Mutations in Actin Subdomain 3 That Impair Thin Filament Regulation by Troponin and Tropomyosin

Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca2+ and myosin. We establish and exploitSaccharomyces cerevisiae actin as a model system to investigate this process. Ca2+-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca2+-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca2+ activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca2+ affinity and a 50% decrease in the MgATPase rate at saturating Ca2+concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca2+, a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca2+ affinity, there was no detectable change in the true Ca2+ affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca2+ affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca2+- and myosin-induced thin filament activation. Thin filament-mediated regulation of striated muscle contraction involves conformational switching among a few quaternary structures, with transitions induced by binding of Ca2+ and myosin. We establish and exploitSaccharomyces cerevisiae actin as a model system to investigate this process. Ca2+-sensitive troponin-tropomyosin binding affinities for wild type yeast actin are seen to closely resemble those for muscle actin, and these hybrid thin filaments produce Ca2+-sensitive regulation of the myosin S-1 MgATPase rate. Yeast actin filament inner domain mutant K315A/E316A depresses Ca2+ activation of the MgATPase rate, producing a 4-fold weakening of the apparent Ca2+ affinity and a 50% decrease in the MgATPase rate at saturating Ca2+concentration. Observed destabilization of troponin-tropomyosin binding to actin in the presence of Ca2+, a 1.4-fold effect, provides a partial explanation. Despite the decrease in apparent MgATPase Ca2+ affinity, there was no detectable change in the true Ca2+ affinity of the thin filament, measured using fluorophore-labeled troponin. Another inner domain mutant, E311A/R312A, decreased the MgATPase rate but did not change the apparent Ca2+ affinity. These results suggest that charged residues on the surface of the actin inner domain are important in Ca2+- and myosin-induced thin filament activation. Cardiac and striated muscle contraction are regulated by conformational changes in the thin filament, which comprises F-actin, tropomyosin, and troponin. Troponin has three subunits: troponin C, which serves as the Ca2+ sensor for the system; troponin I, which inhibits myosin cycling; and troponin T, which binds to tropomyosin, troponin I, troponin C, and F-actin (reviewed in Refs.1Tobacman L.S. Annu. Rev. Physiol. 1996; 58: 447-481Crossref PubMed Scopus (461) Google Scholar, 2Zot A.S. Potter J.D. Annu. Rev. Biophys. Biophys. Chem. 1987; 16: 535-559Crossref PubMed Scopus (447) Google Scholar, 3Leavis P.C. Gergely J. CRC Crit. Rev. Biochem. 1984; 16: 235-305Crossref PubMed Scopus (326) Google Scholar). Structural and biochemical evidence favor a three-state model for the regulation of muscle contraction (4McKillop D.F.A. Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 5Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (387) Google Scholar, 6Lehman W. Vibert P. Uman P. Craig R. J. Mol. Biol. 1995; 251: 191-196Crossref PubMed Scopus (145) Google Scholar, 7Lehman W. Craig R. Vibert P. Nature. 1994; 368: 65-67Crossref PubMed Scopus (275) Google Scholar, 8Lorenz M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar, 9Butters C.A. Tobacman J.B. Tobacman L.S. J. Biol. Chem. 1997; 272: 13196-13202Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 10Geeves M.A. Lehrer S.S. Biophys. J. 1994; 67: 273-282Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 11Landis C.A. Bobkova A. Homsher E. Tobacman L.S. J. Biol. Chem. 1997; 272: 14051-14056Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The inhibited or blocked state occurs in the absence of Ca2+ and allows tropomyosin and troponin to inhibit actin-myosin binding and cycling (4McKillop D.F.A. Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 6Lehman W. Vibert P. Uman P. Craig R. J. Mol. Biol. 1995; 251: 191-196Crossref PubMed Scopus (145) Google Scholar, 10Geeves M.A. Lehrer S.S. Biophys. J. 1994; 67: 273-282Abstract Full Text PDF PubMed Scopus (174) Google Scholar). The Ca2+ state, alternatively viewed as closed or permissive, occurs upon Ca2+ binding to troponin C. It involves tropomyosin movement and possibly other conformational changes, and results in improved myosin binding to the thin filament. The active or open state corresponds to a final shift in the position of the regulatory proteins, which allows myosin to bind strongly to F-actin. This state appears to be required for cross-bridge cycling (4McKillop D.F.A. Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (663) Google Scholar,5Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (387) Google Scholar, 10Geeves M.A. Lehrer S.S. Biophys. J. 1994; 67: 273-282Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 12Geeves M.A. Conibear P.B. Biophys. J. 1995; 68: 194s-201sPubMed Google Scholar, 13Holmes K.C. Biophys. J. 1995; 68: 2s-7sPubMed Google Scholar). Currently there is little detail concerning where on the actin filament the regulatory proteins bind for each of the different states. Average tropomyosin positions relative to actin have been proposed based on three-dimensional reconstructions using electron micrographs of thin filaments or x-ray diffraction of oriented filaments (6Lehman W. Vibert P. Uman P. Craig R. J. Mol. Biol. 1995; 251: 191-196Crossref PubMed Scopus (145) Google Scholar, 7Lehman W. Craig R. Vibert P. Nature. 1994; 368: 65-67Crossref PubMed Scopus (275) Google Scholar, 8Lorenz M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar, 14Lehman W. Craig R. Uman P. Vibert P. Biophys. J. 1996; 70 (abstr.): A15Google Scholar). Only indirect methods such as cross-linking and peptide NMR have been able to suggest where troponin might interact with F-actin (15Levine B.A. Moir A.J.G. Perry S.V. Eur. J. Biochem. 1988; 172: 389-397Crossref PubMed Scopus (60) Google Scholar). Three model systems have been used to generate actin mutants to examine the interactions of actin with myosin and with the regulatory proteins. Actin from the indirect flight muscle of Drosophila provides useful in vivo information as well as the potential for genetically-derived insights (16Molloy J. Kreuz A. Miller R. Tansey T. Maughan D. Adv. Exp. Med. Biol. 1993; 332: 165-171Crossref PubMed Scopus (17) Google Scholar, 17Bing W. Razzaq A. Sparrow J. Marston S.B. J. Biol. Chem. 1998; 273: 15016-15021Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 18Anson M. Drummond D.R. Geeves M.A. Hennessey E.S. Ritchie M.D. Sparrow J.C. Biophys. J. 1997; 68: 1991-2003Abstract Full Text PDF Scopus (23) Google Scholar, 19Molloy J.E. Burns J.E. Sparrow J.C. Tregear R.T. Kendrick-Jones J. White D.C. Biophys. J. 1995; 68: 298s-303sPubMed Google Scholar). Only a limited amount of actin can be purified from this system, but this was used to investigate tropomyosin function in one study (17Bing W. Razzaq A. Sparrow J. Marston S.B. J. Biol. Chem. 1998; 273: 15016-15021Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The other two model systems that have been used to investigate muscle regulation and actin-myosin interactions are Dictyostelium and Saccharomyces cerevisiae (20Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar, 21Cook R.K. Root D. Miller C. Reisler E. Rubenstein P.A. J. Biol. Chem. 1993; 268: 2410-2415Abstract Full Text PDF PubMed Google Scholar, 22Kron S.J. Drubin D.G. Botstein D. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4466-4470Crossref PubMed Scopus (43) Google Scholar, 23Miller C.J. Reisler E. Biochemistry. 1995; 34: 2694-2700Crossref PubMed Scopus (52) Google Scholar, 24Miller C.J. Wong W.W. Bobkova E. Rubenstein P. Reisler E. Biochemistry. 1996; 35: 16557-16565Crossref PubMed Scopus (64) Google Scholar, 25Adams S. Reisler E. Biochemistry. 1993; 32: 5051-5056Crossref PubMed Scopus (12) Google Scholar, 26Kim E. Miller C.J. Reisler E. Biochemistry. 1996; 35: 16566-16572Crossref PubMed Scopus (46) Google Scholar, 27Saeki K. Sutoh K. Wakabayashi T. Biochemistry. 1996; 35: 14465-14472Crossref PubMed Scopus (29) Google Scholar). Both Dictyostelium and S. cerevisiae can yield mg quantities of actin. Although S. cerevisiae actin has the lowest homology to mammalian muscle actin of these two systems, it is still a relatively high 88% (28Gallwitz D. Sures I. