PHOSPHORYLATION OF THE LIGHT-HARVESTING POLYPEPTIDE LHI-ALPHA OF RHODOBACTER-CAPSULATUS AT SERINE AFTER MEMBRANE INSERTION UNDER CHEMOTROPIC AND PHOTOTROPHIC GROWTH-CONDITIONS

The kinetics of protein phosphorylation was studied in cells of phototrophic cultures and in dark-grown cells induced to form the photosynthetic apparatus by lowering of the oxygen tension. Cells of Rhodobacter capsulatus grown in a malate medium with 0.2 mM potassium phosphate were shifted to semiaerobic conditions, and (PO43-)-P-32 or [S-35]Met was added 25 min after induction. The label of both radioactive precursors appeared in the membrane fraction about 20 min after addition. The maximum of P-32 was found after 1 h of labeling in the alpha polypeptide of the light-harvesting (LH) complex I (B870). The LHI alpha protein was phosphorylated after insertion into the membrane. Chloramphenicol inhibited the phosphorylation of LHI alpha but not of phospholipids. The steady-state level of phosphorylation was higher in anaerobic cultures grown at the low light intensity of 2000 lux than in cultures grown at high light intensity of 35 000 lux. The phosphate label did not change significantly during a chase with unlabelled phosphate for 2 h. The phosphoamino acids in LHI alpha were detected with monoclonal antibodies and radioautography of labeled and hydrolyzed LHI alpha. Serine was shown to be the amino acid with the highest phosphate content; threonine and tyrosine were weakly phosphorylated. From the positions of these three amino acids in LHI alpha it was concluded that serine-2, which is exposed on the cytoplasmic side of the membrane, is the main phosphorylated amino acid. P-threonine and P-tyrosine are exposed on the periplasmic surface of the membrane.