Ultra short yeast tropomyosins show novel myosin regulation.
Tropomyosin (Tm) is an alpha-helical coiled-coil actin-binding protein present in all eukaryotes from yeast to man. Its functional role has been best described in muscle regulation; however its much wider role in cytoskeletal actin regulation is still to be clarified. Isoforms vary in size from 284 or 248 amino acids in vertebrates, to 199 and 161 amino acids in yeast, spanning from 7 to 4 actin binding sites respectively. In Saccharomyces cerevisiae, the larger yTm1 protein is produced by an internal 38-amino acid duplication, corresponding to a single actin-binding site. We have produced an ultra-short Tm with only 125 amino acids by removing both of the 38 amino acid repeats from yTm1, with the addition of an Ala-Ser extension used to mimic the essential N-terminal acetylation. This short Tm, and an M1T mutant of it, bind to actin with a similar affinity to most Tms previously studied (K(50%) approximately 0.5 microm). However, an equilibrium fluorescence binding assay shows a much greater inhibition of myosin binding to actin than any previously studied Tm. Actin cosedimentation assays show this is caused by direct competition for binding to actin. The M1T mutant shows a reduced inhibition, probably due to weaker end-to-end interactions making it easier for myosin to displace Tm. All previously characterized Tms, although able to sterically block the myosin-binding site, are able to bind to actin along with myosin. By showing that Tm can compete directly with myosin for the same binding site these new Tms provide direct evidence for the steric blocking model.
Maytum
Robin
R
School of Biological and Chemical Sciences, Queen Mary, University of London, E1 4NS, United Kingdom. r.maytum@qmul.ac.uk
Hatch
Victoria
V
Konrad
Manfred
M
Lehman
William
W
Geeves
Michael A
MA
D000107:Acetylation; D000199:Actins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003599:Cytoskeleton / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000199:Actins; D029701:Saccharomyces cerevisiae Proteins; C490772:TPM1 protein, S cerevisiae; D014335:Tropomyosin; D009218:Myosins