Fig. 8

Phosphorylation of PS-2 occurs within the N-terminal domain

(A) COS-7 cells transfected with either wt PS-2 or with the PS-2 cDNA constructs containing the C-terminal deletion constructs described in Fig. 4 were metabolically labeled with [³⁵S]-methionine or [³²P]-orthophosphate. Cell lysates were immunoprecipitated with antibody TOR519. Recombinant PS proteins derived from all deletion constructs are still phosphorylated indicating that PS-2 phosphorylation occurs between the N-terminus and TM4. Note that only the two higher-molecular weight peptides of the three observed by [³⁵S]-methionine labeling are phosphorylated. wt PS-2 as well as the TM7 derived PS-2 proteins were separated on 8% SDS/urea gels, all other recombinant PS-2 proteins were separated on 10% SDS/urea gels. (B) Hybrid molecules were generated by exchanging the N terminus of PS-1 with that of PS-2 (PS-l/NT PS-2) and vice versa (PS-2/NT PS-1). COS-7 cells were transfected with these cDNA constructs and metabolically labeled with [³⁵S]-methionine or [³²P]-orthophosphate. Strikingly, PS-1 containing the N-terminal domain of PS-2 is heavily phosphorylated, whereas little phosphate incorporation was observed for PS-2 containing the N-terminal domain of PS-1.