標題: | Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase |
作者: | Lee, Ching-Hung Chen, Yen-Wei Huang, Yun-Tzu Pan, Yih-Jiuan Lee, Chien-Hsien Lin, Shih-Ming Huang, Lin-Kun Lo, Yueh-Yu Huang, Yu-Fen Hsu, Yu-Di Yen, Shih-Chung Hwang, Jenn-Kang Pan, Rong-Long 生物資訊及系統生物研究所 Institude of Bioinformatics and Systems Biology |
關鍵字: | Proton-translocating pyrophosphatase;Proton transport;Transmembrane helix;Site-directed mutagenesis;Coupling efficiency;GxxxG-like motif |
公開日期: | 1-十二月-2013 |
摘要: | H+-translocating pyrophosphatase (H+-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH(+)-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H+-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H+-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH(+)-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K+-mediated stimulation of H+-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase. |
URI: | http://dx.doi.org/10.1007/s00232-013-9599-7 http://hdl.handle.net/11536/22923 |
ISSN: | 0022-2631 |
DOI: | 10.1007/s00232-013-9599-7 |
期刊: | JOURNAL OF MEMBRANE BIOLOGY |
Volume: | 246 |
Issue: | 12 |
起始頁: | 959 |
結束頁: | 966 |
顯示於類別: | 期刊論文 |