Mechanism and Significance of Post-Translational Modifications in the Large Subunit of Ribulose Biphosphate Carboxylase/Oxygenase

The functional significance of N-terminal methylation of the processed form of the SS of Rubisco at Met-l will be determined by measuring changes in SS stability, coupled with identification of the N-terminal residue of the SS in in vitro assays and transgenic plants, In vitro studies using intact isolated chloroplasts from both wild type and transgenic tobacco plants expressing sense and antisense constructs of tobacco Rubisco LSMT, will allow a direct determination of alterations in the N-terminal region of the SS by N-terminal modifYing enzymes, as well as changes in stability using recombinantly expressed mature and processed forms of the SS which occur as a consequence of the absence of"Nmethylation of Met-L The potential overall physiological and biochemical ramifications of these changes will be determined by measuring whole plant photosynthesis and growth in transgenic plants, The potential consequences ofN-terminally altered SSs which assemble into holoenzyme will be determined by analyses of Rubisco purified from transgenic plants, The nucleotide and protein sequences responsible for establishing the specificity of Rubisco SS and LS methyltransferase activity will be examined by construction and bacterial expression of recombinant chimeric forms of Rubisco LSMT/SSMT, engineered from their mono and bifunctional counterparts, The interaction between spinach Rubisco SSMT and the SS of Rubisco will be investigated using kinetic and binding analyses with an Iasys affinity biosensor, and the application of more conventional homo- and hetero-bifunctional cross-linking reagents, followed by proteolytic mapping to identifY polypeptide regions of close proximity, and amino acid residues involved in cross-link formation. The functional consequences of the introduction of a trimethyllysyl residue at position 14 in the LS of des (methyl) spinach Rubisco will be investigated by transformation of spinach plants with the pea Rubisco LSMT cDNA. Perturbations in the methylation state of the SS of spinach Rubisco will also be examined by construction of transgenic spinach plants expressing sense and antisense constructs of spinach Rubisco SSMT, thus providing a comparison with transgenic tobacco where the methylation status of the LS may also be affected in addition to the SS, The study of Rubisco LSMT and SSMT is revealing the molecular and biochemical characteristics of what appears to be a new family of protein methyltransferases. The recent findings suggestive of a chloroplast-localized N-end rule system of targeted protein degradation may be the mechanism by which subunit stoichiometry is maintained for the LS and SS of Rubisco, The fundamental role that Rubisco plays in carbon dioxide assimilation by plants underscores its importance in food and fiber production, Additionally, scientific interest in the coordinated gene expression and assembly required for formation of the Rubisco holoenzyme suggests that an understanding of the structural modifications in both the LS and SS, and the associated methyltransferases, may represent an important contribution towards understanding processes affecting the level and perhaps activity of Rubisco in plants, Furthermore, the characterization of Rubisco LSMT and SSMT may identifY new molecular targets for genetic engineering with the potential for improvements in crop productivity or control of plant growth,