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2546-2550Crossref PubMed Scopus (267) Google Scholar, 29Ng R. Abelson J. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 3912-3916Crossref PubMed Scopus (349) Google Scholar). Yeast actin has been used to study myosin and actin interactions (20Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar, 21Cook R.K. Root D. Miller C. Reisler E. Rubenstein P.A. J. Biol. Chem. 1993; 268: 2410-2415Abstract Full Text PDF PubMed Google Scholar, 22Kron S.J. Drubin D.G. Botstein D. Spudich J.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4466-4470Crossref PubMed Scopus (43) Google Scholar, 23Miller C.J. Reisler E. Biochemistry. 1995; 34: 2694-2700Crossref PubMed Scopus (52) Google Scholar, 24Miller C.J. Wong W.W. Bobkova E. Rubenstein P. Reisler E. Biochemistry. 1996; 35: 16557-16565Crossref PubMed Scopus (64) Google Scholar, 25Adams S. Reisler E. Biochemistry. 1993; 32: 5051-5056Crossref PubMed Scopus (12) Google Scholar), and one report using in vitro motility assays that movement is in the presence of the regulatory proteins E. Miller C.J. Reisler E. Biochemistry. 1996; 35: 16566-16572Crossref PubMed Scopus (46) Google Scholar). a study of in vivo of Drubin D.G. Botstein D. 1992; PubMed Google a of two or charged residues in the actin to suggest that charged interactions are important for tropomyosin and troponin binding to actin M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar, B.A. Moir A.J.G. Perry S.V. Eur. J. Biochem. 1988; 172: 389-397Crossref PubMed Scopus (60) Google Scholar), and binding to F-actin R. C.A. Tobacman L.S. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). this of mutants provides a to the actin surface for of with the regulatory proteins. The yeast actin to examine troponin-tropomyosin Despite the yeast actin and mammalian muscle actin, tropomyosin and troponin are to bind to yeast actin and to the actin-myosin used myosin myosin with in a Ca2+-sensitive to results for muscle actin. actin residues and in have been by M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google to interact with the charged to actin the this to be important in tropomyosin, troponin as well as the troponin C, troponin I, and troponin purified from using the of Tobacman and L.S. R. J. Biol. Chem. 1987; Full Text PDF PubMed Google Scholar). S-1 was from muscle using the of and Nature. PubMed Scopus Google Scholar). muscle was from using the as C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). which is was to C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). yeast as a from and to in at for mutant K315A/E316A or for mutant Drubin D.G. Botstein D. 1992; PubMed Google Scholar). type actin was from a as of Saccharomyces is to the S. cerevisiae actin and it can be in quantities (20Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar). Actin was purified from the wild type and mutant using the from (20Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar). was by or with by the tropomyosin or was at with M. Tobacman L.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (46) Google Scholar). and are in for at and at using a for at The of the was with the of the The C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar, J.D. J. Mol. Biol. PubMed Scopus Google Scholar), which the binding of to a was used to the the affinity of one for binding and a of the that two bind to is by the with 50% of actin the tropomyosin or S-1 was measured using the by and J. Biol. Chem. Full Text PDF PubMed Google Scholar). The used F-actin wild type or muscle myosin tropomyosin, and of or the of was to the Ca2+, the was to the at at The for myosin S-1 The Ca2+ was using the for these L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the was used it has a Ca2+ affinity, which of a of Ca2+ The for was by at using the by the in at upon the of The from each to a binding with a The was and the was used to the the these of the The Ca2+ was for the in the actin The MgATPase to in Tobacman and L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which Ca2+ binding to a of troponin was with and with troponin and troponin the by Tobacman and L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). state Ca2+ the F-actin wild type or tropomyosin, troponin with and a with a The at and the was at The by of to The Ca2+ was as L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), the in the actin for and to from L.S. D. J. Biol. Chem. Full Text PDF PubMed Google was with the by and Proc. Natl. Acad. Sci. U. S. A. 1980; 77: PubMed Scopus Google Scholar). state Ca2+ the F-actin wild type or and The at and the was at The by of to The Ca2+ was as L.S. Biochemistry. 