The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100627
ISSN1083-351X
AutoresKatie J. Porter, Lingyan Cao, Yaodong Chen, Allan D. TerBush, Cheng Chen, Harold Erickson, Katherine W. Osteryoung,
Tópico(s)Plant Stress Responses and Tolerance
ResumoBacterial cell and chloroplast division are driven by a contractile "Z ring" composed of the tubulin-like cytoskeletal GTPase FtsZ. Unlike bacterial Z rings, which consist of a single FtsZ, the chloroplast Z ring in plants is composed of two FtsZ proteins, FtsZ1 and FtsZ2. Both are required for chloroplast division in vivo, but their biochemical relationship is poorly understood. We used GTPase assays, light scattering, transmission electron microscopy, and sedimentation assays to investigate the assembly behavior of purified Arabidopsis thaliana (At) FtsZ1 and AtFtsZ2 both individually and together. Both proteins exhibited GTPase activity. AtFtsZ2 assembled relatively quickly, forming protofilament bundles that were exceptionally stable, as indicated by their sustained assembly and slow disassembly. AtFtsZ1 did not form detectable protofilaments on its own. When mixed with AtFtsZ2, AtFtsZ1 reduced the extent and rate of AtFtsZ2 assembly, consistent with its previously demonstrated ability to promote protofilament subunit turnover in living cells. Mixing the two FtsZ proteins did not increase the overall GTPase activity, indicating that the effect of AtFtsZ1 on AtFtsZ2 assembly was not due to a stimulation of GTPase activity. However, the GTPase activity of AtFtsZ1 was required to reduce AtFtsZ2 assembly. Truncated forms of AtFtsZ1 and AtFtsZ2 consisting of only their conserved core regions largely recapitulated the behaviors of the full-length proteins. Our in vitro findings provide evidence that FtsZ1 counterbalances the stability of FtsZ2 filaments in the regulation of chloroplast Z-ring dynamics and suggest that restraining FtsZ2 self-assembly is a critical function of FtsZ1 in chloroplasts. Bacterial cell and chloroplast division are driven by a contractile "Z ring" composed of the tubulin-like cytoskeletal GTPase FtsZ. Unlike bacterial Z rings, which consist of a single FtsZ, the chloroplast Z ring in plants is composed of two FtsZ proteins, FtsZ1 and FtsZ2. Both are required for chloroplast division in vivo, but their biochemical relationship is poorly understood. We used GTPase assays, light scattering, transmission electron microscopy, and sedimentation assays to investigate the assembly behavior of purified Arabidopsis thaliana (At) FtsZ1 and AtFtsZ2 both individually and together. Both proteins exhibited GTPase activity. AtFtsZ2 assembled relatively quickly, forming protofilament bundles that were exceptionally stable, as indicated by their sustained assembly and slow disassembly. AtFtsZ1 did not form detectable protofilaments on its own. When mixed with AtFtsZ2, AtFtsZ1 reduced the extent and rate of AtFtsZ2 assembly, consistent with its previously demonstrated ability to promote protofilament subunit turnover in living cells. Mixing the two FtsZ proteins did not increase the overall GTPase activity, indicating that the effect of AtFtsZ1 on AtFtsZ2 assembly was not due to a stimulation of GTPase activity. However, the GTPase activity of AtFtsZ1 was required to reduce AtFtsZ2 assembly. Truncated forms of AtFtsZ1 and AtFtsZ2 consisting of only their conserved core regions largely recapitulated the behaviors of the full-length proteins. Our in vitro findings provide evidence that FtsZ1 counterbalances the stability of FtsZ2 filaments in the regulation of chloroplast Z-ring dynamics and suggest that restraining FtsZ2 self-assembly is a critical function of FtsZ1 in chloroplasts. Chloroplasts, the photosynthetic organelles in plants, arose from the endosymbiosis of a free-living cyanobacterium (1Gould S.B. Waller R.F. McFadden G.I. Plastid evolution.Annu. Rev. Plant Biol. 2008; 59: 491-517Crossref PubMed Scopus (451) Google Scholar). 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FtsZ1 and FtsZ2 presumably arose through ancient duplication of a single FtsZ gene acquired from the cyanobacterial endosymbiont and have been conserved throughout green algae and land plants (2Miyagishima S.-Y. Nakanishi H. Kabeya Y. Structure, regulation, and evolution of the plastid division machinery. Int. Rev. Cell Mol. Biol.291. 2011: 115-153Google Scholar, 34Stokes K.D. Osteryoung K.W. Early divergence of the FtsZ1 and FtsZ2 plastid division gene families in photosynthetic eukaryotes.Gene. 2003; 320: 97-108Crossref PubMed Scopus (58) Google Scholar, 35Osteryoung K.W. Stokes K.D. Rutherford S.M. Percival A.L. Lee W.Y. Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial FtsZ.Plant Cell. 1998; 10: 1991-2004Crossref PubMed Scopus (289) Google Scholar). 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Conserved dynamics of chloroplast cytoskeletal FtsZ proteins across photosynthetic lineages.Plant Physiol. 2018; 176: 295-306Crossref PubMed Scopus (14) Google Scholar) and direct evidence of coassembly was shown using chimeric AtFtsZ1/AtFtsZ2 proteins (27Yoshida Y. Mogi Y. TerBush A.D. Osteryoung K.W. Chloroplast FtsZ assembles into a contractible ring via tubulin-like heteropolymerization.Nat. Plants. 