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W2037182210 abstract "Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments. The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2. Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding. These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin. Both of these effects are influenced by which tropomyosin isoform is present. Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function. Tropomyosin is an extended coiled-coil protein that influences actin function by binding longitudinally along thin filaments. The present work compares cardiac tropomyosin and the two tropomyosins from Saccharomyces cerevisiae, TPM1 and TPM2, that are much shorter than vertebrate tropomyosins. Unlike cardiac tropomyosin, the phase of the coiled-coil-forming heptad repeat of TPM2 is discontinuous; it is interrupted by a 4-residue deletion. TPM1 has two such deletions, which flank the 38-residue partial gene duplication that causes TPM1 to span five actins instead of the four of TPM2. Each of the three tropomyosin isoforms modulates actin-myosin interactions, with isoform-specific effects on cooperativity and strength of myosin binding. These different properties can be explained by a model that combines opposite effects, steric hindrance between myosin and tropomyosin when the latter is bound to a subset of its sites on actin, and also indirect, favorable interactions between tropomyosin and myosin, mediated by mutually promoted changes in actin. Both of these effects are influenced by which tropomyosin isoform is present. Finally, the tropomyosins have isoform-specific effects on in vitro sliding speed and on the myosin concentration dependence of this movement, suggesting that non-muscle tropomyosin isoforms exist, at least in part, to modulate myosin function. myosin subfragment 1 heavy meromyosin rhodamine phalloidin bovine cardiac tropomyosin P 1,P 5-di(adenosine 5′)-pentaphosphate 4-morpholinepropanesulfonic acid Tropomyosin is a highly elongated coiled-coil protein that binds to actin filaments in both muscle and non-muscle cells. Tropomyosin, which has many isoforms, stabilizes actin filaments against fragmentation, and by its presence on the thin filament has the potential to influence many aspects of F-actin function. In particular, tropomyosins have complex and incompletely understood effects on actin-myosin interactions. One consistent finding is that vertebrate tropomyosins increase the affinity of myosin subfragment-1 for actin (1Murray J.M. Knox M.K. Trueblood C.E. Weber A. Biochemistry. 1982; 21: 906-915Crossref PubMed Scopus (37) Google Scholar, 2Williams D.L. Greene L.E. Biochemistry. 1983; 22: 2770-2774Crossref PubMed Scopus (44) Google Scholar, 3Geeves M.A. Halsall D.J. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1986; 229: 85-95Crossref PubMed Scopus (19) Google Scholar, 4Lehrer S.S. Golitsina N.L. Geeves M.A. Biochemistry. 1997; 36: 13449-13454Crossref PubMed Scopus (81) Google Scholar, 5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), despite steric hindrance between the preferred binding sites for tropomyosin and myosin S11 when they bind to actin separately (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar, 7Holmes K.C. Biophys. J. 1995; 68: 2-7PubMed Google Scholar, 8Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (380) Google Scholar).Saccharomyces cerevisiae has two tropomyosin isoforms, TPM1 and TPM2, both of them substantially shorter than vertebrate tropomyosins (9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar, 10Liu H. Bretscher A. Cell. 1989; 57: 233-242Abstract Full Text PDF PubMed Scopus (187) Google Scholar). The 199-residue TPM1 is the predominant isoform by expression level, and it spans five actin monomers. TPM2 is 161 residues and spans four actin monomers, whereas vertebrate tropomyosins span either six or seven actin monomers. Like other tropomyosins, TPM1 and TPM2 have the classical heptad repeat that is responsible for coiled-coil formation, in which hydrophobic residues are found in the first and fourth positions of successive groups of seven amino acids. Below we show that, unlike other tropomyosins, this motif is interrupted once (TPM2) or twice (TPM1) in the amino acid sequence; the phase of the heptad pattern shifts, due to four residue deletions. Both the short length and the interrupted heptad pattern of yeast tropomyosins suggest they could significantly differ from vertebrate tropomyosins functionally. In the present report we describe the solution properties of TPM1 and TPM2, in comparison to each other and to muscle tropomyosin. The results indicate that yeast and vertebrate tropomyosins act in an isoform-specific manner to modulate myosin binding to actin, in