A novel rhodanese is required to maintain chloroplast translation in Chlamydomonas.
Rhodanese-domain proteins (RDPs) are widespread in plants and other organisms, but their biological roles are mostly unknown. Here we report on a novel RDP from Chlamydomonas that has a single rhodanese domain, and a predicted chloroplast transit peptide. The protein was produced in Escherichia coli with a His-tag, but lacking most of the N-terminal transit peptide, and after purification was found to have rhodanese activity in vitro. It was also used to elicit antibodies for western blot analysis, which showed that the native Chlamydomonas protein migrated slower on SDS gels (apparent M(r) =34 kDa) than its predicted size (27 kDa), and co-fractionated with chloroplasts. To assess function in vivo, the tandem-RNAi approach was used to generate Chlamydomonas strains that had reductions of 30-70% for the mRNA and ~20-40% for the 34-kDa protein. These strains showed reduced growth under all trophic conditions, and were sensitive to even moderate light; properties reminiscent of chloroplast translation mutants. Pulse-labeling in the presence of cycloheximide indicated that chloroplast protein synthesis was broadly reduced in the RNAi strains, and transcript analysis (by RT-PCR and northern blotting) indicated the effect was mainly translational. These results identify a novel rhodanese-like protein that we have named CRLT, because it is required to maintain chloroplast translation.
Section of Molecular Cell and Developmental Biology, School of Biological Sciences, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA.
Liming
L
Luo
David L
DL
Herrin
D000595:Amino Acid Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D060365:Chloroplast Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D018747:RNA, Chloroplast / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013884:Thiosulfate Sulfurtransferase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D060365:Chloroplast Proteins; D010940:Plant Proteins; D018747:RNA, Chloroplast; D012333:RNA, Messenger; D018749:RNA, Plant; D011994:Recombinant Proteins; D013884:Thiosulfate Sulfurtransferase