title: Arabidopsisfructokinase-like protein associations are regulated by ATP.
abstract: TheArabidopsis thalianafructokinase-like proteins FLN1 and FLN2 are required for the differentiation of plastids into photosynthetically competent chloroplasts. However, their specific roles are unknown. FLN1 and FLN2 localize in a multisubunit prokaryotic-type polymerase (plastid-encoded RNA polymerase) complex that transcribes genes encoding components of photosynthesis-related assemblies. Despite sequence identity with fructokinases, which are members of the pfkB (phosphofructokinase B) family of enzymes, kinase activity of FLN1 and FLN2 has not been demonstrated. Homology modeling using pfkB X-ray structures, sequence comparisons, and mutational analyses suggests that FLN proteins may bind their substrates differently from other pfkB proteins. We provide evidence that purified recombinant FLN1 undergoes an ATP-mediated change in binding affinity with both itself and recombinant FLN2. The ATP-mediated change in the affinity of FLN1 for FLN2 is not affected by mutations in conserved active-site residues known to affect catalysis in active pfkB enzymes. In contrast, recombinant FLN2 hetero-oligomerizes independently of ATP concentration. At ATP concentrations that promote FLN1 homomeric interactions, the FLN1-FLN2 hetero-oligomer is the dominant formin vitroWe further present evidence that FLN1 associates with a large protein complex in chloroplasts independently of ATP. Given that ATP levels fluctuate between light-dark cycles in the 1-5 mM range, we propose that changes in FLN1 and FLN2 interactions are biologically meaningful.
authors.affiliation: Department of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, CA 95616, U.S.A.
authors.forename: John W
authors.initials: JW
authors.lastname: Riggs
authors.affiliation: Department of Molecular and Cellular Biology, University of California, 1 Shields Avenue, Davis, CA 95616, U.S.A. jcallis@ucdavis.edu.
authors.forename: Judy
authors.initials: J
authors.lastname: Callis
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002736:Chloroplasts / Q000201:enzymology* / Q000378:metabolism; D017124:Conserved Sequence; D030562:Databases, Protein; D008024:Ligands; D008958:Models, Molecular; D009154:Mutation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D011487:Protein Conformation; D055503:Protein Multimerization; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D040681:Structural Homology, Protein
chemical_list: D029681:Arabidopsis Proteins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; C000594290:FLN1 protein, Arabidopsis; C000605310:FLN2 protein, Arabidopsis; D017853:Phosphotransferases (Alcohol Group Acceptor)
keywords: ATP binding; Arabidopsis thaliana; chloroplasts; fructokinase; pfkB proteins; plant biology