Fig. 6

In vivo phosphorylation of PS-2

(A) Phosphorylation of PS-2 proteins. COS-7 cells transfected with the PS-1 and PS-2 cDNA, respectively, were in vivo labeled with either [³⁵S]-methionine or [³²P]-orthophosphate. Cell lysates were immunoprecipitated with antibody BOS4627. Very little if any phosphate incorporation was observed for the PS-1 protein, whereas PS-2 proteins are strongly phosphorylated. The ∼100-kD protein (*) is also observed in mock-transfected cells labeled with [³²P]-orthophosphate. Arrowheads indicate the putative PS monomers and dimers. Oligomers of higher molecular weight were revealed after longer exposure. (B) Very little phosphate incorporation of mutant PS-1 containing the Ala246Glu mutation (FAD 1) was observed, whereas PS-2 containing the Asnl41Ile mutation (Volga Germans) is phosphorylated similar to wt PS-2. COS-7 cells were transfected with the corresponding cDNA constructs and metabolically labeled with either [³⁵S]-methionine or [³²P]-orthophosphate. Cell lysates were immunoprecipitated with antibody BOS4627. The ∼100-kD protein labeled by an asterisk is also detected in mock transfected cells. Arrowheads indicate the putative PS monomers and dimers. Oligomers of higher molecular weight were revealed after longer exposure. (C) Phosphoamino acid analysis of in vivo phosphorylated PS-2 protein. PS-2 is phosphorylated on serine residues. The position of phospho-serine, -theronine, and -tyrosine standards is indicated. (D) Phosphorylation of PS-2 occurs in kidney 293 cells as well as in CHO cells transfected with the PS-2 cDNA. After metabolic labeling with either [³⁵S]-methionine or [³²P]-orthophosphate, cell lysates were immunoprecipitated with antibody BOS4627. The ∼00-kD protein (*) is also detected in mock-transfected cells. Arrowheads indicate the putative PS monomers and dimers. Oligomers of higher molecular weight were revealed after longer exposure.