Sequences within both the N‐ and C‐terminal domains of phytochrome A are required for PFR ubiquitination and degradation

Abstract

SummaryPhotoconversion of the plant photoreceptor phytochrome A (phyA) from its inactive Pr form to its biologically active Pfr form initiates its rapid proteolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that multiple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro and analyzed the Pfr-specific degradation of the resulting photoreceptors expressed in transgenic tobacco. One important site is within the C-terminal half of the polypeptide as its removal stabilizes oat phyA as Pfr. Within this half is a set of conserved lysines that are potentially required for ubiquitin attachment. Substitution of these lysines did not prevent ubiquitination or breakdown of Pfr, suggesting either that they are not the attachment sites or that other lysines can be used in their absence. A small domain just proximal to the C-terminus is essential for the form-dependent breakdown of the holoprotein. Removal of just six amino acids in this domain generated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric photoreceptors generated from potato PHYA and PHYB, we found that the N-terminal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated and rapidly degraded as Pfr. Taken together, our data demonstrate that, whereas an intact C-terminal

Light-Induced Nuclear Translocation of Endogenous Pea Phytochrome A Visualized by Immunocytochemical Procedures

“…Therefore, we conclude that the loss of PHYA-associated FITC labeling under cR light resulted from enhanced degradation of the apoprotein. These observations are consistent with studies on oat, in which phytochrome also became gradually undetectable by immunostaining during cR irradiation (Mackenzie et al, 1978), and with many previous reports of FR light-absorbing phytochrome (Pfr)-specific degradation of PhyA (Clough et al, 1999). However, the site at which PHYA is degraded is still not determined.…”

Lysine 206 inArabidopsisphytochrome A is the major site for ubiquitin-dependent protein degradation

“…Lysine 206 of Arabidopsis phyA is the main ubiquitination site The N-terminal domain of phyA has been shown to be ubiquitinated and degraded as a result of the biochemical response to light (31,32). Identification of ubiquitination site can be accomplished by either direct analysis of ubiquitinated proteins by mass spectrometry (MS) or protein degradation analysis using lysine mutated proteins (34,35).…”

“…COP1 have been identified as E3 ligases in the proteasome pathway, which with CUL1 is involved in phyA degradation under different growth media conditions (29,30). To define specific ubiquinated lysines in phyA, C-terminal conserved lysines within the region required for Pfr signal transduction of oat phyA were replaced with arginines but these mutant phyA proteins showed the same degradation kinetics as wild-type phyA (31). Recent phyA functional studies showed that the N-terminal domain of phyA is important for ubiquitination and degradation (25,31,32).…”

“…The N-terminal domain of potato phyA is required for phyA degradation through the selective recognition and ubiquitination of Pfr function (31). In addition, a truncated Arabidopsis phyA N-terminal mutant (PHYA686-YEP-DD) was imported into the nucleus and degraded under red light, even though its degradation was a little slower than that of wild-type PHYA-YEP; however, PHYA686-YEP-DD did not initiate far-red HIR signalling responses (25).…”

“…Previous phyA degradation studies (31,32) found that the N-terminal domain of phyA plays an important role in phyA degradation, with some contribution from the C-terminal domain. The N-terminal domain of potato phyA is required for phyA degradation through the selective recognition and ubiquitination of Pfr function (31).…”