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

Abstract

Vacuolar H(+)-pyrophosphatase (V-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 PP(i) hydrolysis. The topology of V-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 V-PPase. In this study, alanine-scanning mutagenesis along TM6 of the mung bean V-PPase was carried out to identify its structural and functional role. Mutants Y299A, A306S and L317A exhibited gross impairment in both PP(i) 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 PP(i) 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.

Roles of the Hydrophobic Gate and Exit Channel in Vigna radiata Pyrophosphatase Ion Translocation

“…2a). E301 is highly conserved in plant H + -PPases, and previous mutagenesis studies have shown that mutation of E301 abolishes PPi hydrolysis and proton translocation; for example, E305Q in AVP1 [25] and E301A in VrH + -PPase [26]. We found that the PPi hydrolysis activity of the E301Q mutant was half that of the wild type, and proton pumping activity was undetectable ( Fig.…”

Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

“…Enzyme Assay and Protein Determination-PP i hydrolysis activity was determined by measuring the release of P i from PP i as delineated previously (7,12,13,26). The reaction medium contained 30 mM Tris/Mes (pH 8.0), 1 mM MgSO 4 , 0.5 mM NaF, 50 mM KCl, 1 mM PP i , 1.5 g/ml gramicidin D, and 20 -30 g/ml microsomal protein.…”

“…The mutated nucleotides were subsequently defined and confirmed by DNA sequencing. The pYVH6 and its relative variants were transformed into the yeast host cell S. cerevisiae strain BJ2168 (MATa, prc-407, prb1-1122, pep4-3, leu2, trp1, ura3, GAL) and cultured according to previous methods (7,12,13). The yeast microsomal membranes enriched in H ϩ -PPases were prepared followed by solubilization and purification with Ni 2ϩ -nitrilotriacetic acid beads as described previously (7,12,13,25).…”

“…Furthermore, the dimeric structure of H ϩ -PPase was visualized by atomic force microscopy (AFM) and electron microscopy, respectively (10,11). Previous studies showed the structure-function relationship of H ϩ -PPase and identified the pivotal domains and residues involved in enzymatic activities and structural integrity (1,(12)(13)(14)(15)(16).…”

Structural basis for the reversibility of proton pyrophosphatase

“…2), a residue, which according to site-directed mutagenesis data, is indispensable for ion translocation. [31][32][33][34] The strategic positive charge on the E301 residue facilitates PPi synthesis by stabilizing at the transition state conformation, the negatively charged hydroxyl ion released: Pi 2¡ C Pi 2¡ ! PPi 3¡ C OH ¡ (Step 2; Fig.…”