The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro

Abstract

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.

Dynamics of the Synechococcus elongatus cytoskeletal GTPase FtsZ yields mechanistic and evolutionary insight into cyanobacterial and chloroplast FtsZs

“…The single FtsZ present in bacteria must impart enough stability for protofilaments to support their scaffolding function within the Z ring while also allowing sufficient GTPase-dependent subunit turnover to drive protofilament and Z-ring dynamics ( 16 ). By contrast, we have shown that the A. thaliana chloroplast proteins AtFtsZ2 and AtFtsZ1, which coassemble, play distinct roles in promoting protofilament stability and dynamics, respectively ( 30 , 47 ). This suggests partial separation of these complementary functions during the evolution of chloroplasts from cyanobacteria.…”

“…In vitro light scattering (LS) assays have shown that EcFtsZ typically undergoes rapid assembly upon addition of GTP, and rapid disassembly as GTP hydrolysis reduces protofilament stability ( 23 , 52 , 53 ). In contrast, we found that A. thaliana FtsZ2 (AtFtsZ2), one of the two FtsZs in A. thaliana chloroplasts ( 41 , 42 ), disassembled very slowly even after GTP was fully consumed, indicating AtFtsZ2 protofilaments are far more stable ( 47 ). Given the evolutionary relationship between cyanobacterial and chloroplast FtsZs ( 41 , 48 , 49 ), we were interested in the dynamic behavior of SeFtsZ and used LS assays ( 47 , 52 ) to investigate its assembly and disassembly dynamics.…”

“…This is close to the Cc of Synechocystis sp. PCC 6803 FtsZ ( 32 ) but higher than those of the A. thaliana chloroplast proteins ( 47 ), indicating the cyanobacterial FtsZ subunits interact with lower affinity ( 45 ).

Figure 1 Se FtsZ hydrolyzes GTP and undergoes GTP-dependent assembly.

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“…Although Synechococcus elongatus PCC 7942 is the most extensively used model system for investigating the regulation of cyanobacterial cell division ( 6 , 10 , 37 , 38 , 39 , 40 ), partly because it is rod shaped like E. coli , FtsZ has yet to be biochemically characterized in this important species. In addition, only a few studies have examined the biochemistry of the chloroplast FtsZs, in part because, unlike bacteria that have only a single FtsZ, most chloroplasts possess two nonredundant FtsZs that coassemble ( 41 , 42 , 43 , 44 , 45 , 46 , 47 ), adding complexity to their functional analysis.…”

A Novel Z-Ring Associated Protein ZapA-Like Protein (PA5407) From Pseudomonas aeruginosa Promotes FtsZ to Form Double Filaments

“…In vitro , fast-growing bacterial FtsZ usually self-assembles into mostly single, dynamic protofilaments ( Chen et al, 2005 ; Chen and Erickson, 2005 ), but through lateral contact, FtsZ rearranges into bundles or sheets in the solution of calcium, high concentration magnesium, or in some crowding environments ( Yu and Margolin, 1997 ; Mukherjee and Lutkenhaus, 1999 ; Chen and Erickson, 2009 ). Some slow-growing bacterial and chloroplast FtsZ could assemble into bundles, and circles directly ( White et al, 2000 ; Chen et al, 2007 , 2017b ; Wang et al, 2019 ; Porter et al, 2021 ). FtsZ filaments are also regulated by some associated proteins.…”