News and Publications

Regulation of Translation and Expression of Recombinant Proteins in Plant Chloroplasts

S.P. Mayfield, M. Beligni, D. Barnes, E. Brown, A. Coragliotti, E. Efuet, K. Espina, S. Franklin, D. Hambly, R. Henry, A. Manuell, B. Ngo, J. Schultz, A. Somanchi, K. Yamaguchi

Gene expression in most organisms is keyed to environmental signals. In plants and algae, expression of key genes is regulated in response to exposure to light. Biochemical and genetic analysis revealed that light activates translation of plant mRNAs and that both RNA elements and their cognate RNA-binding proteins are required for this translational regulation.

Using in vitro selection, site-directed mutagenesis, and reporter gene constructs, we identified several RNA elements required for translation in chloroplasts. We found that a ribosome-binding sequence, similar to sequences found in bacteria, is essential for ribosome association of chloroplast mRNAs that encode proteins bound to thylakoid membranes but is not required for translation of mRNAs that encode soluble proteins. In addition, the ribosome-binding sequence of chloroplast mRNAs is positioned farther upstream than is the sequence of bacterial mRNAs, suggesting a fundamental difference between chloroplasts and bacteria in the mechanism by which a ribosome-binding sequence facilitates initiation of translation. These data suggest that 2 independent mechanisms exist in chloroplasts for regulating mRNA translation. We are examining the chloroplast ribosome and ribosome-associated proteins to identify regulatory components involved in these processes.

Using a proteomic approach, we identified the complete set of chloroplast ribosomal proteins from Chlamydomonas reinhardtii. We also identified a number of ribosome-associated proteins and proteins previously shown to bind specifically to RNA elements contained within the untranslated regions of chloroplast mRNAs. We identified 4 major and several minor proteins that bind to the 5´ untranslated region of the chloroplast psbA mRNA. One of these proteins is homologous to poly(A)-binding proteins (PABPs), which are proteins that facilitate the interaction of mRNAs and ribosomes. Another of the psbA mRNA-binding proteins has homology to protein disulfide isomerases, enzymes involved in the oxidation and reduction of disulfide bonds within proteins. We showed that the chloroplast protein disulfide isomerase can modulate the mRNA-binding activity of the chloroplast PABP by changing the status of the sulfide bonds of cysteine residues within the RNA-binding domains in a redox-dependent manner. The 2 other major psbA-associated proteins are novel RNA-binding proteins.

We also analyzed a number of nuclear mutants deficient in psbA mRNA translation. This genetic analysis indicated that specific members of the RNA-binding complex, such as the PABP protein, are required for initiation of translation of psbA mRNA. This work also revealed that a unique set of proteins, not previously identified as translational components, are required for translation in chloroplasts.

On the basis of these studies, a model for translational activation can be drawn in which redox potential generated by the light reactions of photosynthesis is used by chloroplast protein disulfide isomerase to activate the binding of the chloroplast PABP, and other RNA-binding proteins, to the 5´ untranslated region of the chloroplast psbA mRNA. Binding of these proteins to RNA elements allows increased ribosome association and initiation of translation. Binding of the chloroplast PABP to these mRNAs also allows association of the message with photosynthetic membranes, possibly targeting the mRNA to the correct site of protein insertion.

We are using proteomic and genetic analysis to identify the complete set of proteins and RNA elements required for chloroplast translation. Structural analysis of these RNA-protein complexes is being used to determine how RNA-protein interactions enhance ribosome binding and membrane association and regulate translational activation.

Finally, we developed a reporter system to monitor chloroplast translation in vivo. Using a chloroplast codon optimized green fluorescent protein, we identified chloroplast promoters and 5´ untranslated regions that can support high levels of plastid translation. Using the results of biochemical studies and studies with these reporter constructs, we expressed recombinant antibodies in the chloroplast and showed that these antibodies assemble into functional proteins. Our results indicate that C reinhardtii, and perhaps other algae, offer tremendous potential for the expression of recombinant human therapeutic proteins.

PUBLICATIONS

Barnes, D., Mayfield, S.P. Redox control of posttranscriptional processes in the chloroplast. Antioxidant Redox Signaling, in press.

Franklin, S., Ngo, B., Efuet, E., Mayfield, S.P. Development of a GFP reporter gene for Chlamydomonas reinhardtii chloroplast. Plant J. 30:733, 2002.

Kim, J., Mayfield, S.P. The active site of the thioredoxin-like domain of chloroplast protein disulfide isomerase, RB60, catalyzes the redox regulated binding of chloroplast poly(A)-binding protein, RB47, to the 5´ untranslated region of the psbA in RNA. Plant Cell Physiol., in press.

Yamaguchi, K., Prieto, S., Beligni, M., Haynes, P., McDonald, W., Yates, J., Mayfield, S.P. Proteomic characterization of the small subunit of Chlamydomonas reinhardtii chloroplast ribosome. Plant Cell, in press.