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Structure/function relationships of the plant vacuolar H+- pumps

Reference	BBS/E/C/00011419
Principal Investigator / Supervisor	Dr Ruth Gordon-Weeks
Co-Investigators / Co-Supervisors
Institution	Rothamsted Research
Department	Rothamsted Research Department
Funding type	Research
Value (£)	284,566
Status	Completed
Type	Institute Project
Start date	01/04/1997
End date	31/03/1999
Duration	24 months

Abstract

The plant vacuolar membrane (tonoplast) has two proton pumps responsible for acidification of the vacuolar sap and the generation of a protonmotive force across the tonoplast. One is a V-type ATPase similar to those found on endomembranes of all eukaryotes, the other an inorganic pyrophosphatase (V- PPase) which is unique to plants. The reason for the presence of two proton pumps on this membrane is not known. This project seeks to understand the physiological role of the V- PPase both through studies of its structure-function relations to identify domains responsible for the binding of substrates or regulatory molecules and through manipulation of gene expression. 2 The V- PPase has multiple sites binding different Mg-PPi complexes, and because it is unique to plants it has the potential to be a target for novel herbicides. Structure function studies use covalent, residue-specific inhibitors to label and identify key amino acid residues and the domains containing them. A further strategy uses the chemical modification of substrate analogues which competitively inhibit the V-PPase to develop photoaffinity probes to label and locate the substrate binding site. Following proteolysis and sequencing of labelled polypeptides, the domains can be aligned with the amino acid sequence of the complete catalytic subunit which has been derived elsewhere. Gene knockout studies are in progress, using homologous recombination in the moss plant, Physcomitrella patens, to establish the physiological function of the V- PPase. The role of a particular cytosolic loop in the binding of free Mg and the substrate (Mg2PPi) is to be investigated. Our results with covalent inhibitors are consistent with this loop having important roles in the binding of both substrate and free Mg. Our approach will be to label with N-ethylmaleimide (which reacts only with a Cys in this loop and is available radioactively labelled to high specific activity) and then see if reagents against other residues in the loop modify the degree of NEM labelling. In addition, antibodies against this loop will be raised using synthetic polypeptides and their effect on inhibition by different reagents determined. A study of the inhibition of the V-PPase by bisphosphonate compounds will be completed and submitted for publication. The expression of histidine tagged V- PPase in yeast and purification of the enzyme by immunoprecipitation will be used to enable progress to be made both in the identification of the residues involved in substrate/ligand protectable inhibition by covalent inhibitors and of the domains which interact with photoaffinity labelled probes constructed from substrate analogues. 2 The kinetics of the tobacco V-PPase isoforms will be characterised by heterologous expression in yeast and any differences compared to differences in tissue expression. The effects of altering the expression of the V- PPase by targeted gene disruption using homologous recombination in Physcomitrella patens and antisense strategies in Arabidopsis be analysed, with particular emphasis on stresses that have been shown to affect the expression of the enzyme.

Summary

unavailable

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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