Identification of the major regulatory phosphorylation site in sucrose-phosphate synthase.
Sucrose-phosphate synthase (SPS; EC 2.4.1.14) is regulated in part by reversible protein phosphorylation. When dephospho-SPS is partially purified from illuminated spinach leaves and incubated with [gamma-32P]ATP the enzyme is phosphorylated by a copurifying protein kinase. In this report, 32P-phosphopeptides from tryptic digests of in vitro phosphorylated SPS were purified by metal-ion affinity chromatography and reversed-phase high-performance liquid chromatography. Three distinct 32P-phosphopeptides were resolved. Edman sequencing of the major phosphopeptide (which contained > 80% of the total 32P) identified the amino acid sequence as Ile-Ser-Ser(P)-Val-Glu-Met-Met-Asp-Asn-Trp-Ala-Asn-Thr-Phe-Lys. This sequence corresponds to residues 156 to 170 of the deduced amino acid sequence of spinach SPS [Klein, R. R., Crafts-Brandner, S. J., and Salvucci, M. E. (1993) Planta 190, 498-510, and Sonnewald, U., Quick, W. P., MacRae, E., Krause, K.-P., and Stitt, M. (1993) Planta 189, 174-181]. Identification of the phosphoseryl residue was accomplished by manual Edman sequencing. The two other phosphopeptides, which each contained less than 10% of the total 32P, were not sequenced. An Escherichia coli expressed, 26-kDa fragment of SPS which contains the major phosphorylation site was a substrate for the protein kinase which copurifies with SPS. Two-dimensional peptide mapping analysis of this fragment showed the major phosphopeptide was present but not the other site(s), suggesting that other peptides are derived from a site other than Ser158. These results provide additional indirect evidence for the presence of multiple phosphorylation sites in SPS.
McMichael
R W
RW
U.S. Department of Agriculture, North Carolina State University, Raleigh 27695-7631.
Klein
R R
RR
Salvucci
M E
ME
Huber
S C
SC
D000595:Amino Acid Sequence; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010748:Phosphopeptides / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D010768:Phosphoserine / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014675:Vegetables; D003313:Zea mays
D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D010768:Phosphoserine; D005964:Glucosyltransferases; C022698:sucrose-phosphate synthase