Degradation of Rubisco SSU during oxidative stress triggers aggregation of Rubisco particles in Chlamydomonas reinhardtii.

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.

A proposed mechanism for the inhibitory effects of oxidative stress on Rubisco assembly and its subunit expression.

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 H2O2 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.

RNA binding activity of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit from Chlamydomonas reinhardtii.

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 RNA-binding 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.