Cross-linking within a subunit of coupling factor 1 increases the proton permeability of spinach chloroplast thylakoids.

Abstract

WEISS AND RICHARD E. MCCARTY From the Section of Biochemistry, Molecular and Cell Biology, Cornell Uniuersity, Ithaca, New York 14853 1. The bifunctional maleimide, o-phenylenedimaleimide (OPDM), inhibits photophosphorylation in spinach chloro- plast thylakoids at concentrations as low as 100 nM and is therefore, about 500-fold more effective than N-ethylma- leimide. Thylakoids must be illuminated in the presence of this reagent prior to the assay of photophosphorylation for the irreversible inhibition to be expressed. Uncouplers and adenosine 5’-triphosphate plus inorganic phosphate largely prevent the development of the inhibition. 2. Phosphorylation in thylakoids treated with OPDM in the light is uncoupled from electron flow. The extent of the light-induced uptake of protons is decreased by the light and OPDM treatment, indicating that treated thylakoid membranes have enhanced permeability to protons. N,N’- Dicyclohexylcarbodiimide restores H+ uptake in OPDM- treated thylakoids. 3. N-Phenylmaleimide is only slightly more effective than N-ethylmaleimide as an inhibitor of phosphorylation. Thus, the bifunctionality of OPDM, rather than its hydrophobic- ity, accounts for its potency. Although incubation of thyla- koids with N-ethylmaleimide in the dark does not affect the light-dependent inhibition of phosphorylation by N-ethyl- maleimide, it abolishes the ability of low concentrations of OPDM to inhibit. Moreover, thylakoids incubated with OPDM in the dark followed by removal of the unreacted OPDM show inhibited rates of phosphorylation after illu- mination in the absence of nucleotides or sulfhydryl com- pounds. 4. [‘CIOPDM is incorporated into the y and E subunits of coupling factor 1 (CF,) in thylakoids to the same extent in the light as in the dark. No other chloroplast protein was labeled to a significant extent by the OPDM. Less cysteine reacts with OPDM bound to the y subunit of CF, in thylakoids which had been illuminated in the presence of the OPDM than in thylakoids which had been incubated with OPDM in the dark. 5. Thus, OPDM appears to react with a readily accessible group on the y subunit in the dark. The other maleimide