Modulation of Myosin Function by Isoform-specific Properties ofSaccharomyces cerevisiae and Muscle Tropomyosins

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 isoformspecific 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 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 (1-5), despite steric hindrance between the preferred binding sites for tropomyosin and myosin S1 1 when they bind to actin separately (6 -8). Saccharomyces cerevisiae has two tropomyosin isoforms, TPM1 and TPM2, both of them substantially shorter than vertebrate tropomyosins (9, 10). 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 s...

The Troponin Tail Domain Promotes a Conformational State of the Thin Filament That Suppresses Myosin Activity

“…In parallel with the myosin S1 ATPase results, TnT-(1-153) decreased the heavy meromyosin-propelled sliding speed of actin-tropomyosin filaments, and this inhibitory effect was much larger than the effect of tropomyosin alone. (The illustrated effects of tropomyosin alone resemble findings in an earlier report (31).) For all tested concentrations of heavy meromyosin that were attached to the sliding surface, the observed thin filament sliding speed was decreased when TnT-(1-153) was added to the actin-tropomyosin.…”

Cardiomyopathic Tropomyosin Mutations That Increase Thin Filament Ca2+ Sensitivity and Tropomyosin N-domain Flexibility

“…Instead, it appears that the tendency of the tropomyosin strand to shift position on many rather than few adjacent actins (3) is preserved in the presence of the mutations. The explanation for this apparent discrepancy may be that the cooperative shifting of the tropomyosin strand depends only in part on tropomyosin (or troponin-tropomyosin) stiffness; it also depends on the shape of the free energy profile for tropomyosin contacting all possible positions across the actin filament surface (32,65), and this energy profile depends on actin-tropomyosin interactions (5). In the M-state position, these interactions are very tight, and tropomyosin binds with affinity Ͼ Ͼ 10 9 M Ϫ1 (66).…”

“…Actin (30) and myosin S1 (31) were obtained from rabbit fast skeletal muscle. Recombinant control and mutant bovine tropomyosins were expressed in DE3 cells using vector pET3d, and purified to homogeneity as described (32). A63V or K70T mutations were introduced into cDNA encoding rat striated muscle ␣-tropomyosin by the same PCR-based approach used previously to create other missense mutations (33).…”

Tropomyosin isoforms and reagents

“…10,11 The actin depolymerizing factor (ADF) and filamin compete with Tm for binding to actin filaments [12][13][14][15] in a Tm isoform specific manner. 16 Tms have also been shown to control the activity of myosin motors [17][18][19][20] and specify the intracellular sorting of myosin II in a Tm isoform specific manner. 16,21 Tm and caldesmon can also regulate myosin II activity in smooth muscle cells.…”