Functional Homodimers and Heterodimers of Recombinant Smooth Muscle Tropomyosin

Abstract

Skeletal and smooth muscle tropomyosin (Tm) require acetylation of their N termini to bind strongly to actin. Tm containing an N-terminal alanine serine (AS) extension to mimic acetylation, has been widely used to increase binding. The current study investigates the ability of an N-terminal AS extension to mimic native acetylation for both αα and ββ smooth Tm homodimers. We show that : 1) ASα-Tm binds actin 100 fold tighter than α-Tm, and 2 fold tighter than native smooth αβ-Tm; 2) β-Tm requires an AS extension to bind actin; 3) ASβ-Tm binds actin 10 fold weaker than ASα-Tm. Tm is present in smooth muscle tissues as >95% heterodimer, therefore we studied the binding of recombinant αβ heterodimers with different AS extensions. This study shows that recombinant Tm requires an AS extension on both α and β chains to bind like native Tm and that the α chain contributes more to actin binding than the β chain. Once assembled onto an actin filament all smooth muscle Tms regulate S1 binding to actin Tm in the same way, irrespective of the presence of an AS extension.Tropomyosin (Tm) is an actin binding, α-helical, coiled coil protein dimer which binds along the length of actin filaments in both muscle and non-muscle cells, and thus cooperatively regulates the interaction of actin with myosin heads. Muscle cells contain Tm expressed via two genes Tm1 and Tm2 (α and β), and isoform diversity (smooth and skeletal) results from the alternative splicing of the two genes. Each monomer of the dimer coiled-coil contains 284 amino acid residues (1-3).In skeletal muscle tissue Tm is present predominantly as a mixture of αβ heterodimer and αα homodimer (4-6). ββ is less stable and is much less common in muscle tissues (5,6). Studies involving guanidinium hydrochloride dissociation of native homodimer chains and anion exchange chromatography have shown that smooth muscle contains roughly equal amounts of α and β chains, more than 95% of which is present as heterodimer (7-9). Thermal unfolding studies also show that the heterodimer is preferentially formed at temperatures below its unfolding transition (9). Preference of the heterodimer is not fully understood, however viscosity and thermal unfolding measurements indicate that the strength of the end-to-end interaction of the smooth αβ heterodimer is much higher than that of the αα and ββ homodimers which have about the same strength of end-to-end interaction (7,8,10). The strength of end-toend interactions in the heterodimer might affect the cooperative behaviour of the tropomyosin, and thus might provide a functional advantage for the predominance of heterodimer in native smooth muscle. The ability to be able to assemble in vitro heterodimers would be very valuable. Such ability could be used to monitor the behaviour of a single α or β chain on the dimer via

Acetylation regulates tropomyosin function in the fission yeastSchizosaccharomyces pombe

“…These data are consistent with the finding that only Tms with a low K T (i.e. smooth muscle Tm and fibroblast Tm) are capable of complementing Cdc8 function in vivo, providing strong evidence that unlike the budding yeast Tms, Cdc8 has essential roles in regulating myosin function in addition to stabilising actin filaments (Balasubramanian et al, 1992;Coulton et al, 2006;Maytum et al, 2001).…”

“…We have not yet determined whether any of the nonendogenous Tms are acetylated in fission yeast, but the fact that only smooth Tm can bind to actin in the absence of Nterminal modification whereas Tpm1, Tpm2 and skeletal Tm do not (Coulton et al, 2006;Maytum et al, 2001) may explain why only smooth muscle Tm was able to function in yeast (Fig. 4A,B).…”

N-terminal acetylome analyses and functional insights of the N-terminal acetyltransferase NatB

“…Previous studies have observed that an increment in tropomyosin expression partially restores functional defects in actin stress fibers (31,37). In contrast, Tpm1 overexpression in wildtype HeLa cells significantly reduced the area and number of focal adhesions, in agreement with other expressed tropomyosin isoforms (38) and suggestive of a compromised Tpm1 function, most probably by its reduced Nt-acetylation.…”

“…This recovery was not observed when Tpm1 was overexpressed in cells that were treated with latrunculin B, which caused the disorganization of the actin network and impaired cellular motility. The expression of Tpm1 with a dipeptide N-terminal extension, mimicking the structural effects of Nt-acetylation (37), restored the cytoskeletal and motility defects induced by hNatB depletion (Fig. S4).…”

“…It was previously shown that NatB-mediated Nt-acetylation of Tpm1 is important for the formation of stable yeast F-actin structures (10,31). In the case of Tpm1, Nt-acetylation can be mimicked by the expression of an amino-terminally extended form of Tpm1 in vitro (37). Therefore, and because our proteomics data indicated only a partial reduction in the degree of Nt-acetylation of hNatB substrates when knocking down hNatB, we reasoned that overexpression of Tpm1 in hNatB-depleted HeLa cells may restore the function of actomyosin microfibrils.…”