Abstract
H+-translocating pyrophosphatase (H+-PPase; EC 3.6.1.1) is critical for acidifying lumens by translocating protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH+-PPase) contains 16 transmembrane helices (TMs). The amino acids in TM3 are highly conserved in plants, and the hydrophobicity of TM3 is relatively lower than most other TMs, and. Moreover, TM5 and 6, which are the core TMs involving in H+-PPase functions are in the vicinity of TM3. It is thus speculated that TM3 is related with the H+-PPase activities. To address this possibility, site-directed mutagenesis was applied in this study to determine the role of TM3 in VrH+-PPase. Upon alanine/serine substitution, T138 and S142, whose functional groups face toward the center TMs were shown to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the critical termini of the two GxxxG-like motifs are necessary in maintaining the enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is vital for efficient expression. S153, M161, and A164 are crucial for the K+-mediated stimulation of H+-PPase. Taken together, this study demonstrates that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase.