The Arabidopsis thaliana fructokinase-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 form in vitro. We 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.
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Atrial natriuretic peptide (ANP) is a cardiac hormone released by the atrium in response to stretching forces. ANP, which maintains cardiovascular homeostasis via its diuretic, natriuretic and hypotensive effects, acts through its receptor, GC-A. In this issue of the Biochemical Journal, Miura et al. (pages 1897–1918) established mouse immortalized endothelial cells stably expressing GC-A (SVEC/GC-A) and found that ANP promotes cell spreading and endothelial barrier function via regulation of CCM2, an adaptor protein involved in the pathogenesis of cerebral cavernous malformations, a neurovascular disease characterized by dysfunction of the endothelial barrier. The image shows ANP promotes co-localization of GC-A (green) and CCM2 (red) at membrane ruffles in SVEC/GC-A. Image kindly provided by Koichi Miura
Arabidopsis fructokinase-like protein associations are regulated by ATP
John W. Riggs, Judy Callis; Arabidopsis fructokinase-like protein associations are regulated by ATP. Biochem J 1 June 2017; 474 (11): 1789–1801. doi: https://doi.org/10.1042/BCJ20161077
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