vitro motility, cooperative interactions between myosin and tropomyosin, and the distribution of thin filament conformational states.DISCUSSIONGenetic experiments in S. cerevisiae imply different functions for TPM1 and TPM2. Deletion of the predominant isoform, TPM1, impairs growth rate and vesicular traffic to the cell surface, and these defects are not corrected by overexpression of TPM2. Also, overexpression of TPM2 but not TPM1 changes budding morphology (9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar). We now report biochemical observations that potentially explain thesein vivo observations, i.e. isoform-specific effects of yeast tropomyosins on binding of myosin to actin. Under cellular conditions where myosin number is limiting and few cross-bridges are bound, Figs. 4 and 5 suggest that TPM1-containing filaments would be more resistant to movement and to cross-bridge attachment than TPM2-containing filaments. More generally, both experiments show that myosin-thin filament interactions depend quantitatively on tropomyosin isoforms. In normal cells, individual actin filaments can be expected to contain a mixture of isoforms in amounts reflecting their relative concentrations (Fig. 2 and Ref. 9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar). It is unknown whether these relative amounts vary depending upon environment, cell cycle, and/or subcellular location. Under conditions where myosin is limiting, the amount of TPM2 could be important for cooperative induction of movement or force.The equilibrium constants summarized in Table II make it possible to comment on how myosin binding to the thin filament is related both to the unusual structural features of yeast tropomyosins and to the specific difference between TPM1 and TPM2. First, the extra stammer in TPM1 is expected to produce more flexibility than for TPM2, and this in principle might have decreased the cooperativity of myosin-thin filament binding. Instead, myosin binding was more cooperative to filaments containing TPM1 than to those with TPM2. Furthermore, back and forth shifts in the actin position of the TPM1 strand were unfavorable (Y ≫ 1), despite the two stammers in each TPM1. These results suggest that the intrinsic stiffness of tropomyosin is not the only property governing the cooperativity of the shifts in its position on the actin surface. As an additional mechanism, we suggest that cooperativity of tropomyosin shifts (and therefore cooperativity of myosin-thin filament binding) is due to localized energy minima for tropomyosin position on actin (the M-, C-, and B-state positions) (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar, 8Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (380) Google Scholar), with an energetic penalty (equalingRT lnY 1/2) for each site along the actin filament where the tropomyosin strand crosses from one minimum to another.The 100- to 10,000-fold effect of myosin on vertebrate tropomyosin-actin binding implies either direct contacts between the two proteins or else an indirect interaction mediated by a mutually promoted change in actin. Experiments using tropomyosin mutants in which this process is greatly suppressed suggest (5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) that the interaction is in fact indirect, because the mutations did not simply weaken myosin-thin filament binding as would have been expected if direct interactions were eliminated. Instead, myosin-thin filament binding exhibited exaggerated cooperativity, as is characteristic of a switch in quaternary structure. Therefore, to explain how tropomyosin and myosin promote each other's binding to actin, it was proposed that the changes in actin that accompany myosin binding (47Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (718) Google Scholar, 48Feng L. Kim E. Lee W.-L. Miller C.J. Kuang B. Reisler E. Rubenstein P. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 49Prochniewicz E. Thomas D.D. Biochemistry. 1999; 38: 14860-14867Crossref PubMed Scopus (24) Google Scholar) result in a strengthening of the association of tropomyosin with the actin inner domain (5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Correspondingly, when tropomyosin is bound to the actin inner domain, it was proposed to promote some of the same changes in actin, thereby increasing the fraction of actin monomers in the M-state (schematically indicated by stripes in Fig. 6).The current results suggest that a major difference between muscle tropomyosin and the yeast isoforms is in the equilibrium constant for actin to convert to the M-state once the tropomyosin is located on the actin inner domain. Schematically, this is the process in Fig. 6 that has an equilibrium value indicated by the product KT× L. (See also