Functional investigation of transmembrane helix 3 in Hâº-translocating pyrophosphatase.
J Membr Biol
; 246(12): 959-66, 2013 Dec.
Article
en En
| MEDLINE
| ID: mdl-24121627
ABSTRACT
Hâº-translocating pyrophosphatase (Hâº-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 Hâº-PPase (VrHâº-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 Hâº-PPase functions, are near TM3. It is thus proposed that TM3 is associated with Hâº-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrHâº-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 Kâº-mediated stimulation of Hâº-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PP(i) hydrolysis, proton transport, expression, and K⺠stimulation of Hâº-PPase.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Proteínas de Plantas
/
Pirofosfatasa Inorgánica
/
Dominios y Motivos de Interacción de Proteínas
Idioma:
En
Revista:
J Membr Biol
Año:
2013
Tipo del documento:
Article