PLANT PHYSIOLOGY , Vol 109, Issue 4 1379-1388, Copyright © 1995 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Molecular Dissection of the [epsilon] Subunit of the Chloroplast ATP Synthase of Spinach
J. A. Cruz, B. Harfe, C. A. Radkowski, M. S. Dann and R. E. McCarty
Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218-2658
The gene encoding the [epsilon] subunit (atpE) of the chloroplast ATP
synthase of Spinacia oleracea has been overexpressed in Escherichia coli.
The recombinant protein can be solubilized in 8 M urea and directly diluted
into buffer containing ethanol and glycerol to obtain [epsilon] that is as
biologically active as [epsilon] purified from chloroplast-coupling factor
1 (CF1). Recombinant [epsilon] folded in this manner inhibits the ATPase
activity of soluble and membrane-bound CF1 deficient in [epsilon] and
restores proton impermeability to thylakoid membranes reconstituted with
CF1 deficient in [epsilon]. Site-directed mutagenesis was used to generate
truncations and single amino acid substitutions in the primary structure of
[epsilon]. In the five mutants tested, alterations that weaken ATPase
inhibition by recombinant [epsilon] affect its ability to restore proton
impermeability to a similar extent, with one exception. Substitution of
histidine-37 with arginine appears to uncouple ATPase inhibition and the
restoration of proton impermeabilty. As in the case of E. coli, it appears
that N-terminal truncations of the [epsilon] subunit have more profound
effects than C-terminal deletions on the function of [epsilon]. Recombinant
[epsilon] with six amino acids deleted from the C terminus, which is the
only region of significant mismatch between the [epsilon] of spinach and
the [epsilon] of Pisum sativum, inhibits ATPase activity with a reduced
potency similar to that of purified pea [epsilon]. Four of the six amino
acids are serine or threonine. These hydroxylated amino acids may be
important in [epsilon]-CF1 interactions.
