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

液泡焦磷酸水解酶第六穿膜區參與蛋白質定位與質子傳送

潘羿娟(Yih-Jiuan Pan)

指導教授 ： 潘榮隆

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2011年)

https://doi.org/10.6843/NTHU.2011.00020

摘要

Vacuolar H+-pyrophosphatase (H+-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 H+-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 H+-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean H+-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.

關鍵字

Vacuolar H + -pyrophosphatase ； Site-directed mutagenesis ； Proton translocation ； Tonoplast ； Vacuole ； protein targeting

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