Actomyosin regulatory properties of yeast tropomyosin are dependent upon N-terminal modification.
The yeast tropomyosin 1 gene (TPM1) encodes the major isoform of the two tropomyosins (Tm) found in yeast. The gene has been expressed in E. coli and the protein purified. The gene product (yTm1) is a 199-amino acid protein that has a low affinity for actin compared to the native yTm1 purified from yeast. Mass spectrometry shows that the native protein is acetylated while the recombinant protein is not. A series of yTm1 N-terminal constructs were made with either an Ala-Ser dipeptide extension previously shown to restore actin binding to skeletal muscle Tm or the natural extension found in fibroblast Tm 5a/b. All constructs bound actin tightly and showed similar CD spectra and thermal stability. All constructs induced cooperativity in the equilibrium binding of myosin subfragment 1, to actin but the binding curves differed significantly between the constructs. The apparent cooperative unit size (n) and closed/open equilibrium (K(T)) were determined using a fluorescence titration technique [Maytum et al. (1998) Biophys. J. 74, A347]. The data could be accounted for by changes in K(T) (0.1-1) with no change in n. Values of n were approximately twice the structural unit size (5 actin sites). The presence of yTm on actin had little effect upon the overall affinity of S1 for actin despite showing an ability to regulate the acto-myosin interaction. These results show that the short yTm can aid our understanding of actomyosin regulation and that the N-terminus of Tm has a major influence upon its regulatory properties.
Maytum
R
R
Department of Biosciences, University of Kent at Canterbury, Canterbury, U.K. rmm@ukc.ac.uk
Geeves
M A
MA
Konrad
M
M
D000205:Actomyosin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D029721:Drosophila Proteins; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors / Q000138:chemical synthesis; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics; D014335:Tropomyosin / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
D029721:Drosophila Proteins; D005656:Fungal Proteins; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; C490772:TPM1 protein, S cerevisiae; C097163:Tm2 protein, Drosophila; D014335:Tropomyosin; D000205:Actomyosin