Differential interaction of cardiac, skeletal muscle, and yeast tropomyosins with fluorescent (pyrene235) yeast actin.

To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase, suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, whereas that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca(+2)-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin +/- troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform-specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.

[1]  Icrp.  Chapters 1 and 2 , 2007 .

[2]  E. Hardeman,et al.  Tropomyosin isoforms: divining rods for actin cytoskeleton function. , 2005, Trends in cell biology.

[3]  E. Egelman,et al.  Actin-destabilizing factors disrupt filaments by means of a time reversal of polymerization. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[4]  D. Helfman,et al.  Alterations in tropomyosin isoform expression in human transitional cell carcinoma of the urinary bladder , 2004, International journal of cancer.

[5]  P. Graceffa,et al.  Myosin-Induced Movement of αα, αβ, and ββ Smooth Muscle Tropomyosin on Actin Observed by Multisite FRET , 2004 .

[6]  S. Almo,et al.  Cofilin (ADF) affects lateral contacts in F-actin. , 2004, Journal of molecular biology.

[7]  P. Graceffa,et al.  Myosin-Induced Movement of aa , ab , and bb Smooth Muscle Tropomyosin on Actin Observed by Multisite FRET , 2004 .

[8]  E. Egelman,et al.  ADF/cofilin use an intrinsic mode of F-actin instability to disrupt actin filaments , 2003, The Journal of cell biology.

[9]  S. Almo,et al.  The structure of nonvertebrate actin: Implications for the ATP hydrolytic mechanism , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  S. Lehrer,et al.  Ca(2+)-induced movement of tropomyosin in skeletal muscle thin filaments observed by multi-site FRET. , 2002, Biophysical journal.

[11]  E. Homsher,et al.  Modulation of Myosin Function by Isoform-specific Properties ofSaccharomyces cerevisiae and Muscle Tropomyosins* , 2001, The Journal of Biological Chemistry.

[12]  A. Bretscher,et al.  Ras regulates the polarity of the yeast actin cytoskeleton through the stress response pathway. , 2001, Molecular biology of the cell.

[13]  S. Hitchcock-DeGregori,et al.  Importance of internal regions and the overall length of tropomyosin for actin binding and regulatory function. , 2001, Biochemistry.

[14]  P. Rubenstein,et al.  Tropomyosin-dependent Filament Formation by a Polymerization-defective Mutant Yeast Actin (V266G,L267G)* , 2000, The Journal of Biological Chemistry.

[15]  J. Shaw,et al.  Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions. , 2000, Genetics.

[16]  R Craig,et al.  Tropomyosin and actin isoforms modulate the localization of tropomyosin strands on actin filaments. , 2000, Journal of molecular biology.

[17]  W. Lehman,et al.  An Actin Subdomain 2 Mutation That Impairs Thin Filament Regulation by Troponin and Tropomyosin* , 2000, The Journal of Biological Chemistry.

[18]  T. Wakabayashi,et al.  A230Y mutation of actin on subdomain 4 is sufficient for higher calcium activation of actin-activated myosin adenosinetriphosphatase in the presence of tropomyosin-troponin. , 2000, Biochemistry.

[19]  W. Lehman,et al.  Three-dimensional reconstruction of thin filaments containing mutant tropomyosin. , 2000, Biophysical journal.

[20]  P. Graceffa.  Movement of smooth muscle tropomyosin by myosin heads. , 1999, Biochemistry.

[21]  V. Korman,et al.  Mutations in Actin Subdomain 3 That Impair Thin Filament Regulation by Troponin and Tropomyosin* , 1999, The Journal of Biological Chemistry.

[22]  W. Lehman,et al.  Tropomyosin positions in regulated thin filaments revealed by cryoelectron microscopy. , 1999, Biophysical journal.

[23]  R. Ludescher,et al.  Differential mobility of skeletal and cardiac tropomyosin on the surface of F-actin. , 1999, Biochemistry.

[24]  Wei-Lih Lee,et al.  Fluorescence Probing of Yeast Actin Subdomain 3/4 Hydrophobic Loop 262–274 , 1997, The Journal of Biological Chemistry.