Table II, column Equilibrium toward M-state actin.) The equilibrium for conversion to the M-state is biased toward formation for muscle tropomyosin, because KT ×L = 6 ± 3, i.e. > 1, but is less than 1 and biased against M-state formation for TPM1 and TPM2 (KT × L = 0.63 ± 0.06 and 0.32 ± 0.15, respectively), and even further against M-state formation for actin alone: KT0 = 0.17 (from Ref. 5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Describing these measurements another way, myosin alters actin when it binds, and vertebrate tropomyosins interact with the actin inner domain so as to assist this process. Yeast tropomyosins interact more weakly with the actin inner domain and so provide less assistance to myosin binding.Since the N- and C-terminal regions of TPM1 are homologous to those of TPM2, the functional differences between the two proteins are most likely due to the extra duplicated region present within TPM1. From Fig. 5 and Table II, inclusion of this region significantly alters the equilibria among the various thin filament states. The effects of the duplicated region on L (increased), KT(unchanged), and KT × L (increased) can be explained if the extra region either selectively destabilizes tropomyosin binding to the inner domain of non-M-state actin (i.e. destabilizes tropomyosin binding to the intermediate state in Fig. 6) or else selectively strengthens formation of both the C-state and the M-state. The former explanation is more likely, because it is simpler and also because the latter explanation conflicts with the absence of an effect of myosin S1 on the TPM1 versusTPM2 competition data in Fig. 2. In either case, the result would be a tendency for TPM1 to move away from the inner domain position, producing cooperative myosin binding to the thin filament.Further experiments will be required to determine whether the effects of tropomyosin depend not only on tropomyosin isoform but also on actin and/or myosin isoform. However, the strong myosin-tropomyosin interaction observed with muscle tropomyosin does not seem to depend upon the use of muscle actin, since myosin-decorated yeast actin binds to muscle tropomyosin with very high affinity (37Korman V.L. Tobacman L.S. J. Biol. Chem. 1999; 274: 22191-22196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 38Korman V.L. Hatch V. Dixon K. Craig R. Lehman W. Tobacman L.S. J. Biol. Chem. 2000; 275: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Also preliminary experiments (not shown) suggest little difference in TPM2 binding to muscle versus yeast actin. Finally, the myosin isoform may not make much difference either, since even weak myosin binding to actin strengthens tropomyosin attachment to the actin inner domain (25Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).The Fig. 6 model characterizes the effects of tropomyosins on equilibrium actin-myosin binding, rather than the effects of tropomyosins on thin filament sliding. Nevertheless, the model is qualitatively consistent with the in vitro motility data. The myosin concentration requirement for filament sliding was increased by each of the tropomyosins, was increased more by TPM1 than by TPM2, and was particularly increased by cardiac tropomyosin. Each of these observations is consistent with the steric blocking effect that is part of the model, an effect that may be greater for cardiac tropomyosin because it is located on the actin outer domain (the B-state (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar)). Steric blocking is greater for TPM1 than for TPM2 for a different reason, because its movement to the M-state position is more unfavorable (L is greater). On the other hand, it is difficult to relate the model to the effects of tropomyosins on maximal sliding speed, in part because determining the maximum speeds would require higher myosin concentrations than were tested. More significantly, conclusions are difficult because even at maximal speed the densities of myosin attachment are unknown, as are the distributions of thin filament states (C-, M-, etc.) for any of the sliding filament types. However, the model does suggest qualitatively how tropomyosin might affect the maximal speed. Once tropomyosin is bound to the actin inner domain, it affects actin in a manner that affects myosin. Strong myosin binding is tighter (Fig. 5). Also, the apparently faster maximal sliding (Fig. 4) implies that detachment of cycling cross-bridges is faster in the presence of tropomyosin, perhaps by an acceleration of the rate of ADP release from acto-myosin.In summary TPM1, TPM2, and cardiac tropomyosin differ not only in length but also in continuity of the coiled-coil motif. They modulate myosin binding to actin both in the presence and absence of ATP, and each