The transmembrane domain 6 of vacuolar H+-pyrophosphatase mediates protein targeting and proton transport

Yih Jiuan Pan, Chien Hsien Lee, Shen Hsing Hsu, Yun Tzu Huang, Ching Hung Lee, Tseng Huang Liu, Yen Wei Chen, Shih Ming Lin, Rong Long Pan

研究成果: Article同行評審

14 引文 斯高帕斯(Scopus)

摘要

Vacuolar H+-pyrophosphatase (V-PPase; EC 3.6.1.1) plays a significant role in the maintenance of the pH in cytoplasm and vacuoles via proton translocation from the cytosol to the vacuolar lumen at the expense of PPi hydrolysis. The topology of V-PPase as predicted by TopPred II suggests that the catalytic site is putatively located in loop e and exposed to the cytosol. The adjacent transmembrane domain 6 (TM6) is highly conserved and believed to participate in the catalytic function and conformational stability of V-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean V-PPase was carried out to identify its structural and functional role. Mutants Y299A, A306S and L317A exhibited gross impairment in both PPi hydrolysis and proton translocation. Meanwhile, mutations at L307 and N318 completely abolished the targeting of the enzyme, causing broad cytosolic localization and implicating a possible role of these residues in protein translocation. The location of these amino acid residues was on the same side of the helix wheel, suggesting their involvement in maintaining the stability of enzyme conformation. G297A, E301A and A305S mutants showed declines in proton translocation but not in PPi hydrolysis, consequently resulting in decreases in the coupling efficiency. These amino acid residues cluster at one face of the helix wheel, indicating their direct/indirect participation in proton translocation. Taken together, these data indicate that TM6 is crucial to vacuolar H+-pyrophosphatase, probably mediating protein targeting, proton transport, and the maintenance of enzyme structure.

原文English
頁(從 - 到)59-67
頁數9
期刊Biochimica et Biophysica Acta - Bioenergetics
1807
發行號1
DOIs
出版狀態Published - 2011 1月

All Science Journal Classification (ASJC) codes

  • 生物物理學
  • 生物化學
  • 細胞生物學

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