1987; PubMed Scopus Google Scholar), the in the actin for and to from L.S. D. J. Biol. Chem. Full Text PDF PubMed Google The model that residues in the and of actin, residues from to are interact with tropomyosin in the absence of troponin M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar). of the yeast actin and by Drubin D.G. Botstein D. 1992; PubMed Google produce yeast from which actin can be purified and used to investigate this Both of these mutants for in the of tropomyosin and troponin to thin filaments these mutant Ca2+ of the myosin S-1 the from of each type of yeast actin. thin filaments wild type yeast actin the myosin S-1 in to the of the wild type actin, in the absence of Ca2+ the rate was of the rate seen at saturating the presence of saturating Ca2+, regulated thin filaments actin or K315A/E316A actin 50% of the thin S-1 MgATPase rate with wild type actin. The apparent Ca2+ affinity was for the wild type filament, to results seen with muscle actin 9Butters C.A. Tobacman J.B. Tobacman L.S. J. Biol. Chem. 1997; 272: 13196-13202Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). this apparent Ca2+ affinity was not different from results seen for actin it was 4-fold the apparent Ca2+ affinity seen for K315A/E316A these to on mutant actin that the thin S-1 MgATPase rate in the presence of saturating Ca2+ was K315A/E316A actin was other from these by the absence of troponin-tropomyosin that the myosin S-1 using wild type F-actin and K315A/E316A F-actin for wild type and the mutant the decreased MgATPase rate occurs the regulatory proteins are Ca2+ has a on the thin S-1 MgATPase rate L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, L.S. Biochemistry. 1987; PubMed Scopus Google Scholar), and this is true yeast actin is used L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the was to of the actin on the thin S-1 MgATPase rate for wild type actin the presence of saturating was not to a activation by myosin. myosin S-1 was not these This is based upon not that the MgATPase rate was with myosin S-1 to the used in This results with muscle actin C.A. Tobacman J.B. Tobacman L.S. J. Biol. Chem. 1997; 272: 13196-13202Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). We the apparent Ca2+ affinity by troponin to the binding of Ca2+ to the regulatory of troponin the thin filament L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.D. Potter J.D. J. Biol. Chem. 1980; Full Text PDF PubMed Google Scholar). that the actin no on troponin Ca2+ affinity for wild type for K315A/E316A this was not the a in thin filament regulatory affinity L.S. D. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and this have the of the actin was with the regulation and Ca2+ affinity are by Proc. Natl. Acad. Sci. U. S. A. 1980; 77: PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). The that the apparent Ca2+ affinity was for regulated thin filaments wild type or K315A/E316A and The wild type actin is to for muscle actin Proc. Natl. Acad. Sci. U. S. A. 1980; 77: PubMed Scopus Google Scholar). a MgATPase rate using muscle the actin the Ca2+ for wild type actin K315A/E316A actin was not The are the which are with yeast actin and 21Cook R.K. Root D. Miller C. Reisler E. Rubenstein P.A. J. Biol. Chem. 1993; 268: 2410-2415Abstract Full Text PDF PubMed Google Scholar), for to results with regulatory proteins L.S. Biochemistry. PubMed Scopus Google Scholar), muscle the rate as as with actin E. Biochemistry. 