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GTP-dependent heteropolymer formation and bundling of chloroplast FtsZ1 and FtsZ2.

ABSTRACT: Bacteria and chloroplasts require the ring-forming cytoskeletal protein FtsZ for division. Although bacteria accomplish division with a single FtsZ, plant chloroplasts require two FtsZ types for division, FtsZ1 and FtsZ2. These proteins colocalize to a mid-plastid Z ring, but their biochemical relationship is poorly understood. We investigated the in vitro behavior of recombinant FtsZ1 and FtsZ2 separately and together. Both proteins bind and hydrolyze GTP, although GTPase activities are low compared with the activity of Escherichia coli FtsZ. Each protein undergoes GTP-dependent assembly into thin protofilaments in the presence of calcium as a stabilizing agent, similar to bacterial FtsZ. In contrast, when mixed without calcium, FtsZ1 and FtsZ2 exhibit slightly elevated GTPase activity and coassembly into extensively bundled protofilaments. Coassembly is enhanced by FtsZ1, suggesting that it promotes lateral interactions between protofilaments. Experiments with GTPase-deficient mutants reveal that FtsZ1 and FtsZ2 form heteropolymers. Maximum coassembly occurs in reactions containing equimolar FtsZ1 and FtsZ2, but significant coassembly occurs at other stoichiometries. The FtsZ1:FtsZ2 ratio in coassembled structures mirrors their input ratio, suggesting plasticity in protofilament and/or bundle composition. This behavior contrasts with that of alpha- and beta-tubulin and the bacterial tubulin-like proteins BtubA and BtubB, which coassemble in a strict 1:1 stoichiometry. Our findings raise the possibility that plasticity in FtsZ filament composition and heteropolymerization-induced bundling could have been a driving force for the coevolution of FtsZ1 and FtsZ2 in the green lineage, perhaps arising from an enhanced capacity for the regulation of Z ring composition and activity in vivo.

SUBMITTER: Olson BJ

PROVIDER: S-EPMC2898327 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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GTP-dependent heteropolymer formation and bundling of chloroplast FtsZ1 and FtsZ2.

Olson Bradley J S C BJ Wang Qiang Q Osteryoung Katherine W KW

The Journal of biological chemistry 20100426 27

Bacteria and chloroplasts require the ring-forming cytoskeletal protein FtsZ for division. Although bacteria accomplish division with a single FtsZ, plant chloroplasts require two FtsZ types for division, FtsZ1 and FtsZ2. These proteins colocalize to a mid-plastid Z ring, but their biochemical relationship is poorly understood. We investigated the in vitro behavior of recombinant FtsZ1 and FtsZ2 separately and together. Both proteins bind and hydrolyze GTP, although GTPase activities are low com ...[more]

PMID: 20421292

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Project description:Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and β-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-protofilament tubes named bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may be derived from an early ancestor of the modern eukaryotic microtubule (MT). Previous biochemical studies revealed that BtubA/B polymerization is reversible and that BtubA/B folding does not require chaperones. To better understand BtubA/B filament behavior and gain insight into the evolution of microtubule dynamics, we characterized in vitro BtubA/B assembly using a combination of polymerization kinetics assays and microscopy. Like eukaryotic microtubules, BtubA/B filaments exhibit polarized growth with different assembly rates at each end. GTP hydrolysis stimulated by BtubA/B polymerization drives a stochastic mechanism of filament disassembly that occurs via polymer breakage and/or fast continuous depolymerization. We also observed treadmilling (continuous addition and loss of subunits at opposite ends) of BtubA/B filament fragments. Unlike MTs, polymerization of BtubA/B requires KCl, which reduces the critical concentration for BtubA/B assembly and induces it to form stable mixed-orientation bundles in the absence of any additional BtubA/B-binding proteins. The complex dynamics that we observe in stabilized and unstabilized BtubA/B filaments may reflect common properties of an ancestral eukaryotic tubulin polymer.IMPORTANCE Microtubules are polymers within all eukaryotic cells that perform critical functions; they segregate chromosomes, organize intracellular transport, and support the flagella. These functions rely on the remarkable range of tunable dynamic behaviors of microtubules. Bacterial tubulin A and B (BtubA/B) are evolutionarily related proteins that form polymers. They are proposed to be evolved from the ancestral eukaryotic tubulin, a missing link in microtubule evolution. Using microscopy and biochemical approaches to characterize BtubA/B assembly in vitro, we observed that they exhibit complex and structurally polarized dynamic behavior like eukaryotic microtubules but differ in how they self-associate into bundles and how this bundling affects their stability. Our results demonstrate the diversity of mechanisms through which tubulin homologs promote filament dynamics and monomer turnover.

Dataset Information

GTP-dependent heteropolymer formation and bundling of chloroplast FtsZ1 and FtsZ2.

Publications

GTP-dependent heteropolymer formation and bundling of chloroplast FtsZ1 and FtsZ2.

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