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 inactive Pr form to its biologically active Pfr from 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 domain is essential for phyA degradation, the N-terminal domain is responsible for the selective recognition and ubiquitination of Pfr.     

Red light-induced accumulation of ubiquitin-phytochrome conjugates in both monocots and dicots

Phytochrome is rapidly degraded in vivo after photoconversion from the stable red-absorbing (Pr) form to the far red-absorbing (Pfr) form. Previously, we have shown in etiolated oat seedlings that ubiquitin-phytochrome conjugates (Ub-P) appear after Pfr formation suggesting that oat phytochrome is rapidly degraded by a ubiquitin-dependent proteolytic pathway. Here, we extend this observation to etiolated tissue from other monocotyledonous (corn [Zea mays. (L.)] and rye [Secale cereale (L.)] and dicotyledonous species (pea [Pisum sativum (L,)] and zucchini squash [Cucurbita pepo (L.)]). Following Pfr formation by red light, all four species synthesized a heterogeneous series of Ub-P that appeared and disappeared concomitant with the degradation of the chromoprotein. When Pfr was photoconverted back to Pr by a far-red light pulse, degradation of phytochrome ceased and the levels of Ub-P concomitantly dropped. In pea and zucchini squash, loss of Ub-P after photoconversion of Pfr back to Pr was rapid, occurring with a half-life of approximately 5 to 10 minutes. These data indicate that the accumulation of Ub-P after Pfr formation is a general phenomenon in etiolated seedlings of higher plants and further support the hypothesis that plants degrade Pfr via Ub-P intermediates.     

Kinetic and Thermodynamic Analysis of the Light-induced Processes in Plant and Cyanobacterial Phytochromes

The light-induced processes of the biological photoreceptor phytochrome (recombinant phyA of oat and recombinant CphA from the cyanobacterium Tolypothrix PCC7601) have been investigated in a time-resolved manner in the temperature range from 0 to 30°C. Both proteins were heterologously expressed and assembled in vitro with phycocyanobilin. The Pr state of plant phytochrome phyA is converted to the Pfr state after formation of four intermediates with an overall quantum yield of ∼18%. The reversal reaction (Pfr-to-Pr) shows several intermediates, all of which, even the first detectable one, exhibit already all spectral features of the Pr state. The canonical phytochrome CphA from Tolypothrix showed a similar intermediate sequence as its plant ortholog. Whereas the kinetics for the forward reaction (Pr-to-Pfr) was nearly identical for both proteins, the reverse process (Pr formation) in the cyanobacterial phytochrome was slower by a factor of three. As found for the Pfr-to-Pr intermediates in the plant protein, also in CphA all detectable intermediates showed the spectral features of the Pr form. For both phytochromes, activation parameters for both the forward and the backward reaction pathways were determined.     

Characterization of Tobacco Expressing Functional Oat Phytochrome : Domains Responsible for the Rapid Degradation of Pfr Are Conserved between Monocots and Dicots

Constitutive expression of a chimeric oat phytochrome gene in tobacco (Nicotiana tabacum) results in the accumulation of a functional 124-kilodalton photoreceptor that markedly alters the phenotype of light-grown tobacco (Keller et al. [1989] EMBO J 8: 1005-1012). Here, we provide a detailed phenotypic and biochemical characterization of homozygous tobacco expressing high levels of oat phytochrome. Phenotypic changes include a substantial inhibition of stem elongation, decreased apical dominance, increased leaf chlorophyll content, and delayed leaf senescence. Oat phytochrome synthesized in tobacco is indistinguishable from that present in etiolated oats, having photoreversible difference spectrum maxima at 665 and 730 nanometers, exhibiting negligible dark reversion of phytochrome—far red-absorbing form (Pfr) to phytochrome—red-absorbing form (Pr), and existing as a dimer with an apparent size of approximately 300 kilodaltons. Heterodimers between the oat and tobacco chromoproteins were detected. Endogenous tobacco phytochrome and transgenically expressed oat phytochrome are rapidly degraded in vivo upon photoconversion of Pr to Pfr. Breakdown of both oat and tobacco Pfr is associated with the accumulation of ubiquitin-phytochrome conjugates, suggesting that degradation occurs via the ubiquitin-dependent proteolytic pathway. This result indicates that the factors responsible for selective recognition of Pfr by the ubiquitin pathway are conserved between monocot and dicot phytochromes. More broadly, it demonstrates that the domain(s) within a plant protein responsible for its selective breakdown can be recognized by the degradation machinery of heterologous species.     

