Cross-linking within a Subunit of Coupling Factor 1 Increases the Proton Permeability of Spinach Chloroplast Thylakoids”

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


The bifunctional
maleimide, o-phenylenedimaleimide (OPDM), inhibits photophosphorylation in spinach chloroplast thylakoids at concentrations as low as 100 nM and is therefore, about 500-fold more effective than N-ethylmaleimide. 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 OPDMtreated 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 hydrophobicity, accounts for its potency. Although incubation of thylakoids with N-ethylmaleimide in the dark does not affect the light-dependent inhibition of phosphorylation by N-ethylmaleimide, 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 illumination in the absence of nucleotides or sulfhydryl compounds.
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 * This work was supported by Research Grants GB-44233X and PCM 76-15027 from the National Science Foundation and was taken in part from an Undergraduate Honors Thesis in Biology submitted by M. A. W. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
function of the immobilized OPDM can then react with a group in the y subunit of CF, which is exposed by lightdependent conformational changes of the enzyme in thylakoids, resulting in cross-linking within the y subunit. Since OPDM treatment in the light causes enhanced H+ permeation and uncoupling, this cross-linking may alter the conformation of CF, to expose a proton channel.   which results in the alkylation of one group in the y subunit and another in the l subunit of CF, but has no effect on the light-dependent incorporation of N-ethylmaleimide into the y subunit (2), abolished the ability of low concentrations of OPDM to inhibit phosphorylation in the light (Table IV)   This was found to be the case (Table  VI). Very little OPDM was incorporated into the (Y, ,6, or 6 subunits of CF,, whereas a little more than 0.5 mol of OPDM/mol of CF, was detected in the y subunit and 0.05 to 0.15 mol/mol of CF, was found in the E subunit.
In one experiment, gels of [WIOPDM-CF, were sliced into l-mm sections and radioactivity determined.
Significant radioactivity was found only in the regions