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.