Ubiquitin-phytochrome conjugates. Pool dynamics during in vivo phytochrome degradation

The plant photoreceptor chromoprotein, phytochrome, is rapidly degraded in vivo after photoconversion from a stable red light-absorbing form (Pr) to a far-red light-absorbing form (Pfr). Previously, we demonstrated that during Pfr degradation in etiolated oat seedlings, ubiquitin-phytochrome conjugates, (Ub-P), appear and disappear suggesting that phytochrome is degraded via a ubiquitin-dependent proteolytic pathway (Shanklin, J., Jabben, M., and Vierstra, R. D. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 359-363). Here, we provide additional kinetic and localization data consistent with this hypothesis by exploiting the unique ability to photoregulate phytochrome degradation in vivo. An assay for the quantitation of Ub-P was developed involving immunoprecipitation of total conjugates with anti-ubiquitin antibodies, followed by the detection of Ub-P with anti-phytochrome antibodies. Using this immunoassay, we found that Ub-P will accumulate to approximately 5% of initial phytochrome during Pfr degradation induced by a saturating red light pulse. Reducing the amount of Pfr produced initially by attenuating the red light pulse, lowered the amount of phytochrome degraded in the following dark period and concomitantly reduced the maximal accumulation of Ub-P. Continuous far-red irradiations that maintained only 4% of phytochrome as Pfr induced rapid phytochrome degradation similar to that induced by a red light pulse converting 86% of Pr to Pfr. The appearance and disappearance of Ub-P were similar for each irradiation indicating that Ub-P accumulation is independent of the level of Pfr provided rapid phytochrome degradation is maintained. Pulse-chase studies employing continuous far-red light followed by darkness showed that Ub-P are continuously synthesized during phytochrome degradation and rapidly disappear once degradation ceases. Ub-P also accumulated during "cycled Pr" degradation induced by the transformation of Pr to Pfr and back to Pr. The commitment to degrade cycled Pr and form Ub-P occurred within seconds after Pfr formation making the cause(s) underlying this phenomenon one of the fastest phytochrome reactions known. Within seconds after Pfr formation, a majority of phytochrome is also known to aggregate in vivo (previously defined as sequestered or pelletable), with aggregated phytochrome preferentially lost during phytochrome degradation. In vitro analysis of aggregated phytochrome indicated that they contain most of the Ub-P. Moreover, the appearance of Ub-P in the aggregated and soluble fractions correlated with the time that phytochrome disappeared from that fraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tetranitromethane oxidation of phytochrome chromophore as a function of spectral form and molecular weight

Tetranitromethane bleaches Avena phytochrome. The phytochrome (far-red absorbing form; Pfr) chromophore of 124 kilodalton (kD) phytochrome is oxidized 8 times more rapidly than the red absorbing form (Pr). Proteolysis of the 124 kD molecule to the extensively studied mixture of 118 and 114 kD polypeptides increases the rate of oxidation of Pfr 5-fold without affecting the rate of Pr oxidation. As a result, the Pfr form of 118/114 kD preparations is oxidized at a rate 40 times greater than the Pr form. Further proteolytic degradation of the chromoprotein to 60 kD results in an additional increase in the oxidation rates of both Pr and Pfr. These differences in reactivity to tetranitromethane indicate that the chromophore of Pfr is either intrinsically more chemically reactive and/or physically more accessible than the Pr chromophore and that the reactivity/accessibility of both spectral forms is increased by proteolysis. The enhanced reactivity of the Pfr chromophore after proteolytic cleavage of the 6 to 10 kD polypeptide segment(s) from the 124 kD species is further evidence that these segment(s) affect the environment of the native photoreceptor.     

Light-regulated nuclear import and degradation of Arabidopsis phytochrome-A N-terminal fragments

The photoreceptor phytochrome-A (phyA) regulates germination and seedling establishment by mediating very low fluence (VLFR) and far-red high irradiance (FR-HIR) responses in Arabidopsis thaliana. In darkness, phyA homodimers exist in the biologically inactive Pr form and are localized in the cytoplasm. Light induces formation of the biologically active Pfr form and subsequent rapid nuclear import. PhyA Pfr, in contrast to the Pr form, is labile and has a half-life of ～30 min. We produced transgenic plants in a phyA-201 null background that express the PHYA-yellow fluorescent protein (YFP) or the PHYA686-YFP-dimerization domain (DD) and PHYA686-YFP-DD-nuclear localization signal (NLS) or PHYA686-YFP-DD-nuclear exclusion signal (NES) fusion proteins. The PHYA686-YFP fusion proteins contained the N-terminal domain of phyA (686 amino acid residues), a short DD and the YFP. Here we report that (i) PHYA686-YFP-DD fusion protein is imported into the nucleus in a light-dependent fashion; (ii) neither of the PHYA686 fusion proteins is functional in FR-HIR and nuclear VLFR; and (iii) the phyA-dependent, blue light-induced inhibition of hypocotyl growth is mediated by the PHYA686-YFP-DD-NES but not by the PHYA686-YFP-DD-NLS and PHYA686-YFP-DD fusion proteins. We demonstrate that (i) light induces degradation of all PHYA N-terminal-containing fusion proteins and (ii) these N-terminal domain-containing fusion proteins including the constitutively nuclear PHYA686-YFP-DD-NLS and predominantly cytoplasmic PHYA686-YFP-DD-NES degrade at comparable rates but markedly more slowly than PHYA-YFP, whereas (iii) light-induced degradation of the native phyA is faster compared with PHYA-YFP.     

