Phytochrome regulates phosphorylation of a protein with characteristics of a nucleoside diphosphate kinase in the crude membrane fraction from stem sections of etiolated pea seedlings

Abstract

The molecular mechanism of light signal perception via phytochrome was analysed using the third internodes of etiolated pea seedlings irradiated with red or red followed by far-red light. A crude membrane fraction prepared from the tissue was labelled by [γ-32P]ATP at 4 × 10−8M for 15 s at 0°C, and the proteins were separated by two-dimensional gel electrophoresis. The phosphorylation of a protein with a molecular mass of about 15 kDa in the crude membrane fraction increased with an increase in the intensity of red light irradiation (10, 50 and 100 μmol m−2 s−1) for 20 s. Successive irradiation with red light (100 μmol m−2 s−1 for 20 s) and subsequent irradiation with far-red light reduced the phosphorylation of the protein, depending on the intensity of the far-red light (from 0.1 to 8 μmol m−2 s−1 for 180 s) . A plasma membrane purified from the crude membrane fraction from red light irradiated tissue showed a rapid phosphorylation of the 15 kDa protein by 4 × 10−8M [γ-32P]ATP at 0°C for 7 s, and subsequent addition of ATP, GTP, ADP or GDP at 10−5 or 10−6 M efficiently removed the phosphoryl group of the 15 kDa protein. The 15 kDa protein was autophosphorylated in the gel following separation by sodium dodecylsulphate (SDS) polyacrylamide gel electrophoresis. The partially purified 15 kDa protein included nucleoside diphosphate kinase (NDP kinase) activity, as well as cross-reactivity with an antibody specific to rat NDP kinase as assayed by immunostaining and cross-reactivity with an antibody specific to rice NDP kinase as assayed by immunoprecipitation.