2016; 2: 16095Crossref PubMed Google Scholar). Colocalization of FtsZ1 and FtsZ2 in other species also supports their coassembly (6Vitha S. McAndrew R.S. Osteryoung K.W. FtsZ ring formation at the chloroplast division site in plants.J. Cell Biol. 2001; 153: 111-120Crossref PubMed Scopus (221) Google Scholar). FtsZ1 and FtsZ2 differ in several important ways. Both possess a highly conserved globular core region responsible for GTP binding and hydrolysis in all FtsZs, flanked by more variable N- and C-terminal regions (41de Boer P. Crossley R. Rothfield L. 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Similarly, the FtsZ2 CTP tethers the chloroplast Z ring to the inner envelope membrane through interaction with plant-specific membrane proteins (40Schmitz A.J. Glynn J.M. Olson B.J.S.C. Stokes K.D. Osteryoung K.W. Arabidopsis FtsZ2-1 and FtsZ2-2 are functionally redundant, but FtsZ-based plastid division is not essential for chloroplast partitioning or plant growth and development.Mol. Plant. 2009; 2: 1211-1222Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 49Maple J. Aldridge C. Møller S.G. Plastid division is mediated by combinatorial assembly of plastid division proteins.Plant J. 2005; 43: 811-823Crossref PubMed Scopus (110) Google Scholar, 50Zhang M. Chen C. Froehlich J.E. TerBush A.D. Osteryoung K.W. Roles of Arabidopsis PARC6 in coordination of the chloroplast division complex and negative regulation of FtsZ assembly.Plant Physiol. 2016; 170: 250-262Crossref PubMed Scopus (25) Google Scholar). FtsZ1 lacks the CTP and does not interact directly with any known membrane protein. Therefore, its localization to the Z ring is presumed to be a consequence of its coassembly with FtsZ2 (49Maple J. Aldridge C. Møller S.G. Plastid division is mediated by combinatorial assembly of plastid division proteins.Plant J. 2005; 43: 811-823Crossref PubMed Scopus (110) Google Scholar, 50Zhang M. Chen C. Froehlich J.E. TerBush A.D. Osteryoung K.W. Roles of Arabidopsis PARC6 in coordination of the chloroplast division complex and negative regulation of FtsZ assembly.Plant Physiol. 2016; 170: 250-262Crossref PubMed Scopus (25) Google Scholar). The two proteins also differ in their dynamic properties, as shown by fluorescence recovery after photobleaching (FRAP) experiments in which the AtFtsZ proteins were expressed in heterologous yeast systems. While both proteins form homopolymeric filaments and/or rings that undergo subunit exchange in such systems, AtFtsZ2 filaments are much less dynamic than AtFtsZ1 or coassembled filaments (24TerBush A.D. Osteryoung K.W. Distinct functions of chloroplast FtsZ1 and FtsZ2 in Z-ring structure and remodeling.J. Cell Biol. 2012; 199: 623-637Crossref PubMed Scopus (42) Google Scholar, 27Yoshida Y. Mogi Y. TerBush A.D. Osteryoung K.W. Chloroplast FtsZ assembles into a contractible ring via tubulin-like heteropolymerization.Nat. Plants. 2016; 2: 16095Crossref PubMed Google Scholar). These studies, in combination with mutant analysis in Arabidopsis, have led to proposals that FtsZ2 imparts structural stability to the Z ring while FtsZ1 opposes this stability and promotes Z-ring turnover dynamics (24TerBush A.D. Osteryoung K.W. Distinct functions of chloroplast FtsZ1 and FtsZ2 in Z-ring structure and remodeling.J. Cell Biol. 2012; 199: 623-637Crossref PubMed Scopus (42) Google Scholar, 26Johnson C.B. Shaik R. Abdallah R. Vitha S. Holzenburg A. FtsZ1/FtsZ2 turnover in chloroplasts and the role of ARC3.Microsc. Microanal. 2015; 21: 313-323Crossref PubMed Scopus (15) Google Scholar, 27Yoshida Y. Mogi Y. TerBush A.D. Osteryoung K.W. Chloroplast FtsZ assembles into a contractible ring via tubulin-like heteropolymerization.Nat. Plants. 2016; 2: 16095Crossref PubMed Google Scholar, 39Yoder D.W. Kadirjan-Kalbach D. Olson B.J.S.C. Miyagishima S.-Y. DeBlasio S.L. Hangarter R.P. Osteryoung K.W. Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo.Plant Cell Physiol. 2007; 48: 775-791Crossref PubMed Scopus (48) Google Scholar). Complementary in vitro studies are essential for further understanding of how these proteins cooperate biochemically in the chloroplast Z ring. Here, we used purified AtFtsZ1 and AtFtsZ2 to test their self-assembly behavior in vitro and elucidate how their interactions contribute to their cellular roles. Toward this end we compared the GTPase activities, formation of protofilaments, and assembly kinetics of AtFtsZ1 and AtFtsZ2 separately and in mixture. We provide biochemical evidence that FtsZ1 counterbalances the stabilizing properties of FtsZ2 by restraining its assembly into protofilaments. In a previous analysis, bacterially expressed AtFtsZ proteins were insoluble and had to be renatured (51Olson B.J.S.C. Wang Q. Osteryoung K.W. GTP-dependent heteropolymer formation and bundling of chloroplast FtsZ1 and FtsZ2.J. Biol. Chem. 2010; 285: 20634-20643Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Here we optimized expression and purification of soluble, His-tagged AtFtsZ1 and AtFtsZ2 (Fig. 1B) to investigate their in vitro enzymatic and assembly properties. 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