Essential Cell Division Protein FtsZ Assembles into One Monomer-thick Ribbons under Conditions Resembling the Crowded Intracellular Environment
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m305230200
ISSN1083-351X
AutoresJosé M. González, Mercedes Jiménez, Marisela Vélez, Jesús Mingorance, José M. Andreu, Miguel Vicente, Germán Rivas,
Tópico(s)Bacterial biofilms and quorum sensing
ResumoExperimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked. Experimental conditions that simulate the crowded bacterial cytoplasmic environment have been used to study the assembly of the essential cell division protein FtsZ from Escherichia coli. In solutions containing a suitable concentration of physiological osmolytes, macromolecular crowding promotes the GTP-dependent assembly of FtsZ into dynamic two-dimensional polymers that disassemble upon GTP depletion. Atomic force microscopy reveals that these FtsZ polymers adopt the shape of ribbons that are one subunit thick. When compared with the FtsZ filaments observed in vitro in the absence of crowding, the ribbons show a lag in the GTPase activity and a decrease in the GTPase rate and in the rate of GTP exchange within the polymer. We propose that, in the crowded bacterial cytoplasm under assembly-promoting conditions, the FtsZ filaments tend to align forming dynamic ribbon polymers. In vivo these ribbons would fit into the Z-ring even in the absence of other interactions. Therefore, the presence of mechanisms to prevent the spontaneous assembly of the Z-ring in non-dividing cells must be invoked. The FtsZ protein is conserved in most of the prokaryotic organisms and several organelles and plays a central role in microbial and organelle division forming a ring at the division site (1Bi E. Lutkenhaus J. Nature. 1991; 354: 161-164Crossref PubMed Scopus (1158) Google Scholar, 2Vitha S. McAndrew R.S. Osteryoung K.W. J. Cell Biol. 2001; 153: 111-119Crossref PubMed Scopus (235) Google Scholar). It is structurally related to the eukaryotic cytoskeletal tubulin (3Löwe J. Amos L.A. Nature. 1998; 391: 203-206Crossref PubMed Scopus (719) Google Scholar, 4Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1808) Google Scholar), binds guanine nucleotides, and has GTPase activity (5de Boer P.A.J. Crossley R. Rothfield L.I. Nature. 1992; 359: 254-256Crossref PubMed Scopus (451) Google Scholar, 6Mukherjee A. Dai K. Lutkenhaus J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1053-1057Crossref PubMed Scopus (265) Google Scholar, 7RayChaudhuri D. Park J.T. Nature. 1992; 359: 251-254Crossref PubMed Scopus (341) Google Scholar). In the presence of GDP, the FtsZ monomers undergo a reversible non-cooperative magnesium-linked association to form linear oligomers (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 9Sossong Jr., T.M. Brigham-Burke M.R. Hensley P. Pearce Jr., K.H. 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Biochemistry. 1989; 28: 6513-6518Crossref PubMed Scopus (70) Google Scholar, 24Lindner R.A. Ralston G.B. Biophys. Chem. 1997; 66: 57-66Crossref PubMed Scopus (59) Google Scholar). In the case of FtsZ, it has been previously shown that crowding facilitates the formation of magnesium-dependent GDP-FtsZ oligomers (25Rivas G. Fernández J.A. Minton A.P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3150-3155Crossref PubMed Scopus (154) Google Scholar). We have investigated the effects of macromolecular crowding upon the more complex and physiologically relevant FtsZ assembly processes taking place in the presence of GTP, which is the most abundant form of the nucleotide in the cell. By means of analytical centrifugation, light scattering, optical, electron, and atomic force microscopy and assays of biochemical activity performed in solutions containing a suitable content of the main physiological osmolytes (see “Materials and Methods” (18Record Jr., T.M. Courtenay E.S. Cayley S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 190-194Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar)), we have found that high concentrations of unrelated macromolecules, resembling the crowded Escherichia coli cytoplasm, favor the formation of two-dimensional FtsZ polymer arrays (ribbons). This self-organization process, which is GTP-dependent, retards significantly the FtsZ GTPase activity, the polymer assembly/disassembly dynamics, and the nucleotide exchange within the polymer, when