isoform has distinct effects. Their different properties can be explained by a model that combines both steric effects of tropomyosin on myosin binding to actin and also indirect interactions between tropomyosin and myosin, mediated by mutually produced changes in actin. The distinguishable effects of each tropomyosin on the behavior of myosin suggest a biochemical rationale for the existence of tropomyosin isoforms in non-muscle cells. Tropomyosin is a highly elongated coiled-coil protein that binds to actin filaments in both muscle and non-muscle cells. Tropomyosin, which has many isoforms, stabilizes actin filaments against fragmentation, and by its presence on the thin filament has the potential to influence many aspects of F-actin function. In particular, tropomyosins have complex and incompletely understood effects on actin-myosin interactions. One consistent finding is that vertebrate tropomyosins increase the affinity of myosin subfragment-1 for actin (1Murray J.M. Knox M.K. Trueblood C.E. Weber A. Biochemistry. 1982; 21: 906-915Crossref PubMed Scopus (37) Google Scholar, 2Williams D.L. Greene L.E. Biochemistry. 1983; 22: 2770-2774Crossref PubMed Scopus (44) Google Scholar, 3Geeves M.A. Halsall D.J. Proc. R. Soc. Lond. Ser. B Biol. Sci. 1986; 229: 85-95Crossref PubMed Scopus (19) Google Scholar, 4Lehrer S.S. Golitsina N.L. Geeves M.A. Biochemistry. 1997; 36: 13449-13454Crossref PubMed Scopus (81) Google Scholar, 5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), despite steric hindrance between the preferred binding sites for tropomyosin and myosin S11 when they bind to actin separately (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar, 7Holmes K.C. Biophys. J. 1995; 68: 2-7PubMed Google Scholar, 8Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (380) Google Scholar). Saccharomyces cerevisiae has two tropomyosin isoforms, TPM1 and TPM2, both of them substantially shorter than vertebrate tropomyosins (9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar, 10Liu H. Bretscher A. Cell. 1989; 57: 233-242Abstract Full Text PDF PubMed Scopus (187) Google Scholar). The 199-residue TPM1 is the predominant isoform by expression level, and it spans five actin monomers. TPM2 is 161 residues and spans four actin monomers, whereas vertebrate tropomyosins span either six or seven actin monomers. Like other tropomyosins, TPM1 and TPM2 have the classical heptad repeat that is responsible for coiled-coil formation, in which hydrophobic residues are found in the first and fourth positions of successive groups of seven amino acids. Below we show that, unlike other tropomyosins, this motif is interrupted once (TPM2) or twice (TPM1) in the amino acid sequence; the phase of the heptad pattern shifts, due to four residue deletions. Both the short length and the interrupted heptad pattern of yeast tropomyosins suggest they could significantly differ from vertebrate tropomyosins functionally. In the present report we describe the solution properties of TPM1 and TPM2, in comparison to each other and to muscle tropomyosin. The results indicate that yeast and vertebrate tropomyosins act in an isoform-specific manner to modulate myosin binding to actin, in vitro motility, cooperative interactions between myosin and tropomyosin, and the distribution of thin filament conformational states. DISCUSSIONGenetic experiments in S. cerevisiae imply different functions for TPM1 and TPM2. Deletion of the predominant isoform, TPM1, impairs growth rate and vesicular traffic to the cell surface, and these defects are not corrected by overexpression of TPM2. Also, overexpression of TPM2 but not TPM1 changes budding morphology (9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar). We now report biochemical observations that potentially explain thesein vivo observations, i.e. isoform-specific effects of yeast tropomyosins on binding of myosin to actin. Under cellular conditions where myosin number is limiting and few cross-bridges are bound, Figs. 4 and 5 suggest that TPM1-containing filaments would be more resistant to movement and to cross-bridge attachment than TPM2-containing filaments. More generally, both experiments show that myosin-thin filament interactions depend quantitatively on tropomyosin isoforms. In normal cells, individual actin filaments can be expected to contain a mixture of isoforms in amounts reflecting their relative concentrations (Fig. 2 and Ref. 9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar). It is unknown whether these relative amounts vary depending upon environment, cell cycle, and/or subcellular location. Under conditions where myosin is limiting, the amount of TPM2 could be important for cooperative induction of movement or force.The equilibrium constants summarized in