Mass spectrometric characterization of oat phytochrome A: isoforms and posttranslational modifications.
At least four mRNAs for oat phytochrome A (phyA) are present in etiolated oat tissue. The complete amino acid sequences of two phyA isoforms (A3 and A4) and the N-terminal amino acid sequence of a third isoform (A5) were deduced from cDNA sequencing (Hershey et al., 1985). In the present study, heterogeneity of phyA on a protein level was studied by tryptic mapping using electrospray ionization mass-spectrometry (ESIMS). The total tryptic digest of iodoacetamide-modified phyA was fractionated by gel filtration chromatography followed by reversed-phase high-performance liquid chromatography. ESIMS was used to identify peptides. Amino acid sequences of the peptides were confirmed or determined by collision-induced dissociation mass spectrometry (CID MS), MS/MS, or by subdigestion of the tryptic peptides followed by ESIMS analysis. More than 97% of the phyA3 sequence (1,128 amino acid residues) was determined in the present study. Mass-spectrometric analysis of peptides unique to each form showed that phyA purified from etiolated oat seedling is represented by three isoforms A5, A3, and A4, with ratio 3.4:2.3:1.0. Possible light-induced changes in phytochrome in vivo phosphorylation site at Ser7 (Lapko VN et al., 1997, Biochemistry 36:10595-10599) as well at Ser17 and Ser598 (known as in vitro phosphorylation sites) were also analyzed. The extent of phosphorylation at Ser7 appears to be the same for phyA isolated from dark-grown and red-light illuminated seedlings. In addition to Ser7, Ser598 was identified as an in vivo phosphorylation site in oat phyA. Ser598 phosphorylation was found only in phyA from the red light-treated seedlings, suggesting that the protein phosphorylation plays a functional role in the phytochrome A-mediated light-signal transduction.
Lapko
V N
VN
Department of Chemistry, University of Nebraska-Lincoln, 68588-0304, USA.
Jiang
X Y
XY
Smith
D L
DL
Song
P S
PS
D000595:Amino Acid Sequence; D018554:Avena / Q000737:chemistry*; D002850:Chromatography, Gel; D013058:Mass Spectrometry; D008954:Models, Biological; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010766:Phosphorylation; D010834:Phytochrome / Q000737:chemistry*; D051942:Phytochrome A; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational
D051942:Phytochrome A; D020033:Protein Isoforms; D010834:Phytochrome