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. 2020 Apr 7;9:e56418. doi: 10.7554/eLife.56418

Figure 2. IMPDH1 phosphorylation sites identified in a phosphoproteomic study of dark/light-adapted bovine retinas.

(A) Mass spectra of identified IMPDH1 phosphorylated peptides. Residue numbers refer to the canonical bovine isoform β (514aa) [Uniprot A0JNA3]. For peptide 154–169 only one phosphorylation was detected, that could not be unequivocally assigned to T159 or S160. Phosphorylation at peptides 413–422 and 475–480 was at S416 and S477, respectively. (B) Peak areas of the precursor ions corresponding to the identified phosphopeptides of IMPDH1. Samples 1–3 are dark-adapted retinas; 4–6 light-exposed retinas (biological replicates). T159/S160 are preferentially phosphorylated in light, S477 in dark, and S416 indistinctly. (C) Ribbon diagram of IMPDH in its tetrameric conformation [pdb: 1jcn], showing the TIM barrel catalytic domain in coral, and the regulatory Bateman domain with two copies of cystathione beta-synthase (CBS) sequence in slate blue and green. (D) Monomer conformation [pdb: 1jcn] showing T159 and S160 at CBS1 in the Bateman domain; and S477 at the COOH-terminus. Disease associated mutations, depicted in red, are proximal to T159/S160 (Asn198; Arg224; Asp226) or to S477 (His372). (E) The catalytic domain of AgIMPDH, with the C319 loop in coral, the COOH-terminus of an adjacent monomer in blue and the mobile flap in green. IMP depicted in space fill model [pdb:4xfi]. (F) Alignment of IMPDH from T. foetus (prokaryotic), A. gossypii (filamentous fungus), H. sapiens, M. musculus and B. taurus IMPDH1 canonical isoforms. Phosphosites highlighted in yellow.

Figure 2.

Figure 2—figure supplement 1. Dark/light fold-change of peak areas from control phosphopeptides from phosducin, GRK1 and rhodopsin shown as a quality control of the phosphoproteomic analysis.

Figure 2—figure supplement 1.

Mass spectra of phosphorylated peptides of: phosducin 72–82 peptide MSVQEYELIHK [Uniprot F1MSE1]; GRK1 480–491 peptide NIQDVGAFSTVK [Uniprot P28327]; and rhodopsin 326–348 peptide NPLGDDEASTTVSKTETSQVAPA [Uniprot P02699]. For each peptide, the peak area of the precursor peptide ion was extracted and plotted for each sample. Samples 1–3 are dark-adapted retinas; 4–6 light-exposed retinas (biological replicates). Light/dark log2 fold-change values were: phosducin S73 (−1.39; p=0.01); GRK1 S488/T489 (2.70; p=0.08); Rhodopsin T336 (2.49; p=0); and Rhodopsin T340/T342 (5.53; p=0).