Table II make it possible to comment on how myosin binding to the thin filament is related both to the unusual structural features of yeast tropomyosins and to the specific difference between TPM1 and TPM2. First, the extra stammer in TPM1 is expected to produce more flexibility than for TPM2, and this in principle might have decreased the cooperativity of myosin-thin filament binding. Instead, myosin binding was more cooperative to filaments containing TPM1 than to those with TPM2. Furthermore, back and forth shifts in the actin position of the TPM1 strand were unfavorable (Y ≫ 1), despite the two stammers in each TPM1. These results suggest that the intrinsic stiffness of tropomyosin is not the only property governing the cooperativity of the shifts in its position on the actin surface. As an additional mechanism, we suggest that cooperativity of tropomyosin shifts (and therefore cooperativity of myosin-thin filament binding) is due to localized energy minima for tropomyosin position on actin (the M-, C-, and B-state positions) (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar, 8Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (380) Google Scholar), with an energetic penalty (equalingRT lnY 1/2) for each site along the actin filament where the tropomyosin strand crosses from one minimum to another.The 100- to 10,000-fold effect of myosin on vertebrate tropomyosin-actin binding implies either direct contacts between the two proteins or else an indirect interaction mediated by a mutually promoted change in actin. Experiments using tropomyosin mutants in which this process is greatly suppressed suggest (5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) that the interaction is in fact indirect, because the mutations did not simply weaken myosin-thin filament binding as would have been expected if direct interactions were eliminated. Instead, myosin-thin filament binding exhibited exaggerated cooperativity, as is characteristic of a switch in quaternary structure. Therefore, to explain how tropomyosin and myosin promote each other's binding to actin, it was proposed that the changes in actin that accompany myosin binding (47Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (718) Google Scholar, 48Feng L. Kim E. Lee W.-L. Miller C.J. Kuang B. Reisler E. Rubenstein P. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 49Prochniewicz E. Thomas D.D. Biochemistry. 1999; 38: 14860-14867Crossref PubMed Scopus (24) Google Scholar) result in a strengthening of the association of tropomyosin with the actin inner domain (5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Correspondingly, when tropomyosin is bound to the actin inner domain, it was proposed to promote some of the same changes in actin, thereby increasing the fraction of actin monomers in the M-state (schematically indicated by stripes in Fig. 6).The current results suggest that a major difference between muscle tropomyosin and the yeast isoforms is in the equilibrium constant for actin to convert to the M-state once the tropomyosin is located on the actin inner domain. Schematically, this is the process in Fig. 6 that has an equilibrium value indicated by the product KT× L. (See also Table II, column Equilibrium toward M-state actin.) The equilibrium for conversion to the M-state is biased toward formation for muscle tropomyosin, because KT ×L = 6 ± 3, i.e. > 1, but is less than 1 and biased against M-state formation for TPM1 and TPM2 (KT × L = 0.63 ± 0.06 and 0.32 ± 0.15, respectively), and even further against M-state formation for actin alone: KT0 = 0.17 (from Ref. 5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Describing these measurements another way, myosin alters actin when it binds, and vertebrate tropomyosins interact with the actin inner domain so as to assist this process. Yeast tropomyosins interact more weakly with the actin inner domain and so provide less assistance to myosin binding.Since the N- and C-terminal regions of TPM1 are homologous to those of TPM2, the functional differences between the two proteins are most likely due to the extra duplicated region present within TPM1. From Fig. 5 and Table II, inclusion of this region significantly alters the equilibria among the various thin filament states. The effects of the duplicated region on L (increased), KT(unchanged), and KT × L (increased) can be explained if the extra region either selectively destabilizes tropomyosin binding to the inner domain of non-M-state actin (i.e. destabilizes tropomyosin binding to the intermediate state in Fig. 6) or else selectively strengthens formation of both the C-state and the M-state. The former explanation is more likely, because it is simpler and also because the latter explanation conflicts with the absence