过SDS-PAGE和蛋白质印迹分析测试其在中性pH下的生长情况、组装能力以及在铜菲咯啉存在下的交联情况。亚基a的四个不同半胱氨酸突变体分别与亚基c'的十个不同独特半胱氨酸突变体进行两两测试。观察到aS728C/c'I142C、aA731C/c'E145C、aA738C/c'F143C、aA738C/c'L147C和aL739C/c'L147C这几对有强交联。在分析的40对中的另外13对中观察到部分交联。当排列在螺旋轮图上时，结果表明含有Arg-735的亚基a的TM7的螺旋面与以Val-146为中心、由Glu-145和Leu-147界定的亚基c'的TM4的螺旋面相互作用。结果与这两个跨膜片段中的一个或两个可能的旋转灵活性以及垂直于膜的一些移动灵活性一致。
