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

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.

Keywords: GTPase; biophysics; cell division; cytokinesis; cytoskeleton; tubulin.

FIGURE 1.

Sequence of the FATT tag segment of our redesigned vector pHFATTC. The vector expresses an N-terminal His tag, the FATT domain, a 3C protease site, and a linker, ahead of the NdeI·BamHI sites (HM … GS) that are used to insert the target protein sequence.

FIGURE 2.

A and B, negative stain EM showed that 10 μm GsFtsZA assembled into large straight pf bundles in the presence of either 500 μm GTP (A) or 500 μm GDP (B). C, GsFtsZB (10 μm) assembled similar bundles in the presence of 500 μm GTP. D and E, assembly kinetics of GsFtsZA (D) and GsFtsZB (E) at the indicated concentrations were assayed by light scattering in 500 μm GTP (black) or GDP (red).

FIGURE 3.

A, assembly of 10 μm GsFtsZA in 50–1000 μm GDP. Assembly was strongly dependent on GDP concentration. B, assembly of 10 μm GsFtsZA was identical in 50 and 500 μm GTP. C, SDS-PAGE of the supernatant (S) and pellet (P) of 10 μm GsFtsZA assembled in 50–500 μm GDP and 50 mm GTP, after centrifugation at 85,000 rpm for 30 min. D, assembly of 10 μm GsFtsZA in 50 μm GTP followed for 3 h. The GsFtsZA pf bundles started to disassemble only after 2 h. E, assembly of 10 μm GsFtsZB in 50 and 200 μm GTP (note that the scale in D is min, and that in E is s).

FIGURE 4.

A, the GTPase activity of GsFtsZA and GsFtsZB assayed separately at 25 °C. GTP was 500 μm. B, the GTPase activity of GsFtsZA mixed with increasing ratios of GsFtsZB. The x axis gives the concentration of GsFtsZA, and the ratio of A:B is indicated above the fitted lines. Each assay was done by stepwise increase in FtsZ concentration, which gave a single measurement at each step, precluding error bars. However, the accuracy of the assay is demonstrated by the excellent fit of the ensemble of points to the straight lines.

FIGURE 5.

A, the assembly-disassembly kinetics of 5 μm GsFtsZA, 10 μm GsFtsZB, and their mixture in 50 μm GTP. B, the assembly-disassembly kinetics of 5 μm GsFtsZA with increasing GsFtsZB in 50 μm GTP. C–E, EM of 5 μm GsFtsZA plus 10 μm GsFtsZB at different times after adding 50 μm GTP. The polymers are mostly gone at 10 min.

FIGURE 6.

A, GsFtsZA-mVenus-mts expressed in E. coli from plasmid pJSB2 formed helices and ring structures. B, GsFtsZB-mCerulean-mts expressed from plasmid pET11 was diffuse and assembled into small spots or patches. C–E, co-expression of GsFtsZA-mVenus-mts (C, red) and GsFtsZB-mCerulean without mts (D, green) gave a small fraction of cells with balanced expression of both GsFtsZA and GsFtsZB. These cells showed co-localization of both proteins to spiral structures (E, orange).