of an effect of myosin S1 on the TPM1 versusTPM2 competition data in Fig. 2. In either case, the result would be a tendency for TPM1 to move away from the inner domain position, producing cooperative myosin binding to the thin filament.Further experiments will be required to determine whether the effects of tropomyosin depend not only on tropomyosin isoform but also on actin and/or myosin isoform. However, the strong myosin-tropomyosin interaction observed with muscle tropomyosin does not seem to depend upon the use of muscle actin, since myosin-decorated yeast actin binds to muscle tropomyosin with very high affinity (37Korman V.L. Tobacman L.S. J. Biol. Chem. 1999; 274: 22191-22196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 38Korman V.L. Hatch V. Dixon K. Craig R. Lehman W. Tobacman L.S. J. Biol. Chem. 2000; 275: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Also preliminary experiments (not shown) suggest little difference in TPM2 binding to muscle versus yeast actin. Finally, the myosin isoform may not make much difference either, since even weak myosin binding to actin strengthens tropomyosin attachment to the actin inner domain (25Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar).The Fig. 6 model characterizes the effects of tropomyosins on equilibrium actin-myosin binding, rather than the effects of tropomyosins on thin filament sliding. Nevertheless, the model is qualitatively consistent with the in vitro motility data. The myosin concentration requirement for filament sliding was increased by each of the tropomyosins, was increased more by TPM1 than by TPM2, and was particularly increased by cardiac tropomyosin. Each of these observations is consistent with the steric blocking effect that is part of the model, an effect that may be greater for cardiac tropomyosin because it is located on the actin outer domain (the B-state (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar)). Steric blocking is greater for TPM1 than for TPM2 for a different reason, because its movement to the M-state position is more unfavorable (L is greater). On the other hand, it is difficult to relate the model to the effects of tropomyosins on maximal sliding speed, in part because determining the maximum speeds would require higher myosin concentrations than were tested. More significantly, conclusions are difficult because even at maximal speed the densities of myosin attachment are unknown, as are the distributions of thin filament states (C-, M-, etc.) for any of the sliding filament types. However, the model does suggest qualitatively how tropomyosin might affect the maximal speed. Once tropomyosin is bound to the actin inner domain, it affects actin in a manner that affects myosin. Strong myosin binding is tighter (Fig. 5). Also, the apparently faster maximal sliding (Fig. 4) implies that detachment of cycling cross-bridges is faster in the presence of tropomyosin, perhaps by an acceleration of the rate of ADP release from acto-myosin.In summary TPM1, TPM2, and cardiac tropomyosin differ not only in length but also in continuity of the coiled-coil motif. They modulate myosin binding to actin both in the presence and absence of ATP, and each isoform has distinct effects. Their different properties can be explained by a model that combines both steric effects of tropomyosin on myosin binding to actin and also indirect interactions between tropomyosin and myosin, mediated by mutually produced changes in actin. The distinguishable effects of each tropomyosin on the behavior of myosin suggest a biochemical rationale for the existence of tropomyosin isoforms in non-muscle cells. Genetic experiments in S. cerevisiae imply different functions for TPM1 and TPM2. Deletion of the predominant isoform, TPM1, impairs growth rate and vesicular traffic to the cell surface, and these defects are not corrected by overexpression of TPM2. Also, overexpression of TPM2 but not TPM1 changes budding morphology (9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar). We now report biochemical observations that potentially explain thesein vivo observations, i.e. isoform-specific effects of yeast tropomyosins on binding of myosin to actin. Under cellular conditions where myosin number is limiting and few cross-bridges are bound, Figs. 4 and 5 suggest that TPM1-containing filaments would be more resistant to movement and to cross-bridge attachment than TPM2-containing filaments. More generally, both experiments show that myosin-thin filament interactions depend quantitatively on tropomyosin isoforms. In normal cells, individual actin filaments can be expected to contain a mixture of isoforms in amounts reflecting their relative concentrations (Fig. 2 and Ref. 9Drees B. Brown C. Barrell B.G. Bretscher