compared with the same parameters measured in the FtsZ filaments formed in dilute solutions. We conclude that in the bacterial interior FtsZ may tend to spontaneously arrange into one-subunit-thick ribbons and suggest, that in the non-dividing cell, their formation must be modulated by other mechanisms that regulate Z-ring assembly. Reagents—Guanine nucleotides, GDP and GTP, were from Sigma and Roche Molecular Biochemicals, respectively. The nucleotide analog GMPCPP, 1The abbreviations used are: GMPCPP, guanosine-5′-[(α,β)-methyleno]triphosphate; AFM, atomic force microscopy; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein. which hydrolyzes more slowly than GTP, was purchased from Jena Bioscience. Fluorescein 5′-isothiocyanate (FITC) was from Molecular Probes Inc. Other analytical grade chemicals were from Merck or Sigma. Macromolecular crowders (dextran T70 and Ficoll 70, both with average molar weights of 70,000) were obtained commercially from Amersham Biosciences. They were used without further purification after extensive dialysis against the corresponding working buffer. The final concentration of the equilibrated stock solutions of crowder was determined refractometrically using 0.134 and 0.140 ml g–1 as the specific refractive index increments at 620 nm of Ficoll and dextran, respectively (26Weast R.C. CRC Handbook of Chemistry and Physics. 63rd ed. CRC Press Inc., Boca Raton, FL1982Google Scholar). Protein Purification—E. coli FtsZ was purified by the calcium-induced precipitation method following the procedure described previously (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The protein concentration was measured using the BCA assay (Pierce), with spectrophotometrically calibrated FtsZ standards (see Ref. 8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and bovine serum albumin as secondary standard, which gave a 0.80 ± 0.03 color ratio of FtsZ/albumin (in agreement with Ref. 27Lu C. Erickson H.P. Methods Enzymol. 1998; 298: 305-313Crossref PubMed Scopus (26) Google Scholar). Physiological Buffer and Enzymatic GTP-regenerating System— Most of the in vitro studies of FtsZ (reviewed in Refs. 17Addinall S.G. Holland B. J. Mol. Biol. 2002; 318: 219-236Crossref PubMed Scopus (115) Google Scholar and 28Scheffers D.J. Driessen A.J.M. FEBS Lett. 2001; 25254: 1-5Google Scholar; see also Refs. 12Lu C. Stricker J. Erickson H.P. Cell Motil. Cytoskeleton. 1998; 40: 71-86Crossref PubMed Scopus (165) Google Scholar, 13Mingorance J. Rueda S. Gómez-Puertas P. Valencia A. Vicente M. Mol. Microbiol. 2001; 41: 83-91Crossref PubMed Scopus (81) Google Scholar, 14Mukherjee A. Lutkenhaus J. J. Bacteriol. 1999; 181: 823-832Crossref PubMed Google Scholar) have been carried out in chloride-containing buffers at potassium concentrations that are at the lower end or below the physiological range of this cation. Moreover, many of these studies have been done at slightly acidic pH (6.5). These buffers do not mimic the main intracellular ionic environment of the bacterial interior. For example, chloride is present only at very low concentration in the cytoplasm of E. coli. At moderate to high external osmolarities, potassium (ranging from 0.2 to 0.9 m) and glutamate (from 0.03 to 0.2 m) ions are concentrated in the cytoplasm of E. coli to prevent dehydration and maintain turgor pressure (18Record Jr., T.M. Courtenay E.S. Cayley S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 190-194Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). It has been reported that several of these physiological osmolytes (i.e. glutamate) are modulators of non-covalent DNA-protein interactions in vitro (18Record Jr., T.M. Courtenay E.S. Cayley S. Guttman H.J. Trends Biochem. Sci. 1998; 23: 190-194Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 29Leirmo S. Harrison C. Cayley D.S. Burgess R.R. Record M.T.J. Biochemistry. 1987; 26: 2095-2101Crossref PubMed Scopus (200) Google Scholar). Therefore, most of the experiments described in this work were done in KGA buffer (25 mm Hepes/acetate, pH 7.4, plus 100 mm potassium glutamate, 300 mm potassium acetate, and 5 mm magnesium acetate), which contains the major osmolyte species present in bacterial cytoplasm, supplemented with guanine nucleotides as noted. This buffer with a high potassium content, which increases the intrinsic GTPase activity of E. coli FtsZ and therefore the rate of disassembly of FtsZ filaments (for a recent review see Ref. 28Scheffers D.J. Driessen A.J.M. FEBS Lett. 2001; 25254: 1-5Google Scholar), makes it difficult to get stable FtsZ polymers during the time-scale (>10 min) of most of the experiments performed in this work (mainly those done in the absence of crowding agents, see below). In these experiments the GTP was regenerated by adding to the solution an enzymatic regeneration system (1 unit/ml acetate kinase plus 15 mm acetyl phosphate, both from Sigma), as adapted from tubulin/microtubules studies (30Purich D.L. Terry B.J. MacNeal R.K. Karr T.L. Methods Enzymol. 1982; 85: 416-433Crossref PubMed Scopus (7) Google Scholar). Under these conditions FtsZ polymers were stable in solution during at least 1 h at 30 °C (at 1 g/liter FtsZ). Higher acetyl phosphate concentrations (>50 mm) interfered with FtsZ filament formation (three-dimensional polymer bundles are favored; see AFM results) and with negative staining electron microscopy. Alternatively, in some experiments GTP was replaced by GMPCPP, a GTP analog more slowly hydrolyzable (15Romberg L. Simon M. Erickson H.P. J. Biol. Chem. 2001; 276: 11743-11753Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). FITC Labeling of FtsZ—The labeling of FtsZ with the fluorescent dye fluorescein isothiocyanate (FITC) was carried out as follows. FtsZ (4–6 g/liter) was dialyzed against 20 mm Hepes/HCl, pH 8.0, buffer with 50 mm KCl, 5 mm MgCl2, and 1 mm EDTA. To minimize perturbations on FtsZ association/assembly properties due to the labeling, the protein was previously polymerized at 30 °C upon addition of 20 mm calcium and 2 mm GTP (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). To this preparation a 40-fold excess of FITC was added, and the mixture was incubated for 15 min at 30 °C. The precipitate was resuspended in cold in 50 mm Tris/HCl, pH 7.4, buffer with 100 mm KCl, and the free FITC was removed by gel filtration. The precipitation of FtsZ was reversible, and the degree of labeling was 0.9 ± 0.2 mol of FITC per mol of FtsZ. Analytical Centrifugation—The sedimentation equilibrium and sedimentation velocity experiments in the presence of low concentrations of nucleotide ( 95% of FtsZ filaments were recovered in the soluble fraction. Therefore this assay discriminates between filaments and ribbons in a quantitative manner. The crowding-induced FtsZ ribbons were also monitored by two scattering assays: 90° light scattering (as described above), which detected both the FtsZ filaments and ribbon polymers, and turbidity (33Andreu J.M. Timasheff S.N. Methods Enzymol. 1986; 130: 47-69Crossref PubMed Scopus (79) Google Scholar) in an Ultrospec 3000pro UV-visible spectrophotometer (Amersham Biosciences). The latter is also a specific method for monitoring FtsZ ribbons (because of their size) in the presence of macromolecular crowders, but it does not detect the assembly/disassembly of FtsZ filaments (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Electron Microscopy—The FtsZ polymers (protein concentration ranging from 0.05 to 1 g/liter), in the absence or in the presence of high concentrations of macromolecular crowders (dextran T70 or Ficoll 70), were visualized by electron microscopy after negative staining with 2% uranyl acetate, using either a JEOL-1200 or a LEO 910 electron microscope. Atomic Force Microscopy—AFM images were taken with an Atomic Force Microscope (Nonatec Electrónica, Madrid, Spain) operated in the jump mode (34Moreno-Herrero F. de Pablo P.J. Fernández-Sánchez R. Colchero J. Gómez-Herrero A. Baró A. Appl. Phys. Lett. 2002; 81: 2620-2622Crossref Scopus (41) Google Scholar). The scanning piezo measurement was calibrated using silicon calibrating gratings. Silicon nitride tips (DI instruments) with constant force of 0.2 N7M were used. A drop of the solution with the FtsZ polymers (formed upon addition of 0.1 mm GMPCPP to the protein in KGA buffer with 100–200 g/liter Ficoll) was incubated over freshly cleaved circular pieces of mica glued onto a Teflon surface, and, after a few minutes, the samples were extensively washed with working buffer and allowed to dry for 15 min under a stream of air. Optical Microscopy—The FtsZ samples labeled with the fluorescent dye FITC were placed directly onto a glass slide, and the polymers were visualized using a Zeiss Axioscope epifluorescence microscope equipped with the FITC filter set and 40×/63×/100× oil immersion objectives. Images were captured with a Photometrics charge-coupled device camera. Other Analytical Procedures—The total nucleotide content of the protein preparations was measured spectrophotometrically after protein extraction with perchloric acid (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 35Díaz J.F. Andreu J.M. Biochemistry. 