Abstract
Trypsin treatment of spinach chloroplast thylakoids in the light but not in the dark, results in a highly active Mg2+-ATPase and an uncoupling of photophosphorylation. These light-dependent effects are due to a modification of coupling factor 1 (CF1). CF1 purified from thylakoids treated with trypsin in the light contained a clipped beta subunit and a partially clipped gamma subunit, whereas that from thylakoids treated in the dark with trypsin contained only the clipped beta subunit. CF1 containing this modified gamma subunit also retained a high level of Ca2+-ATPase activity in solution. These results suggest that the gamma subunit becomes highly sensitive to trypsin only when the CF1 is in an active conformation. A similar hypersensitivity to proteases of the gamma subunit in highly purified CF1 is seen only after the enzyme is activated (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). The conversion of the enzyme to its active form, both on the membrane and in solution, therefore, seems to involve conformational changes that expose the gamma subunit to proteolysis.
Highlights
Mg+-ATPaseand an uncoupling of photophosphoryl- Mg2'-ATPase of thylakoids
The requirement for illumination in the activation of the Mg"-ATPase of thylakoids may be ascribed to energy-dependent changes in the conformation of CF1' that expose critical regions of the enzyme to attack by thiols or assayed at pH 7.9 at 20 "C in a solution (2.0 ml) which contained 1 mM Tricine/NaOH, 50 mM NaCl, 5 m~ MgC12,20 p~ pyocyanine, or 20 p~ PMS, and thylakoids equivalent to 100 p g of chlorophyll/ml
Trypsin has anumber of effects onthe thylakoid membrane, including cleavage of the light-harvesting chlorophyll-binrling protein [24], inhibition of NADP' [32] reduction, uncoupling [4,19],and activation of M$"ATPase activity [2, 4].In this study using low concentrations of trypsin, a clear difference with respect to ATPase activity and phosphorylation inhibition was observed whether the thylakoids were exposed to trypsin in the light or dark
Summary
Taining this modified y subunit retained a high level of Ca2+-ATPaseactivity in solution. Thylakoids (0.15mgof chlorophyll/mU were incubated with various concentrations of trypsin a t room temperature in a mixture which contained 50 mM Tricine/NaOH (pH 8.0), 50 mM NaCl, 5 mM MgC12, 1 m~ ATP,andeither 20 pM pyocyanine or 20 p~ PMS. Light-dependent proton uptake [8] was [2] in the light, thylakoids hydrolyze ATP in the dark at appreciable rates This ATPhydrolysis requires coupling factor 1which is part of the proton-translocating ATPase of chloroplasts. The requirement for illumination in the activation of the Mg"-ATPase of thylakoids may be ascribed to energy-dependent changes in the conformation of CF1' that expose critical regions of the enzyme to attack by thiols or assayed at pH 7.9 at 20 "C in a solution (2.0 ml) which contained 1 mM Tricine/NaOH, 50 mM NaCl, 5 m~ MgC12,20 p~ pyocyanine, or 20 p~ PMS, and thylakoids equivalent to 100 p g of chlorophyll/ml. Soybean trypsin inhibitor, and adenine nucleotides werefromSigmaand TPCK-trypsin, fromWorthington.32Pi was purchased from ICN
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