A. J. Cell Biol. 1995; 128: 383-392Crossref PubMed Scopus (120) Google Scholar). It is unknown whether these relative amounts vary depending upon environment, cell cycle, and/or subcellular location. Under conditions where myosin is limiting, the amount of TPM2 could be important for cooperative induction of movement or force. The equilibrium constants summarized in Table II make it possible to comment on how myosin binding to the thin filament is related both to the unusual structural features of yeast tropomyosins and to the specific difference between TPM1 and TPM2. First, the extra stammer in TPM1 is expected to produce more flexibility than for TPM2, and this in principle might have decreased the cooperativity of myosin-thin filament binding. Instead, myosin binding was more cooperative to filaments containing TPM1 than to those with TPM2. Furthermore, back and forth shifts in the actin position of the TPM1 strand were unfavorable (Y ≫ 1), despite the two stammers in each TPM1. These results suggest that the intrinsic stiffness of tropomyosin is not the only property governing the cooperativity of the shifts in its position on the actin surface. As an additional mechanism, we suggest that cooperativity of tropomyosin shifts (and therefore cooperativity of myosin-thin filament binding) is due to localized energy minima for tropomyosin position on actin (the M-, C-, and B-state positions) (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar, 8Vibert P. Craig R. Lehman W. J. Mol. Biol. 1997; 266: 8-14Crossref PubMed Scopus (380) Google Scholar), with an energetic penalty (equalingRT lnY 1/2) for each site along the actin filament where the tropomyosin strand crosses from one minimum to another. The 100- to 10,000-fold effect of myosin on vertebrate tropomyosin-actin binding implies either direct contacts between the two proteins or else an indirect interaction mediated by a mutually promoted change in actin. Experiments using tropomyosin mutants in which this process is greatly suppressed suggest (5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) that the interaction is in fact indirect, because the mutations did not simply weaken myosin-thin filament binding as would have been expected if direct interactions were eliminated. Instead, myosin-thin filament binding exhibited exaggerated cooperativity, as is characteristic of a switch in quaternary structure. Therefore, to explain how tropomyosin and myosin promote each other's binding to actin, it was proposed that the changes in actin that accompany myosin binding (47Kouyama T. Mihashi K. Eur. J. Biochem. 1981; 114: 33-38Crossref PubMed Scopus (718) Google Scholar, 48Feng L. Kim E. Lee W.-L. Miller C.J. Kuang B. Reisler E. Rubenstein P. J. Biol. Chem. 1997; 272: 16829-16837Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 49Prochniewicz E. Thomas D.D. Biochemistry. 1999; 38: 14860-14867Crossref PubMed Scopus (24) Google Scholar) result in a strengthening of the association of tropomyosin with the actin inner domain (5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Correspondingly, when tropomyosin is bound to the actin inner domain, it was proposed to promote some of the same changes in actin, thereby increasing the fraction of actin monomers in the M-state (schematically indicated by stripes in Fig. 6). The current results suggest that a major difference between muscle tropomyosin and the yeast isoforms is in the equilibrium constant for actin to convert to the M-state once the tropomyosin is located on the actin inner domain. Schematically, this is the process in Fig. 6 that has an equilibrium value indicated by the product KT× L. (See also Table II, column Equilibrium toward M-state actin.) The equilibrium for conversion to the M-state is biased toward formation for muscle tropomyosin, because KT ×L = 6 ± 3, i.e. > 1, but is less than 1 and biased against M-state formation for TPM1 and TPM2 (KT × L = 0.63 ± 0.06 and 0.32 ± 0.15, respectively), and even further against M-state formation for actin alone: KT0 = 0.17 (from Ref. 5Tobacman L.S. Butters C.A. J. Biol. Chem. 2000; 275: 27587-27593Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Describing these measurements another way, myosin alters actin when it binds, and vertebrate tropomyosins interact with the actin inner domain so as to assist this process. Yeast tropomyosins interact more weakly with the actin inner domain and so provide less assistance to myosin binding. Since the N- and C-terminal regions of TPM1 are homologous to those of TPM2, the functional differences between the two proteins are most likely due to the extra duplicated region present within TPM1. From Fig. 5 and Table II, inclusion of this region significantly alters the equilibria among the various thin