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

Photoconversion of the plant photoreceptor phytochrome A (phyA) from its in active Pr form to its biologically active Pfr form initiates its rapid prot eolysis. Previous kinetic and biochemical studies implicated a role for the ubiquitin/26S proteasome pathway in this breakdown and suggested that mult iple domains within the chromoprotein are involved. To further resolve the essential residues, we constructed a series of mutant PHY genes in vitro an d analyzed the Pfr-specific degradation of the resulting photoreceptors exp ressed in transgenic tobacco. One important site is within the C-terminal h alf of the polypeptide as its removal stabilizes oat phyA as Pfr. Within th is half is a set of conserved lysines that are potentially required for ubi quitin attachment. Substitution of these lysines did not prevent ubiquitina tion or breakdown of Pfr, suggesting either that they are not the attachmen t 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 breakdo wn of the holoprotein. Removal of just six amino acids in this domain gener ated a chromoprotein that was not rapidly degraded as Pfr. Using chimeric p hotoreceptors generated from potato PHYA and PHYB, we found that the hi-ter minal half of phyA is also required for Pfr-specific breakdown. Only those chimeras containing the N-terminal sequences from phyA were ubiquitinated a nd rapidly degraded as Pfr. Taken together, our data demonstrate that, wher eas an intact C-terminal domain is essential for phyA degradation, the N-te rminal domain is responsible for the selective recognition and ubiquitinati on of Pfr.