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首页> 外文期刊>Journal of cellular biochemistry. >Topology, glycosylation and conformational changes in the membrane domain of the vacuolar H+-ATPase a subunit
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Topology, glycosylation and conformational changes in the membrane domain of the vacuolar H+-ATPase a subunit

机译：液泡H + -ATPase亚基膜结构域的拓扑，糖基化和构象变化

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摘要

Published topological models of the integral membrane a subunit of the vacuolar proton-translocating ATPase complex have not been in agreement with respect to either the number of transmembrane helices within the integral membrane domain, or their limits and orientations within the lipid bilayer. In the present work we have constructed a predictive model of the membrane insertion of the yeast a subunit, Vph1p, from a consensus of seven topology prediction algorithms. The model was tested experimentally using epitope tagging, green fluorescent protein fusion, and protease accessibility analysis in purified yeast vacuoles. Results suggest that a consensus prediction of eight transmembrane helices with both the amino-terminus and carboxyl-terminus in the cytoplasm is correct. Characterization of two glycosylation sites within the homologous mouse a subunit membrane domain further corroborates this topology. Moreover, the model takes into account published data on cytoplasmic and luminal accessibility of specific amino acids. Changes in the degree of protease accessibility in response to the V-ATPase substrate, MgATP, and the V-ATPase-specific inhibitor, concanamycin A, suggest that functional conformational changes occur in the large cytoplasmic loop between TM6 and TM7 of Vph1p. These data substantially confirm one topological model of the V-ATPase a subunit and support the notion that conformational changes occur within the membrane domain, possibly involving previously proposed axial rotation and/or linear displacement of TM7 in the proton transport cycle.
机译：关于整体膜结构域内的跨膜螺旋的数目，或它们在脂质双层中的界限和方向，关于液泡质子转移ATP酶复合物的亚膜整体膜的一个已发表的拓扑模型尚未达成一致。在目前的工作中，我们根据七个拓扑预测算法的共识，构建了酵母亚基Vph1p膜插入的预测模型。使用表位标记，绿色荧光蛋白融合和纯化的酵母液泡中的蛋白酶可及性分析，对模型进行了实验测试。结果表明，在细胞质中具有氨基末端和羧基末端的八个跨膜螺旋的共识预测是正确的。同源小鼠亚基膜结构域内两个糖基化位点的特征进一步证实了这种拓扑结构。此外，该模型考虑了有关特定氨基酸的细胞质和腔内可及性的公开数据。响应V-ATPase底物MgATP和V-ATPase特异性抑制剂伴刀豆球蛋白A的蛋白酶可及程度的变化表明，功能构象变化发生在Vph1p的TM6和TM7之间的大细胞质环中。这些数据基本上证实了V-ATPase a亚基的一种拓扑模型，并支持了在膜结构域内发生构象变化的观点，这可能涉及质子运输周期中先前提出的TM7轴向旋转和/或线性位移。

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