A UNIQUE PROTEIN-KINASE ACTIVITY IS RESPONSIBLE FOR THE PHOSPHORYLATION OF THE 26-KDA AND 14-KDA PROTEINS BUT NOT OF THE 67-KDA PROTEIN IN THE CHLOROPLAST ENVELOPE MEMBRANES OF SPINACH

Protein-phosphorylation activity has been reported in chloroplast envelope membranes of several species. In spinach (Spinacia oleracea L.), we found three major phosphoproteins after incubation in vitro of envelope membranes in the presence of [gamma-P-32]ATP. A 67-kDa phosphoprotein was associated with both inner and outer envelope membranes whereas 26- and 14-kDa proteins were observed in the inner membrane. Although the phosphorylation of the 67-kDa protein is likely to take place via its phosphoglucomutase activity (Salvucci et al., 1990, Plant Physiol. 93, 105-109), the mechanism by which P-32 is incorporated into the 26- and 14-kDa proteins remains to be elucidated. To this aim, we have compared the conditions under which phosphorylation occurs in these three proteins. The effects of Mg2+, Ca2+, pH, ATP and H7 [1-(5-isoquinolinesulfonyl)-2-methylpiperazine], a specific inhibitor of protein-kinase C. as well as pulse-chase experiments with cold ATP, showed that the phosphorylation mechanism was identical for the 26- and 14-kDa proteins but quite different for the 67-kDa one. The protein kinase involved in the phosphorylation of the 26- and 14-kDa proteins was Ca2+-dependent, which was not the case of the 67-kDa protein. In addition, the use of a Triton X-114 phase-separation treatment indicated that both the 26- and 14-kDa proteins exhibited strong hydrophobic properties, in contrast to the hydrophilic character of the 67-kDa phosphoprotein. As indicated by analyses of phosphoamino acids, the three proteins were exclusively phosphorylated on serine residues. Furthermore, a treatment of envelopes by phospholipase C prior to the phosphorylation process inhibited P-31 incorporation into the three phosphoproteins to different extents (61%, 50% and 29% inhibition for the 67-, 14- and 26-kDa proteins, respectively). These results show that phosphatidylcholine and or phosphatidylglycerol but not phosphatidylinositol were involved in this phosphorylation process.