Mapping the functional domains within the carboxyl terminus of alpha-tropomyosin encoded by the alternatively spliced ninth exon.
Tropomyosins are highly conserved, coiled-coil actin binding proteins found in most eukaryotic cells. Striated and smooth muscle alpha-tropomyosins differ by the regions encoded by exons 2 and 9. Unacetylated smooth tropomyosin expressed in Escherichia coli binds actin with high affinity, whereas unacetylated striated tropomyosin requires troponin, found only in striated muscle, for strong actin binding. The residues encoded by exon 9 cause these differences (Cho, Y.-J., and Hitchcock-DeGregori, S. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10153-10157). We mapped the functional domains encoded by the alpha-tropomyosin exon 9a (striated muscle-specific) and 9d (constitutively expressed), by measuring actin binding and regulation of the actomyosin MgATPase by tropomyosin exon 9 chimeras and truncation mutants expressed in E. coli. We have shown that: 1) the carboxyl-terminal nine residues define the actin affinity of unacetylated tropomyosin; 2) in the presence of Ca2+, the entire exon 9a is required for troponin to promote fully high affinity actin binding; 3) the first 18 residues encoded by exon 9a are critical for the interaction of troponin with tropomyosin on the thin filament, even in the absence of Ca2+. The results give new insight into the structural requirements of tropomyosin for thin filament assembly and regulatory function.
Hammell
R L
RL
Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Hitchcock-DeGregori
S E
SE
D000205:Actomyosin / Q000378:metabolism; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli; D005091:Exons; D014644:Genetic Variation; D007700:Kinetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D010957:Plasmids; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D014335:Tropomyosin; D000205:Actomyosin; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium