Poster: Organelle Biogenesis
Abs #
1144: Iron-sulfur cluster formation in plastids: characterization of a chloroplast nifs-like protein.
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Presenter: |
Pilon, Marinus , pilon@lamar.colostate.edu |
Authors | Pilon, Marinus (A) Garifullina, Gulnara (A) Abdel-Ghany, Salah (A) Tufan, Hale (A) Ackley, Ashley (A) Bodecker, Jared (A) Pilon-Smits, Elizabeth (A) | | Affiliations: |
(A): Colorado State University
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Fe-S clusters are essential cofactors in photosynthesis. Fe-S cluster formation in the mitochondria of eukaryotes involves orthologs of the genes encoded by the nif cluster of nitrogen fixing bacteria. The bacterial nifS gene encodes a cysteine desulfurase, required to produce the sulfur of iron-sulfur clusters. Using seleno-cysteine as a substrate, we found that about 30% of the Nifs activity in green tissue in Arabidopsis thaliana is present in chloroplasts. In A. thaliana two NifS-like proteins are encoded that could be responsible for these activities. We have cloned a cDNA that encodes the precursor of the putative chloroplast NifS-like protein. The protein was over-produced in E. coli and purified. The enzyme contains a PLP cofactor, and is a dimer. Western blots indicate that AtCpNifS is found in the chloroplast stroma. The protein is expressed in all major tissues but light promotes expression in shoots. Northern analysis supports these conclusions. The purified enzyme is active on both selenocysteine and cysteine substrates, however the activity on cysteine is about 300x lower. The relatively high activity on selenocysteine and the low activity on cysteine of the chloroplastic NifS-like protein may reflect the need for regulation of this enzyme in chloroplasts, which may be needed since this organelle is the site of cysteine synthesis. Alternatively it may reflect a possible role in selenium metabolism/tolerance in plants. Antisense lines with a modest reduction in expression (~ 25% less) already show dramatic phenotypes when exposed to low concentrations of selenite but not on normal media. Lines with a stronger reduction in AtCpNifS expression show severe growth phenotypes suggesting an essential role of this protein in plants.
