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

Yaodong Chen,Katie Porter,Masaki Osawa,Anne Marie Augustus,Sara L Milam,Chandra Joshi,Katherine W Osteryoung,Harold P Erickson

doi:10.1074/jbc.m116.767715

Yaodong Chen, Katie Porter + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m116.767715

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Abstract

FtsZ 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.

Highlights

FtsZ 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
We examined the biochemical properties of chloroplast FtsZs from the thermophilic red alga Galdieria sulphuraria, designated here GsFtsZA and GsFtsZB, the first study of the red algal proteins
Expression of Soluble GsFtsZ Using the FATT Tag Vector— GsFtsZA and GsFtsZB were insoluble when expressed in our routine pET11-E. coli system

Summary

Assembly of Chloroplast FtsZA and FtsZB

As in Arabidopsis, the FtsZ with the C-terminal peptide may interact with a membrane protein to tether the Z ring, whereas the one without may play a regulatory role Whatever this role is, it is important enough to have caused gene duplication independently in the green and red chloroplast lineages and in mitochondria. Smith et al [20] reported assembly of long cylinders, ϳ16 nm in diameter, by both AtFtsZ1 and AtFtsZ2 These are very different from the pfs of bacterial FtsZ and are difficult to reconcile with the established structural biology of FtsZ. Smith et al [20] used a yeast expression system that apparently gave soluble AtFtsZs, but as noted above, the polymers they reported were very different from the pfs of bacterial FtsZ. We provide biochemical evidence that GsFtsZA assembles well as the primary scaffold and that GsFtsZB co-assembly enhances polymer disassembly and dynamics

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Experimental Procedures