Partial purification and peptide mapping of ubiquitin-phytochrome conjugates from oat

Abstract

Phytochrome is rapidly degraded in vivo following photoconversion from the relatively stable red light absorbing form Pr to the far red light absorbing form Pfr. In etiolated seedlings from several species, this photoconversion also induces the accumulation of ubiquitin-phytochrome conjugates (Ub-P), suggesting that Pfr is degraded via a ubiquitin-dependent proteolytic pathway. To understand why Pfr is preferentially conjugated with ubiquitin, Ub-P were partially purified and characterized with the ultimate goal of mapping ubiquitin attachment sites. Ub-P were partially purified by poly(ethylene imine) and ammonium sulfate precipitations followed by hydroxyapatite chromatography and separated from unmodified phytochrome by size-exclusion chromatography. Up-P had an apparent native molecular size of approximately 600 kDa, substantially larger than that of the unmodified Pfr dimer (365 kDa). Ub-P retained the property of spectral photoreversibility. The initial digestion patterns of Ub-P were similar to unmodified phytochrome. In an attempt to identify ubiquitin attachment site(s), Ub-P were probed with a library of anti-oat phytochrome monoclonal antibodies. Of the 16 different anti-phytochrome monoclonal antibodies tested, 3 (O76C, O19F, and O311B) poorly recognized all size classes of Ub-P, indicating that the corresponding epitopes were masked either directly or indirectly as a result of ubiquitin ligation. These epitopes are located between residuesmore » 90 and 180 (O76C), 558 and 668 (O19F), and 747 and 830 (O311B) of oat phytochrome. Because the regions recognized by O19F and O311B are near the C-terminal domain containing at least one ubiquitin attachment site, and near amino acid residues that become more accessible when the chromoprotein is in the Pfr form, these regions may be important in the Pfr-dependent ubiquitination of phytochrome.« less