1988; PubMed Scopus Google Scholar). Thin filament was used to changes in the interactions with the regulatory proteins that be detectable by a change in affinities for the mutant actin with wild type actin. the binding of the to type of yeast actin in the presence or absence of The at the binds to wild type and K315A/E316A F-actin The of Ca2+ decrease in affinity of the regulatory for F-actin, a decrease to that seen for muscle actin C.A. Bobkova A. Homsher E. Tobacman L.S. J. Biol. Chem. 1997; 272: 14051-14056Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). the of Ca2+, the affinity of the for wild type F-actin which is the affinity seen for the mutant, other the charged residues at and binding affinity for actin by This is the 4-fold in the apparent Ca2+ affinity seen in the The change in binding is to the change in Ca2+ affinity. The wild type in suggest that yeast actin the troponin-tropomyosin in the absence of troponin in S. Cardiac troponin-tropomyosin affinity for yeast actin is to by the methods using muscle actin in the absence of Ca2+, and in the presence of Ca2+ A. A. C.A. Tobacman L.S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in C.A. Tobacman L.S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google The assays are at to produce a rate, the binding be at high to from in a that binding M. Tobacman L.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (46) Google Scholar). It is that this high the of charged actin residues and on binding to actin, and the of the the of on the relative binding affinities of to wild type mutant yeast actin, the muscle tropomyosin was used of tropomyosin, it is to and a of can be C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). muscle to in the absence of actin, binding high is used to C.A. Tobacman L.S. J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google a not for tropomyosin in assays did not the of the actin that tropomyosin to the mutant F-actin to the wild type actin open The that the of the actin did not at This is to one high the of the on the binding of the regulatory to actin. The actin affinity of tropomyosin is for the K315A/E316A F-actin. is the of the actin on tropomyosin binding to yeast actin filaments in the absence of troponin. This was in the presence of binding is in the absence of troponin to be measured used in the presence of troponin The results that the binding of tropomyosin to actin occurs with a of the wild type actin and a of for the K315A/E316A actin filament Although these a of the the of the two affinities which is the in the presence of troponin and three-dimensional reconstructions of muscle thin filaments the position of tropomyosin on the actin filament to be the these two W. Craig R. Vibert P. Nature. 1994; 368: 65-67Crossref PubMed Scopus (275) Google Scholar, 8Lorenz M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar, 14Lehman W. Craig R. Uman P. Vibert P. Biophys. J. 1996; 70 (abstr.): A15Google Scholar). in the of muscle actin M. Tobacman L.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (46) Google Scholar), the presence of myosin S-1 tropomyosin to bind to yeast actin of yeast actin, the binding of tropomyosin is binding is to the of the on affinity these but J. 1998; PubMed Scopus Google from three-dimensional reconstructions and biochemical is that regulation of striated and muscle contraction can be by a three-state model for the thin filament (4McKillop D.F.A. Geeves M.A. Biophys. J. 1993; 65: 693-701Abstract Full Text PDF PubMed Scopus (663) Google Scholar, 5Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (387) Google Scholar, 6Lehman W. Vibert P. Uman P. Craig R. J. Mol. Biol. 1995; 251: 191-196Crossref PubMed Scopus (145) Google Scholar, 7Lehman W. Craig R. Vibert P. Nature. 1994; 368: 65-67Crossref PubMed Scopus (275) Google Scholar, 8Lorenz M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar, 9Butters C.A. Tobacman J.B. Tobacman L.S. J. Biol. Chem. 1997; 272: 13196-13202Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 10Geeves M.A. Lehrer S.S. Biophys. J. 1994; 67: 273-282Abstract Full Text PDF PubMed Scopus (174) Google Scholar, 11Landis C.A. Bobkova A. Homsher E. Tobacman L.S. J. Biol. Chem. 1997; 272: 14051-14056Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Actin mutants one to the actin surface for residues that interact with the regulatory proteins and these three this evidence that yeast actin can as a model system for this process. thin filaments wild type yeast actin myosin S-1 The presence of myosin S-1 binding of tropomyosin to yeast actin Both of these are to in by the of muscle actin L.S. Biochemistry. PubMed Scopus Google of yeast actin M. Tobacman L.S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (46) Google Scholar, L.S. Biochemistry. 