Localization of cytosolically oriented maleimide-reactive domain of vacuolar H(+)-pyrophosphatase.

Abstract

The vacuolar H(+)-pyrophosphatase (V-PPase) of plant cells is subject to substrate (Mg2PPi)-protectable, free PPi-potentiated irreversible inhibition by the sulfhydryl reagent N-ethylmaleimide (NEM). Inhibition by NEM approximates pseudo-first order kinetics and double-log plots of the first order rate constant for inactivation versus NEM concentration yield a straight line relationship with a slope of approximately unity. Since NEM and the membrane-impermeant cysteine reagent 3-(N-maleimidylpropionyl)biocytin (MPB) inhibit the V-PPase with similar kinetics and compete for a common binding site on the M(r) = 66,000 substrate-binding subunit, a single residue located in a cytosolically disposed extramembranous domain is inferred to undergo covalent modification in both cases. Selective labeling of the V-PPase of vacuolar membrane vesicles with [14C]NEM, purification of the M(r) = 66,000 subunit, and its digestion with V8 protease generates multiple peptide fragments. Of the bands identified after electrophoresis of the digests on Tris-Tricine gels, only one, migrating at M(r) = 14,000 (V814K), contains 14C label. Gas-phase sequence analysis of this band after electrotransfer to Immobilon PSQ yields two overlapping sequences (V814K2 and V814K2) which unambiguously align with the carboxyl-terminal segment of the M(r) = 66,000 subunit. Both V814K1 and V814K2 encompass only 1 cysteine residue at position 634 which is conserved between the V-PPases from Arabidopsis thaliana, Beta vulgaris (isoforms 1 and 2), and Hordeum vulgare. On the basis of these findings, the strict conservation of the sequence of the V-PPase from multiple plant sources, and the identical kinetics of interaction of the enzymes from Vigna and Beta with NEM and MPB, Cys634 of putative hydrophilic loop X is concluded to be the cytosolically oriented residue whose alkylation by maleimides is responsible for inactivation of the V-PPase. The significance of these results with respect to earlier speculations concerning the identity of the catalytic site and topology of the V-PPase is discussed.