[25]  E. Homsher,et al.  The Active State of the Thin Filament Is Destabilized by an Internal Deletion in Tropomyosin* , 1997, The Journal of Biological Chemistry.

[26]  P. Rubenstein,et al.  The Effects of Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop on the Dynamics and Stability of Yeast G-actin* , 1997, The Journal of Biological Chemistry.

[27]  P. Rubenstein,et al.  Beryllium Fluoride and Phalloidin Restore Polymerizability of a Mutant Yeast Actin (V266G,L267G) with Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop* , 1997, The Journal of Biological Chemistry.

[28]  J. Lin,et al.  Tropomyosin isoforms in nonmuscle cells. , 1997, International review of cytology.

[29]  L. Tobacman,et al.  Opposite Effects of Myosin Subfragment 1 on Binding of Cardiac Troponin and Tropomyosin to the Thin Filament* , 1996, The Journal of Biological Chemistry.

[30]  L. Tobacman,et al.  Thin filament-mediated regulation of cardiac contraction. , 1996, Annual review of physiology.

[31]  K. Holmes,et al.  An atomic model of the unregulated thin filament obtained by X-ray fiber diffraction on oriented actin-tropomyosin gels. , 1995, Journal of molecular biology.

[32]  B. Barrell,et al.  Tropomyosin is essential in yeast, yet the TPM1 and TPM2 products perform distinct functions , 1995, The Journal of cell biology.

[33]  S. Lehrer.  Pyrene excimer fluorescence as a probe of protein conformational change. , 1995, Sub-cellular biochemistry.

[34]  S. Hitchcock-DeGregori,et al.  Requirement of amino-terminal modification for striated muscle alpha-tropomyosin function. , 1994, The Journal of biological chemistry.

[35]  X. Chen,et al.  Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect , 1993, The Journal of cell biology.

[36]  Y. Ishii,et al.  Kinetics of the "on-off" change in regulatory state of the muscle thin filament. , 1993, Archives of biochemistry and biophysics.

[37]  M. Geeves,et al.  Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. , 1993, Biophysical journal.

[38]  K. Willadsen,et al.  Cooperative interactions between adjacent troponin-tropomyosin complexes may be transmitted through the actin filament. , 1993, The Journal of biological chemistry.

[39]  L. Tobacman,et al.  Analysis of troponin-tropomyosin binding to actin. Troponin does not promote interactions between tropomyosin molecules. , 1992, The Journal of biological chemistry.

[40]  D. Botstein,et al.  Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[41]  P. Rubenstein,et al.  Removal of the amino-terminal acidic residues of yeast actin. Studies in vitro and in vivo. , 1992, The Journal of biological chemistry.

[42]  M. Dante,et al.  Multifunctional yeast high-copy-number shuttle vectors. , 1992, Gene.

[43]  S. Hitchcock-DeGregori,et al.  Relationship between alternatively spliced exons and functional domains in tropomyosin. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[44]  W. Kabsch,et al.  Atomic model of the actin filament , 1990, Nature.

[45]  A. Bretscher,et al.  Disruption of the single tropomyosin gene in yeast results in the disappearance of actin cables from the cytoskeleton , 1989, Cell.

[46]  A. Bretscher,et al.  Purification of tropomyosin from Saccharomyces cerevisiae and identification of related proteins in Schizosaccharomyces and Physarum. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[47]  R. Adelstein,et al.  Mechanism of regulation of cardiac actin-myosin subfragment 1 by troponin-tropomyosin. , 1986, Biochemistry.

[48]  J. Shay,et al.  Cell and Muscle Motility , 1981, Springer US.

[49]  J. Potter.  [22] Preparation of troponin and its subnits , 1982 .

[50]  J. Potter.  Preparation of troponin and its subunits. , 1982, Methods in enzymology.

[51]  J. Abelson,et al.  Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[52]  L. Smillie,et al.  A comparison of the amino acid sequences of rabbit skeletal muscle alpha- and beta-tropomyosins. , 1980, The Journal of biological chemistry.

[53]  H. Huxley,et al.  Muscular Contraction and Cell Motility , 1973, Nature.