1987; PubMed Scopus Google Scholar). muscle actin and wild type yeast actin have affinities for regulated thin filaments have the Ca2+ affinity at the regulatory which actin is These that the regulatory proteins interact with yeast actin in a to muscle actin. Actin residues and are in the in a proposed by M. Poole K.J.V. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google to bind to tropomyosin in the absence of troponin. that tropomyosin binds to filaments of the K315A/E316A as with wild type This that these residues are in tropomyosin but this be The myosin S-1 assays apparent for regulated thin filaments K315A/E316A that was of the apparent Ca2+ affinity of wild type thin in thin filament there was a the affinity of the for the mutant actin was of the affinity for the wild type actin. a for this was the in the the two The high for the binding might the of the two charged actin This was by the binding of tropomyosin to actin, which can be a of muscle tropomyosin is The results that not the in the binding affinities for the wild type actin filament the mutant actin filament. the and Ca2+ binding to F-actin. to the Ca2+ of the myosin S-1 4-fold by the there be a change in the other that is is the suggest that the actin has a on Ca2+ binding to the thin filament is in and the on the Ca2+ of the of the on process. but for such a is by in which a was myosin S-1 MgATPase and troponin with Ca2+ C.A. Tobacman J.B. Tobacman L.S. J. Biol. Chem. 1997; 272: 13196-13202Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). This that using muscle Ca2+ binding to two was to the myosin S-1 MgATPase rate. This be in the presence of the actin It is that the decreased myosin S-1 with K315A/E316A actin the regulatory proteins We suggest that these charged residues in actin interact with the regulatory proteins in the presence of Ca2+ suggest that strongly the and transitions the and the myosin-induced state of the thin filament, and that to the myosin-induced state is required for cross-bridge the of the on actin residues and might the of the regulatory to actin-myosin is that residues and the from the myosin-induced state to the Ca2+ state of the thin filament. to of cross-bridge This not the MgATPase but a report using in vitro motility to examine the yeast actin mutant Bobkova E. Homsher E. Reisler E. Biophys. J. 1997; (abstr.): Scholar). These that motility for F-actin and wild type F-actin but in the presence of the regulatory proteins the was for the thin filaments with the wild cross-bridge The are not to suggest that troponin and tropomyosin interact with of actin. Thin filament reconstructions suggest that tropomyosin the other and the interactions of troponin with actin two actin are K. Sutoh K. Wakabayashi T. Biochemistry. 1996; 35: 14465-14472Crossref PubMed Scopus (29) Google used Dictyostelium actin mutants to that with tropomyosin in the state of the thin filament. (17Bing W. Razzaq A. Sparrow J. Marston S.B. J. Biol. Chem. 1998; 273: 15016-15021Abstract Full Text Full Text PDF PubMed Scopus (44) Google used the limited amount of actin that be purified to that actin mutant has in vitro motility in the presence of tropomyosin (17Bing W. Razzaq A. Sparrow J. Marston S.B. J. Biol. Chem. 1998; 273: 15016-15021Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). The results of and these suggest that of actin in interactions with the regulatory proteins. yeast actin with the regulatory proteins in a to muscle actin and can as a model system for muscle the on the actin residues and or and by to inhibits myosin the regulatory proteins are the mutant, there is a 4-fold decrease in the apparent Ca2+ affinity as well as a 50% MgATPase in the presence of saturating the K315A/E316A tropomyosin binding to actin, it is on the inner domain of F-actin These results suggest that charged residues on the surface of the actin inner domain are important for thin filament activation. We Drubin for the of the K315A/E316A and yeast We Reisler for and for of this