LOCALIZATION OF CYTOSOLICALLY ORIENTED MALEIMIDE-REACTIVE DOMAIN OF VACUOLAR H-PYROPHOSPHATASE()

The vacuolar H+-pyrophosphatase (V-PPase) of plant cells is subject to substrate (Mg2PPi)-protectable, free PPi-potentiated irreversible inh ibition by the sulfhydryl reagent N-ethylmaleimide (NEM). inhibition b y NEM ap proximates pseudo-first order kinetics and double-log plots o f the first order rate constant for inactivation versus NEM concentrat ion yield a straight line relationship with a slope of approximately u nity. Since NEM and the membrane-impermeant cysteine reagent 3-(N-male -imidylpropionyl)biocytin (MPB) inhibit the V-PPase with similar kinet ics and compete for a common binding site on the M(r) = 66,000 substra te-binding subunit, a single residue located in a cytosolically dispos ed extramembranous domain is inferred to undergo covalent modification in both cases. Selective labeling of the V-PPase of vacuolar membrane vesicles with [C-14]NEM, purification of the M(r) = 66,000 subunit, a nd 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 (V8(14K)), contain s C-14 label. Gas phase sequence analysis of this band after electrotr ansfer to Immobilon P-SQ yields two overlapping sequences (V8(14K2) an d V8(14K2)) which unambiguously align with the carboxyl-terminal segme nt of the M(r) = 66,000 subunit. Both V8(14K1) and V8(14K2) 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 conse rvation of the sequence of the V-PPase from multiple plant sources, an d the identical kinetics of interaction of the enzymes from Vigna and Beta with NEM and MPB, Cys(634) of putative hydrophilic loop X is conc luded to be the cytosolically oriented residue whose alkylation by mal eimides is responsible for inactivation of the V-PPase. The significan ce of these results with respect to earlier speculations concerning th e identity of the catalytic site and topology of the V-PPase is discus sed.