| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 280, Issue 23, 22549-22554, June 10, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710
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 µM (the critical concentration (Cc)) to 3 µM. At concentrations greater than 3 µM, an enhanced FRET signal was observed with both GTP and GDP, indicating additional assembly above this second Cc. This second Cc varied with Mg2+ concentration, whereas the 0.7 µM Cc 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.
Received for publication, January 25, 2005 , and in revised form, March 23, 2005.
* This work was supported by National Institutes of Health Grant GM66014. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 919-684-6385; E-mail: h.erickson{at}cellbio.duke.edu.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Lan, A. Dajkovic, D. Wirtz, and S. X. Sun Polymerization and Bundling Kinetics of FtsZ Filaments Biophys. J., October 15, 2008; 95(8): 4045 - 4056. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Miraldi, P. J. Thomas, and L. Romberg Allosteric Models for Cooperative Polymerization of Linear Polymers Biophys. J., September 1, 2008; 95(5): 2470 - 2486. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Srinivasan, M. Mishra, L. Wu, Z. Yin, and M. K. Balasubramanian The bacterial cell division protein FtsZ assembles into cytoplasmic rings in fission yeast Genes & Dev., July 1, 2008; 22(13): 1741 - 1746. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Osawa, D. E. Anderson, and H. P. Erickson Reconstitution of Contractile FtsZ Rings in Liposomes Science, May 9, 2008; 320(5877): 792 - 794. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dajkovic, A. Mukherjee, and J. Lutkenhaus Investigation of Regulation of FtsZ Assembly by SulA and Development of a Model for FtsZ Polymerization J. Bacteriol., April 1, 2008; 190(7): 2513 - 2526. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Chen and H. P. Erickson In Vitro Assembly Studies of FtsZ/Tubulin-like Proteins (TubZ) from Bacillus Plasmids: EVIDENCE FOR A CAPPING MECHANISM J. Biol. Chem., March 28, 2008; 283(13): 8102 - 8109. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Huecas, O. Llorca, J. Boskovic, J. Martin-Benito, J. M. Valpuesta, and J. M. Andreu Energetics and Geometry of FtsZ Polymers: Nucleated Self-Assembly of Single Protofilaments Biophys. J., March 1, 2008; 94(5): 1796 - 1806. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Huecas, C. Schaffner-Barbero, W. Garcia, H. Yebenes, J. M. Palacios, J. F. Diaz, M. Menendez, and J. M. Andreu The Interactions of Cell Division Protein FtsZ with Guanine Nucleotides J. Biol. Chem., December 28, 2007; 282(52): 37515 - 37528. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Chen, D. E. Anderson, M. Rajagopalan, and H. P. Erickson Assembly Dynamics of Mycobacterium tuberculosis FtsZ J. Biol. Chem., September 21, 2007; 282(38): 27736 - 27743. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Diaz-Espinoza, A. P. Garces, J. J. Arbildua, F. Montecinos, J. E. Brunet, R. Lagos, and O. Monasterio Domain folding and flexibility of Escherichia coli FtsZ determined by tryptophan site-directed mutagenesis Protein Sci., August 1, 2007; 16(8): 1543 - 1556. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. J. Hyde, J. Parisot, A. McNichol, and B. B. Bonev Nisin-induced changes in Bacillus morphology suggest a paradigm of antibiotic action PNAS, December 26, 2006; 103(52): 19896 - 19901. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Kerr, H. Levine, T. J. Sejnowski, and W.-J. Rappel Division accuracy in a stochastic model of Min oscillations in Escherichia coli PNAS, January 10, 2006; 103(2): 347 - 352. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Osawa and H. P. Erickson Probing the domain structure of FtsZ by random truncation and insertion of GFP Microbiology, December 1, 2005; 151(12): 4033 - 4043. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
