In Chlamydomonas reinhardtii, a light-induced oxidative stress shifts the glutathione pool toward its oxidized form, resulting in a translational arrest of the large subunit (LSU) of Rubisco. We show here that the translational arrest of LSU is tightly coordinated with cessation of Rubisco assembly, and both processes take place after a threshold level of reactive oxygen species is reached. As a result, the small subunit is also eliminated by rapid degradation. We previously showed that the amino terminus of the LSU could bind RNA in a sequence-independent manner, as it shares a structural similarity with the RNA recognition motif. This domain becomes exposed only under oxidizing conditions, thus restricting the RNA-binding activity.Here we show that in vitro, thiol groups of both subunits become oxidized in the presence of oxidized glutathione. The structural changes are mediated by oxidized glutathione, whereas only very high concentrations of H 2 O 2 confer similar results in vitro. Changes in the redox state of the LSU thiol groups are also observed in vivo, in response to a physiological light shock caused by transfer of cells from low light to high light. We propose that during a photooxidative stress, oxidation of thiol groups occurs already in nascent LSU chains, perhaps hindering their association with chaperones. As a result, their RNA recognition motif domain becomes exposed and will bind any RNA in its vicinity, including its own transcript. Due to this binding the ribosome stalls, preventing the assembly of additional ribosomes on the transcript. Polysome analysis using Suc gradients indeed shows that the rbcL RNA is associated with the polysomal fraction at all times but shifts toward fractions that contain smaller polysomes and monosomes during oxidative stress. Thus, translational arrest of the LSU most likely occurs at a postinitiation stage.Rubisco is responsible for CO 2 fixation during photosynthesis. In vascular plants and green algae, it exists as a holoenzyme composed of eight large subunits (LSUs; 55 kD) encoded by the chloroplast rbcL gene and eight small subunits (SSUs; 15 kD) produced by a nuclear family of rbcS genes (Spreitzer, 1993). SSU precursors are processed during entry into the chloroplast and are then assembled with the LSUs to yield the holoenzyme. Assembly of the oligomeric protein (approximately 500 kD) is mediated by cpn60 and cpn10 (Gatenby and Ellis, 1990), and a complex of the LSU and cpn60 serves as an intermediate of the assembly process. The chloroplast cpn60 is a homolog of bacterial groEL, and prokaryotic subunits of Rubisco expressed in Escherichia coli can be successfully assembled into a holoenzyme (Goloubinoff et al., 1989). A role for additional chaperone molecules, in accumulation of mature Rubisco complexes, was recently described (Brutnell et al., 1999).Many protein complexes in the chloroplast are composed of multiple polypeptides, which are expressed in a tightly coordinated manner, as removal of one subunit can have an effect on expression of the other ...
Transfer of the green algae Chlamydomonas reinhardtii from low light to high light generated an oxidative stress that led to a dramatic arrest in the synthesis of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The translational arrest correlated with transient changes in the intracellular levels of reactive oxygen species and with shifting the glutathione pool toward its oxidized form (Irihimovitch, V., and Shapira, M. (2000) J. Biol. Chem. 275, 16289 -16295). Here we examined how the redox potential of glutathione affected the RNA-protein interactions with the 5-untranslated region of rbcL. This RNA region specifically binds a group of proteins with molecular masses of 81, 62, 51, and 47 kDa in UV-cross-linking experiments under reducing conditions. Binding of these proteins was interrupted by exposure to oxidizing conditions (GSSG), and a new protein of 55 kDa was shown to interact with the RNA. The 55-kDa protein comigrated with Rubisco LSU in one-and two-dimensional gels, and its RNA binding activity was further verified by using the purified protein in UV-cross-linking experiments under oxidizing conditions. However, the LSU of purified and oxidized Rubisco bound to RNA in a sequence-independent manner. A remarkable structural similarity was found between the amino-terminal domain of Rubisco LSU in C. reinhardtii and the RNA binding domain, a highly prevailing motif among RNAbinding proteins. It appears from the crystal structure of Rubisco that the amino terminus of LSU is buried within the holoenzyme. We propose that under oxidizing conditions it is exposed to the surface and can, therefore, bind RNA. Accordingly, a recombinant form of the polypeptide domain that corresponds to the amino terminus of LSU was found to bind RNA in vitro with or without GSSG.When plants and algae absorb light energy that exceeds the level of electron carrier saturation they generate reactive oxygen species (ROS), 1 that cause a variety of cellular and molecular damage. This phenomenon is referred to as photoinhibition and is common to all photosynthetic organisms (1-3). Recovery from photoinhibition can be achieved by decreasing the chlorophyll content and by activating a variety of antioxidant pathways that involve ascorbate and glutathione (4, 5).Ribulose-1,5-bisphosphate carboxylase (Rubisco) is the key enzyme in photosynthetic carbon assimilation. In Chlamydomonas reinhardtii and in land plants the enzyme is composed of eight large subunits (LSU) encoded by the chloroplast rbcL gene and eight small subunits encoded by the nuclear rbcS gene family. Assembly of the holoenzyme is mediated by the chloroplast chaperonins cpn60 and cpn10 (6, 7). We previously showed that transfer of the green algae C. reinhardtii from low light (70 mol m Ϫ2 s Ϫ1 ) to high light (700 mol m Ϫ2 s Ϫ1 ) generates an oxidative stress that leads to photoinhibition and a dramatic arrest in the synthesis of the LSU of Rubisco (8). These light-induced effects were found to be transient, with cell recovery taking pla...
Oxidative stress in plants and green algae has multiple damaging effects, and leads to the degradation of Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco). We recently showed for the green algae Chlamydomonas reinhardtii that in response to a photo-oxidative stress, nascent synthesis of its chloroplast encoded large subunit (LSU) stops. In parallel, newly synthesized small subunits (SSU) that are encoded by the nucleus are rapidly degraded, thus assembly of new holoenzyme particles is inhibited. Here we show that under extreme oxidizing conditions, the steady-state level of the SSU is also reduced. Cleavage of the LSU under oxidizing conditions is well established, and we show, using sucrose gradients, that the resulting fragments of the LSU co-exist as parts of the holoenzyme. In parallel, we demonstrate the selective in-vivo formation of high-density aggregates of Rubisco particles, in response to oxidative stress. Given the known tendency of unassembled LSUs to aggregate, we propose that the rapid elimination of the SSU during oxidative stress along with the fragmentation of the LSU and formation of intra-protein disulfide bridges, leads to the observed aggregation of Rubisco particles. Indeed, we note here a substantially decreased ratio of SSU in the aggregated Rubisco particles. We also observed that this aggregation marks the viability threshold of C. reinhardtii cells exposed to oxidative stress.
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