Acidic Residues Necessary for Pyrophosphate-energized Pumping and Inhibition of the Vacuolar H+-pyrophosphatase byN,N′-Dicyclohexylcarbodiimide

Abstract

On the basis of a revised topological model of the vacuolar H؉ -pyrophosphatase (V-PPase; EC 3.6.1.1) derived from the analysis of four published sequences using two structure-predicting programs, TopPred II and MEMSAT, eight acidic amino acid residues located near or within transmembrane ␣-helices were identified. The codons specifying these amino acids in the cDNA encoding the V-PPase from Arabidopsis thaliana were singly mutated to examine their involvement in pyrophosphate (PP i The membranes constituting the vacuolysosomal complex of plant cells are unusual in possessing an H ϩ translocating inorganic pyrophosphatase (V-PPase 1 ; EC 3.6.1.1) (2). The VPPase bears no systematic resemblance to soluble PPases at the sequence level (3, 4) and is considered to belong to a fourth class of H ϩ -phosphohydrolase distinct from the F-, P-and VATPases (4). Moreover, unlike the V-ATPase, which is ubiquitous in the membranes bounding the acidic intracellular compartments of all eukaryotic cells, the V-PPase appears to be restricted to plants and a few species of phototrophic bacteria (2, 5). Notwithstanding the intrinsic evolutionary interest of this phenomenon, it poses a problem: the lack of sequencedivergent homologs from phylogenically remote organisms. Because all published V-PPase sequences are from the same group of organisms, vascular plants, and exhibit greater than 85% sequence identity at the amino acid level (6), most attempts to identify conserved amino acid residues of potential mechanistic significance by sequence alignment procedures have been unproductive. Crucial, therefore, has been the development of methods for the expression of functional pump in the yeast, Saccharomyces cerevisiae (7,8). When constructs of the yeast-Escherichia coli shuttle vector pYES2, containing the entire open reading frame of the cDNA (AVP; Ref. 9) encoding the M r 66,000 substrate-binding subunit 2 of the V-PPase from Arabidopsis thaliana are employed to transform S. cerevisiae, endomembrane-associated enzyme active in PP i -dependent H ϩ translocation is generated (7). Since the heterologously expressed pump is indistinguishable from the native plant enzyme, thereby establishing the sufficiency of AVP for the elab-* This work was supported by Grant MCB93-05281 from the National Science Foundation (NSF) and Grant DE-FG02-91ER20055 from the Department of Energy (DOE) (both to P. A. R.) and DOE/NSF/USDA Triagency Plant Training Grant DE-FG02-94ER20162 awarded to the Plant Science Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.)‡ To whom correspondence should be addressed. E-mail: parea@sas.upenn.edu. 1 The abbreviations used are: V-PPase, vacuolar H ϩ -pyrophosphatase; DCCD, N,NЈ-dicyclohexylcarbodiimide; Mes, 4-morpholineethanesulfonic acid; NEM, N-ethylmaleimide; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; V-, F-, and P-ATPases,...

Up-regulation of a H + -pyrophosphatase (H + -PPase) as a strategy to engineer drought-resistant crop plants

“…The E229D gain-of-function mutant (AVP1D) of the Arabidopsis H ϩ -PPase AVP1 has been shown to coordinately increase both PP i hydrolytic activity and PP i -dependent H ϩ -translocation when expressed in yeast (21). A construct containing the Arabidopsis AVP1D gene was introduced into the genome of Lycopersicon esculentum (cv.…”

“…The pRG395 plasmid was generated by cloning the AVP1D gene downstream of a tandem repeat of the 35S promoter of Cauliflower mosaic virus as described (17). Of note, the AVP1D gene is the E229D gain-of-function mutant of the AVP1 gene that has a coordinated increase of both PP i hydrolytic activity and PP i -dependent H ϩ -translocation (21). T 1 XAVP1D plants were screened on 100 mg͞liter kanamycin selection medium and then transferred to soil.…”

Elucidating the Role of Conserved Glutamates in H+-pyrophosphatase of Rhodospirillum rubrum

“…The glutamate residues substituted in this study and mersalyl-reactive cysteine residues (18) are indicated. Similar models have been suggested by other investigators (19,21,22). Single letter amino acid abbreviations are used with position numbers.…”

“…This approach was successfully employed to identify membrane-embedded charged residues contributing to dicyclohexylcarbodiimide binding in H ϩ -PPases (19), reactive Cys residues (18,20), and those residues that determine the potassium requirement of H ϩ -PPases (16). The role of conserved residues possibly located in the cytosol is more difficult to analyze, because their substitution results in an inactive enzyme (21).…”

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.…”