Knock-down of a novel organelle-targeted protein with rhodanese and glycine-rich domains reveals its importance in photoautotrophic and heterotrophic growth in Chlamydomonas
Liming Luo and David L. Herrin
Molecular Cell and Developmental Biology, School of Biological Sciences, University of Texas at Austin, Austin, TX, 78712, USA
Rhodanese domains are found widely in all three major branches of life, and have been found to have multiple biochemical functions. However, the biological roles of many rhodanese-domain proteins, especially those in chloroplasts and mitochondria, are unknown, or their substrates remain elusive. Mapping of 2 nuclear suppressors of defective chloroplast introns that were previously isolated in our lab (Li et al. 2001), lead to our focusing on a novel rhodanese-domain gene with EST evidence of alternative polyadenylation in the 3' UTR. Bioinformatic analysis of the predicted protein sequence gave strong signatures for an N-terminal chloroplast-targeting peptide, and an orthologous, centrally-located rhodanese domain. Also, a glycine-rich, highly basic region at the C-terminus is physicochemically similar to RNA-binding regions found in certain proteins. This gene was knocked down using the tandem RNAi system of Cerutti and colleagues, producing strains that had reductions of 31% to 76% in the mRNA level. These strains showed reduced rates of photoautotrophic and heterotrophic growth, compared to control strains, and they exhibited strong light sensitivity, not unlike chloroplast translation mutants. Preliminary western blots with antiserum for the protein produced in E. coli indicate that the cellular protein co-migrates on SDS gels with the mature protein (~26 kDa) and about 4 kDa faster than the full-length precursor (which was also produced in E. coli and migrates at ~30 kDa). These blots also indicated that the cellular protein level is reduced in the RNAi strains. The results indicate that this novel rhodanese-domain protein is targeted to one or both organelles, and that it may be necessary for chloroplast gene expression, most likely translation. Thus, it is also likely that clones with a severe depletion of the mRNA (elicited by the RNAi) probably did not survive, and that this is an essential gene.
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