1993; 32: 2747-2755Crossref PubMed Scopus (414) Google Scholar). Specific nucleotides were measured by means of ionic-pair high-performance liquid chromatography (35Díaz J.F. Andreu J.M. Biochemistry. 1993; 32: 2747-2755Crossref PubMed Scopus (414) Google Scholar). The GTPase turnover rate was determined by measuring released inorganic phosphate using the malachite green-molybdate reagent (36Hoenig M. Lee R.J. Ferguson D.C. J. Biochem. Biophys. Methods. 1989; 19: 249-252Crossref PubMed Scopus (42) Google Scholar, 37Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1821) Google Scholar). The incorporation of GTP into FtsZ polymers was measured by a nitrocellulose filter-binding assay using [γ-32P]GTP, as described in a previous study (13Mingorance J. Rueda S. Gómez-Puertas P. Valencia A. Vicente M. Mol. Microbiol. 2001; 41: 83-91Crossref PubMed Scopus (81) Google Scholar). FtsZ in Its GDP-bound State in Physiological Buffer Has a Weak Tendency to Oligomerize—We have studied the influence of physiological concentrations of osmolytes present in the E. coli cytoplasm (KGA buffer; see “Materials and Methods”) on the magnesium-induced oligomerization of E. coli GDP-FtsZ. In a previous study, the behavior of GDP-FtsZ in a Tris, pH 7.4, 50 mm KCl buffer was best described by a Mg2+-linked indefinite self-association of FtsZ monomers, with a weakening of the monomer-monomer interaction upon increasing ionic strength (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Analytical ultracentrifugation experiments (sedimentation equilibrium and velocity) were carried out in the presence of GDP. Fig. 1 shows the dependence of the state of association of FtsZ in KGA buffer upon protein concentration. The results indicate that the predominant species over the concentration range studied (0.1–2.5 g/liter) has an average size corresponding to an FtsZ dimer, with a trend to form monomer and higher oligomers at, respectively, decreasing or increasing protein concentrations. In the inset of Fig. 1 the sedimentation velocity ls-g*(s) distribution of FtsZ (1 g/liter) in KGA buffer is plotted. This sedimentation coefficient distribution corresponds to a symmetrical peak with an average sedimentation coefficient of 5.2 S, which is compatible with an average FtsZ dimer (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). These data are in agreement with a previous report showing that, over a broad range of protein concentrations, increasing the ionic strength from 50 to 500 mm KCl lowers the average molar mass and sedimentation coefficient of GDP-FtsZ (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The weak dependence upon protein concentration of the FtsZ self-association in KGA buffer precludes a complete quantitative analysis. The isodesmic association that previously best described the formation of FtsZ oligomers from monomers (dashed line in Fig. 1; see Ref. 8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) may also apply in the present work (performed in KGA buffer) but with a much weaker association constant (dotted line in Fig. 1). We conclude that in solutions that reproduce the main intracellular osmolarity, FtsZ in its GDP-bound state has a weak tendency to form high order oligomers. FtsZ in the Presence of GTP in Physiological Buffer Assembles into Dynamic Filaments—At 30 °C in KGA buffer FtsZ polymers formed quickly upon addition of 1 mm GTP as monitored by 90° light scattering (Fig. 2A). Under the same conditions addition of GDP failed to induce polymerization (Fig. 2A). Given the high concentration of potassium ions in the buffer the GTPase activity of FtsZ is activated, causing the FtsZ polymers to rapidly disassemble following GTP consumption, as reported in other publications (8Rivas G. López A. Mingorance J. Ferrándiz M.J. Zorrilla S. Minton A.P. Vicente M. Andreu J.M. J. Biol. Chem. 2000; 275: 11740-11749Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 14Mukherjee A. Lutkenhaus J. J. Bacteriol. 