The Chloroplast Tubulin Homologs FtsZA and FtsZB from the Red Alga Galdieria sulphuraria Co-assemble into Dynamic Filaments

Abstract

Edited by Velia M. FowlerFtsZ is a homolog of eukaryotic tubulin and is present in almost all bacteria and many archaea, where it is the major cytoskeletal protein in the Z ring, required for cell division. Unlike some other cell organelles of prokaryotic origin, chloroplasts have retained FtsZ as an essential component of the division machinery. However, chloroplast FtsZs have been challenging to study because they are difficult to express and purify. To this end, we have used a FATT tag expression system to produce as soluble proteins the two chloroplast FtsZs from Galdieria sulphuraria, a thermophilic red alga. GsFtsZA and GsFtsZB assembled individually in the presence of GTP, forming large bundles of protofilaments. GsFtsZA also assembled in the presence of GDP, the first member of the FtsZ/tubulin superfamily to do so. Mixtures of GsFtsZA and GsFtsZB assembled protofilament bundles and hydrolyzed GTP at a rate approximately equal to the sum of their individual rates, suggesting a random co-assembly. GsFtsZA assembly by itself in limiting GTP gave polymers that remained stable for a prolonged time. However, when GsFtsZB was added, the co-polymers disassembled with enhanced kinetics, suggesting that the GsFtsZB regulates and enhances disassembly dynamics. GsFtsZA-mts (where mts is a membrane-targeting amphipathic helix) formed Z ring-like helices when expressed in Escherichia coli. Co-expression of GsFtsZB (without an mts) gave co-assembly of both into similar helices. In summary, we provide biochemical evidence that GsFtsZA assembles as the primary scaffold of the chloroplast Z ring and that GsFtsZB co-assembly enhances polymer disassembly and dynamics.

Conserved Dynamics of Chloroplast Cytoskeletal FtsZ Proteins Across Photosynthetic Lineages

“…Because FtsZ1 and FtsZ2 invariably colocalize in chloroplasts Vitha et al, 2001), heteropolymers probably represent the physiologically relevant protofilament configuration in vivo. Like the bacterial proteins, FtsZ1 and FtsZ2 from higher plants possess assembly-stimulated GTPase activity (Lohse et al, 2006;Olson et al, 2010;Smith et al, 2010), as do FtsZA and FtsZB from Galdieria sulphuraria (Chen et al, 2017). In most in vitro studies, chloroplast FtsZ protofilaments associated laterally into bundles, as shown by transmission electron microscopy.…”

“…Both proteins are widely conserved across all plants and green algae (Chloroplastida) with sequenced genomes (Miyagishima et al, 2011;Vieler et al, 2012;Grosche and Rensing, 2017), and we have shown they coassemble in heteropolymers (Olson et al, 2010;Yoshida et al, 2016). Two FtsZ families, FtsZA and FtsZB, are also present in red algae and likewise form heteropolymers (Miyagishima and Kabeya, 2010;TerBush et al, 2013;Chen et al, 2017). Cyanophora paradoxa, a glaucophyte alga, contains only a single plastidic FtsZ (Sato et al, 2005;Leger et al, 2015).…”

The Molecular Machinery of Chloroplast Division

“…This is suggested partly by experiments showing that coassembled GsFtsZA/GsFtsZB protofilaments were much more dynamic than GsFtsZA protofilaments in vitro (Chen et al, 2017). Additionally, mutation of a conserved residue required for GTP hydrolysis drastically reduced turnover of AtFtsZ2 and GsFtsZA homofilaments in S. pombe, as expected based on the GTPase-dependent turnover of bacterial FtsZ described above.…”

“…Recently, FtsZA and FtsZB from the red alga Galdieria sulphuraria were also shown to copolymerize in vitro (Chen et al, 2017), indicating that FtsZ heteropolymerization is a conserved feature and may be the physiologically relevant state. Studies of FtsZ dynamics in heterologous yeast systems are beginning to provide insight into the functional significance of heteropolymerization.…”

“…We refer readers to other reviews for information on the division of other plastid types, including secondary plastids, and for a more evolutionary perspective (for review, see Miyagishima, 2011;Miyagishima et al, 2011Miyagishima et al, , 2014Pyke, 2016). (Olson et al, 2010;Chen et al, 2017); protofilaments may possibly associate laterally to form the Z ring at the division site (Lutkenhaus and Du, 2017). Positioning of the Z ring is confined to the midzone by the chloroplast Min system, comprising ARC3, MCD1, MinD, and MinE, which inhibits Z-ring assembly at nondivision sites.…”