filament states. The effects of the duplicated region on L (increased), KT(unchanged), and KT × L (increased) can be explained if the extra region either selectively destabilizes tropomyosin binding to the inner domain of non-M-state actin (i.e. destabilizes tropomyosin binding to the intermediate state in Fig. 6) or else selectively strengthens formation of both the C-state and the M-state. The former explanation is more likely, because it is simpler and also because the latter explanation conflicts with the absence of an effect of myosin S1 on the TPM1 versusTPM2 competition data in Fig. 2. In either case, the result would be a tendency for TPM1 to move away from the inner domain position, producing cooperative myosin binding to the thin filament. Further experiments will be required to determine whether the effects of tropomyosin depend not only on tropomyosin isoform but also on actin and/or myosin isoform. However, the strong myosin-tropomyosin interaction observed with muscle tropomyosin does not seem to depend upon the use of muscle actin, since myosin-decorated yeast actin binds to muscle tropomyosin with very high affinity (37Korman V.L. Tobacman L.S. J. Biol. Chem. 1999; 274: 22191-22196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 38Korman V.L. Hatch V. Dixon K. Craig R. Lehman W. Tobacman L.S. J. Biol. Chem. 2000; 275: 22470-22478Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Also preliminary experiments (not shown) suggest little difference in TPM2 binding to muscle versus yeast actin. Finally, the myosin isoform may not make much difference either, since even weak myosin binding to actin strengthens tropomyosin attachment to the actin inner domain (25Rosol M. Lehman W. Craig R. Landis C. Butters C. Tobacman L.S. Biophys. J. 2000; 78: 908-917Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The Fig. 6 model characterizes the effects of tropomyosins on equilibrium actin-myosin binding, rather than the effects of tropomyosins on thin filament sliding. Nevertheless, the model is qualitatively consistent with the in vitro motility data. The myosin concentration requirement for filament sliding was increased by each of the tropomyosins, was increased more by TPM1 than by TPM2, and was particularly increased by cardiac tropomyosin. Each of these observations is consistent with the steric blocking effect that is part of the model, an effect that may be greater for cardiac tropomyosin because it is located on the actin outer domain (the B-state (6Lehman W. Hatch V. Korman V.L. Rosol M. Thomas L.T. Maytum R. Geeves M.A. Van Eyk J.E. Tobacman L.S. Craig R. J. Mol. Biol. 2000; 302: 593-606Crossref PubMed Scopus (215) Google Scholar)). Steric blocking is greater for TPM1 than for TPM2 for a different reason, because its movement to the M-state position is more unfavorable (L is greater). On the other hand, it is difficult to relate the model to the effects of tropomyosins on maximal sliding speed, in part because determining the maximum speeds would require higher myosin concentrations than were tested. More significantly, conclusions are difficult because even at maximal speed the densities of myosin attachment are unknown, as are the distributions of thin filament states (C-, M-, etc.) for any of the sliding filament types. However, the model does suggest qualitatively how tropomyosin might affect the maximal speed. Once tropomyosin is bound to the actin inner domain, it affects actin in a manner that affects myosin. Strong myosin binding is tighter (Fig. 5). Also, the apparently faster maximal sliding (Fig. 4) implies that detachment of cycling cross-bridges is faster in the presence of tropomyosin, perhaps by an acceleration of the rate of ADP release from acto-myosin. In summary TPM1, TPM2, and cardiac tropomyosin differ not only in length but also in continuity of the coiled-coil motif. They modulate myosin binding to actin both in the presence and absence of ATP, and each isoform has distinct effects. Their different properties can be explained by a model that combines both steric effects of tropomyosin on myosin binding to actin and also indirect interactions between tropomyosin and myosin, mediated by mutually produced changes in actin. The distinguishable effects of each tropomyosin on the behavior of myosin suggest a biochemical rationale for the existence of tropomyosin isoforms in non-muscle cells. We thank Drs. Carolyn Cohen and Jerry Brown for comments regarding discontinuities in coiled-coil heptad repeats." @default.
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W2037182210 title "Modulation of Myosin Function by Isoform-specific Properties ofSaccharomyces cerevisiae and Muscle Tropomyosins" @default.
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