1999; 181: 823-832Crossref PubMed Google Scholar, 38Mukherjee A. Lutkenhaus J. EMBO J. 1998; 17: 462-469Crossref PubMed Scopus (310) Google Scholar). To overcome the polymer instability, when needed, an enzymatic GTP-regenerating system was added to the preparations (see “Materials and Methods”). Under these conditions, the polymers at 1 g/liter protein were stable for 1 h (Fig. 2A) and could be visualized in electron micrographs of negatively stained preparations as filaments of variable length and curvature with a thickness of 5–6 nm (Fig. 3, top row, center). FtsZ filaments with a thickness of 6–8 nm were also found upon incubation of the protein with GMPCPP, an analog of GTP that is hydrolyzed by FtsZ much more slowly than GTP (not shown).Fig. 3Electron microscopy analysis of FtsZ assembly in the absence and in the presence of macromolecular crowders. Top row, left-hand side: electron micrograph of negative stained FtsZ (1.0 g/liter) samples in 50 mm Tris-HCl, 100 mm NaCl, pH 7.4, buffer (which does not contain potassium and does not promote FtsZ assembly, see Ref. 13Mingorance J. Rueda S. Gómez-Puertas P. Valencia A. Vicente M. Mol. Microbiol. 2001; 41: 83-91Crossref PubMed Scopus (81) Google Scholar) plus 1 mm GTP and 200 g/liter Ficoll 70. The other electron micrographs correspond to negatively stained FtsZ samples in KGA buffer. Top row, center: FtsZ (0.5 g/liter) plus 1 mm GTP. Top row, right-hand side: FtsZ (0.2 g/liter) plus 2 mm GTP and 220 g/liter Ficoll 70. Bottom row, left-hand side: FtsZ (0.1 g/liter) plus 2 mm GTP and 200 g/liter dextran T70. Bottom row, center: FtsZ (0.5 g/liter) plus 0.5 mm GTP and 200 g/liter Ficoll 70 after 1-h incubation at 30 °C. Bottom row, right-hand side: FtsZ (0.5 g/liter) plus 0.1 mm GMPCPP and 200 g/liter Ficoll 70. In all cases, the bar scale is 100 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The soluble and polymerized fractions of FtsZ formed in the presence of the GTP-regenerating system were separated by high speed centrifugation and quantitated (see “Materials and Methods”). Filament formation depended on the concentration of FtsZ showing an apparent critical concentration for filament formation (C fil) of 0.05 ± 0.01 g/liter (1.25 μm, Fig. 2B). Similar samples were analyzed by 90° light scattering resulting in a very similar value for C fil (0.04 ± 0.01 g/liter; Fig. 2C, closed squares). Light scattering analysis were also performed in the absence of the GTP-regenerating system, and the C fil value determined using the initial scattering increments was practically identical to that obtained in the presence of the GTP-regenerating system (Fig. 2C, open squares). The experimental value found for C fil when GTP was replaced by GMPCPP was slightly lower (0.03 ± 0.01 g/liter; Fig. 2C, closed triangles). These results indicate that FtsZ filament formation occurs in a cooperative fashion (33Andreu J.M. Timasheff S.N. Methods Enzymol. 1986; 130: 47-69Crossref PubMed Scopus (79) Google Scholar). The functioning of a cooperative mechanism of FtsZ polymerization has been proposed as well by others (14Mukherjee A. Lutkenhaus J. J. Bacteriol. 1999; 181: 823-832Crossref PubMed Google Scholar, 38Mukherjee A. Lutkenhaus J. EMBO J. 1998; 17: 462-469Crossref PubMed Scopus (310) Google Scholar, 39Caplan M. Erickson H.P. J. Biol. Chem. 2003; 278: 13784-13788Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 40White L.L. Ross L.J. Reynolds R.C. Steitz L.E. Moore G.D. Borhani D.W. J. Bacteriol. 2000; 182: 4028-4034Crossref PubMed Scopus (111) Google Scholar). 2S. Huecas and J. M. Andreu, submitted manuscript. In contrast, in the presence of GDP most of the FtsZ protein (over 98%) was recovered in the soluble fraction as would be expected if the dimer were the most abundant form of the GDP-bound FtsZ in solution (see above, Fig. 1). Macromolecular Crowding Favors the GTP-dependent Formation of Dynamic FtsZ Polymers—The bacterial cytoplasm is a highly crowded environment, a condition that has not been analyzed when studying the formation of FtsZ filaments. To approximate the intracellular conditions we have studied the polymerization of FtsZ in the presence of high concentrations of inert macromolecular crowders (Ficoll or dextran, both with an average molar weight of 70,000, see “Materials and Methods”), as has been used for the study of other systems (20Ellis R.J. Trends Biochem. Sci. 2001; 26: 597-6
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