A PHOTORECEPTOR SYSTEM REGULATING in vivo AND in vitro PHOSPHORYLATION OF A PEA PLASMA MEMBRANE PROTEIN

Abstract
We have continued to characterize the blue light-regulated phosphorylation of a 120 kDa pea plasma membrane protein thought to be involved in sensory transduction for phototropism (Short and Briggs, 1990, Plant Physiol. 92 , 179–185). By incubating pea stem sections in ³²P-phosphate, we show that the 120 kDa protein is phosphorylated in vivo only after blue light irradiation and that the photosensitive fluence range matches that for phototropism. Blue light induces phosphorylation of the protein in vitro as well, but the fluences required to elicit the response are at least 30-fold higher. Triton solubilization of the plasma membrane vesicles does not further alter the fluence-response relationship. Very little turnover was detected over 20 min phosphorylation time courses or by pulse-chase experiments on unirradiated, blue light pulse-irradiated, or continuously irradiated membranes. Experiments with a dark period intervening between irradiation and addition of adenosine triphosphate show the light-induced change to persist for several minutes at 30°C. Agents that disrupt the normal photochemistry of flavins preferentially inhibit the light-induced enhancement of phosphorylation, suggesting a flavin chromophore. However, exogenous free flavins do not affect the sensitivity of the response. Staphylococcus aureus V-8 proteolysis of the phosphorylated protein from membranes subjected to a range of fluences before phosphorylation shows that the radiolabel on each of three peptides increases in proportion to the phosphorylation level of the undigested polypeptide. These studies may be valuable for assessing the nature of the photoreceptor and for unravelling the early sensory transduction steps in phototropism.