title: Functional investigation of transmembrane helix 3 in H⁺-translocating pyrophosphatase.
abstract: 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 (PP(i)) 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 PP(i) hydrolysis, proton transport, expression, and K⁺ stimulation of H⁺-PPase.
authors.forename: Ching-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Yen-Wei
authors.initials: YW
authors.lastname: Chen
authors.forename: Yun-Tzu
authors.initials: YT
authors.lastname: Huang
authors.forename: Yih-Jiuan
authors.initials: YJ
authors.lastname: Pan
authors.forename: Chien-Hsien
authors.initials: CH
authors.lastname: Lee
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Lin
authors.forename: Lin-Kun
authors.initials: LK
authors.lastname: Huang
authors.forename: Yueh-Yu
authors.initials: YY
authors.lastname: Lo
authors.forename: Yu-Fen
authors.initials: YF
authors.lastname: Huang
authors.forename: Yu-Di
authors.initials: YD
authors.lastname: Hsu
authors.forename: Shih-Chung
authors.initials: SC
authors.lastname: Yen
authors.forename: Jenn-Kang
authors.initials: JK
authors.lastname: Hwang
authors.forename: Rong-Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D015870:Gene Expression; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007477:Ions / Q000378:metabolism; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010940:Plant Proteins; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D007477:Ions; D010940:Plant Proteins; D043564:Inorganic Pyrophosphatase; D007930:Leucine