Tm (tropomyosin) is an evolutionarily conserved α-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilizing actin filaments, and regulating myosin function. The fission yeast Schizosaccharomyces pombe possesses a single essential Tm, Cdc8, which can be acetylated on its N-terminal methionine residue to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with N-terminal substitutions to explore how stability of the Cdc8 overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown that the stability of the N-terminal of the Cdc8 α-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased N-terminal stability, dimers of which are capable of forming Tm polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild-type, and was unable to regulate actomyosin interactions. The results of the present paper are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin filament, consistent with a co-operative model for Tm binding to actin.
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Research Article|
August 12 2011
Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind co-operatively to actin and regulate myosin
Daniel A. East;
Daniel A. East
*School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
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Duncan Sousa;
Duncan Sousa
†Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, U.S.A.
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Stephen R. Martin;
Stephen R. Martin
‡Division of Physical Biochemistry, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
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Thomas A. Edwards;
Thomas A. Edwards
§Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, U.K.
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William Lehman;
William Lehman
†Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118, U.S.A.
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Daniel P. Mulvihill
Daniel P. Mulvihill
1
*School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, U.K.
1To whom correspondence should be addressed (email d.p.mulvihill@kent.ac.uk).
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Publisher: Portland Press Ltd
Received:
August 18 2010
Revision Received:
June 08 2011
Accepted:
June 09 2011
Accepted Manuscript online:
June 09 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 438 (2): 265–273.
Article history
Received:
August 18 2010
Revision Received:
June 08 2011
Accepted:
June 09 2011
Accepted Manuscript online:
June 09 2011
Citation
Daniel A. East, Duncan Sousa, Stephen R. Martin, Thomas A. Edwards, William Lehman, Daniel P. Mulvihill; Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind co-operatively to actin and regulate myosin. Biochem J 1 September 2011; 438 (2): 265–273. doi: https://doi.org/10.1042/BJ20101316
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