Rapid in vitro assembly dynamics and subunit turnover of FtsZ demonstrated by fluorescence resonance energy transfer

Abstract

We have developed an assay for the assembly of FtsZ based on fluorescence resonance energy transfer (FRET). We mutated an innocuous surface residue to cysteine and labeled separate pools with fluorescein (donor) and tetramethylrhodamine (acceptor). When the pools were mixed and GTP was added, assembly produced a FRET signal that was linearly proportional to FtsZ concentration from 0.7 microm (the critical concentration (C(c))) to 3 microm. At concentrations greater than 3 microm, an enhanced FRET signal was observed with both GTP and GDP, indicating additional assembly above this second C(c). This second C(c) varied with Mg(2+) concentration, whereas the 0.7 microm C(c) did not. We used the FRET assay to measure the kinetics of initial assembly by stopped flow. The data were fit by the simple kinetic model used previously: monomer activation, a weak dimer nucleus, and elongation, although with some differences in kinetic parameters from the L68W mutant. We then studied the rate of turnover at steady state by pre-assembling separate pools of donor and acceptor protofilaments. When the pools were mixed, a FRET signal developed with a half-time of 7 s, demonstrating a rapid and continuous disassembly and reassembly of protofilaments at steady state. This is comparable with the 9-s half-time for FtsZ turnover in vivo and the 8-s turnover time of GTP hydrolysis in vitro. Finally, we found that an excess of GDP caused disassembly of protofilaments with a half-time of 5 s. Our new data suggest that GDP does not exchange into intact protofilaments. Rather, our interpretation is that subunits are released following GTP hydrolysis, and then they exchange GDP for GTP and reassemble into new protofilaments, all on a time scale of 7 s. The mechanism may be related to the dynamic instability of microtubules.

Scheme I

Fig. 1. FRET assay of FtsZ assembly

a, the spectra of donor only, acceptor only, donor-acceptor (D-A) without GTP, and donor-acceptor with GTP. FtsZ was at 6 μM, and spectra were recorded 5 min after mixing. b—d, the donor fluorescence of FtsZ recorded at 516 nm is plotted versus the concentration of FtsZ. The three curves show different concentrations of Mg²⁺: 2.5 mM (b), 5mM (c), and 10 mM (d).

Fig. 2

a and b, negatively stained EM images are shown of FtsZ-F268C at 2 μM (a) and 10 μM (b) in MMK buffer following 3 min of assembly. Protofilaments are almost all single-stranded at 2 μM, but at 10 μM, some are associated in pairs or small bundles (bar = 100 nm). C, the length distribution of FtsZ filaments assembled as in a is shown. The majority of filaments are in the range of ∼80–160 nm or ∼20–40 subunits.

Fig. 3. The kinetics of FtsZ initial assembly detected by FRET

Assembly was initiated in the stopped-flow device by mixing with GTP, giving the indicated concentrations of FtsZ. The solid lines show the fitting to the dimer nucleation model.

Fig. 4. The kinetics of subunit exchange when pools of preassembled donor and acceptor FtsZ protofilaments were mixed in 5mM Mg²⁺ (a) or in 2.5 mM EDTA (b)

Note the longer time scale in b. The curves are fits with a single exponential (decay constant 9.3 and 127 s with and without Mg²⁺). FtsZ was at 6 μM.

Fig. 5. The disassembly kinetics of FtsZ polymer following the addition of excess GDP

a, in MMK buffer, the decay constant for disassembly is 6 s. b, in MEK buffer, the decay constant for disassembly is 21 s. c, the disassembly of FtsZ-L68W assayed by the decrease in tryptophan fluorescence. The average decay constant is ∼120 s. FtsZ was at 6 μM.