Tandem mass spectrometry reveals that three photosystem II proteins of spinach chloroplasts contain N-acetyl-O-phosphothreonine at their NH2 termini.
Photosystem II cores of spinach contain four phosphoproteins (8.3, 32, 34, and 44 kDa). Tryptic digestion of core particles released four phosphopeptides which were purified by affinity chromatography on Fe3+-chelating Sepharose and reverse-phase high pressure liquid chromatography. One peptide, derived from the 8.3-kDa protein, has been found to be the NH2 terminus of the psbH gene product (Michel, H. P., and Bennett, J. (1987) FEBS Lett. 212, 103-108). The other three peptides were found to be blocked at the NH2 terminus. We now report the use of tandem mass spectrometry to obtain the sequence of the three other peptides, to locate the phosphorylated residue, and to identify the blocking group. The three peptides correspond to the NH2 termini of D1, D2, and CPa-2; and each begins with N-acetyl-O-phosphothreonine. Comparison with sequences deduced from cloned genes indicates that D1 and D2 have lost their initiating N-formylmethionyl residues. The result for D1 contradicts the view that translation of D1 begins at the second AUG of the mRNA (Bloom, M., Brot, N., Cohen, B. N., and Weissbach, H. (1986) Methods Enzymol. 118, 309-315) and supports the view that processing of pre-D1 to its mature form involves loss of amino acids from the COOH terminus (Marder, J. B., Goloubinoff, P., and Edelman, M. (1984) J. Biol. Chem. 259, 3900-3908). In contrast, CPa-2 is processed at the NH2 terminus by cleaving off the first 14 amino acids. These results also establish that the NH2 termini of D1, D2, and CPa-2 are exposed to the stromal side of the thylakoids.
D000595:Amino Acid Sequence; D002734:Chlorophyll / Q000032:analysis*; D002736:Chloroplasts / Q000032:analysis*; D002846:Chromatography, Affinity; D007834:Lasers; D045342:Light-Harvesting Protein Complexes; D013058:Mass Spectrometry; D010449:Peptide Mapping; D010769:Phosphothreonine / Q000032:analysis*; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins / Q000032:analysis*; D013912:Threonine / Q000031:analogs & derivatives*
D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D010769:Phosphothreonine; D002734:Chlorophyll; D013912:Threonine