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.
Cohen
Idan
I
Department of Life Sciences, Ben-Gurion University of the Negev, Beer Sheva 84105, Israel.
Knopf
Joel A
JA
Irihimovitch
Vered
V
Shapira
Michal
M
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D015971:Gene Expression Regulation, Enzymologic; D008027:Light; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000378:metabolism*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*
D021122:Protein Subunits; D012273:Ribulose-Bisphosphate Carboxylase