The 20S/26S proteasomal pathway of protein degradation in muscle tissue

Similar to all other eukaryotic cells and tissues muscle tissue contains the proteolytic system of 20S/26S proteasomes with the 20S proteasome existing predominantly in a latent state. Unlike with the mammalian enzymein vitro transition from the latent to the activated state of the 20S proteasomes isolated from muscle of several fish species and from lobster can be achieved by heat shock. It is very likely that the activated state of the 20S proteasome corresponds to the physiologically active form of the enzyme since only that one is able to attack sarcoplasmic and myofibrillar proteins to any significant extent. As perfusion of rat hindquarters with presumptive low molecular mass activators like free fatty acids does not result in an activation of the muscle proteasome other — possibly protein activators — may serve this purposein vivo. The 26S proteasome complex may be regarded as such a proteasome/activator complex. The 26S proteasome complex has the ability to degrade protein (-ubiquitin-conjugates) by an ATP-consuming reaction. Since increased amounts of ubiquitinated proteins as well as an enhanced activity of the ATP (-ubiquitin)-dependent proteolytic system have been measured in rat muscle tissue during various catabolic conditions, it is not unlikely that this pathway is responsible for catalysis of muscle protein breakdown.Abbreviations Bz benzoyl - PGPH peptidylglutamylpeptide hydrolysing - Suc succinyl - Z benzyloxycarbonyl     

Arabidopsis phytochrome a is modularly structured to integrate the multiple features that are required for a highly sensitized phytochrome

Phytochrome is a red (R)/far-red (FR) light-sensing photoreceptor that regulates various aspects of plant development. Among the members of the phytochrome family, phytochrome A (phyA) exclusively mediates atypical phytochrome responses, such as the FR high irradiance response (FR-HIR), which is elicited under prolonged FR. A proteasome-based degradation pathway rapidly eliminates active Pfr (the FR-absorbing form of phyA) under R. To elucidate the structural basis for the phyA-specific properties, we systematically constructed 16 chimeric phytochromes in which each of four parts of the phytochrome molecule, namely, the N-terminal extension plus the Per/Arnt/Sim domain (N-PAS), the cGMP phosphodiesterase/adenyl cyclase/FhlA domain (GAF), the phytochrome domain (PHY), and the entire C-terminal half, was occupied by either the phyA or phytochrome B sequence. These phytochromes were expressed in transgenic Arabidopsis thaliana to examine their physiological activities. Consequently, the phyA N-PAS sequence was shown to be necessary and sufficient to promote nuclear accumulation under FR, whereas the phyA sequence in PHY was additionally required to exhibit FR-HIR. Furthermore, the phyA sequence in PHY alone substantially increased the light sensitivity to R. In addition, the GAF phyA sequence was important for rapid Pfr degradation. In summary, distinct structural modules, each of which confers different properties to phyA, are assembled on the phyA molecule.     

Ubiquitin-independent proteolytic functions of the proteasome

The discovery of the 20S proteasome (multicatalytic proteinase complex) was followed by the recognition that this multisubunit macromolecule is the proteolytic core of the 26S proteasome. Most of the research on extralysosomal proteolysis has concentrated on the role of the 26S proteasome in the ubiquitin-dependent proteolytic pathway. However, little attention has been directed toward the possible involvement of the proteasome in ubiquitin-independent proteolysis. In the past few years, many publications have provided evidence that both the 20S proteasome and the 26S proteasome can degrade some proteins in an ubiquitin-independent manner. Furthermore, it is becoming clear that demonstration of ubiquitin-protein conjugates after exposure of cells to proteasome inhibitors does not eliminate the possibility that the same protein can also be degraded by the proteasome without ubiquitination. The possible mechanisms of degradation of an unmodified protein by the 20S proteasome are discussed. These include targeting, protein unfolding, and opening of the gated channel to the catalytic sites. It is reasonable to assume that in the future the number of proteins recognized as substates of the ubiquitin-independent pathway will continue to increase, and that the metabolic significance of this pathway will be clarified.