Evidence for a direct but sequential binding of titin to tropomyosin and actin filaments.
Titin is a giant molecule that spans half a sarcomere, establishing several specific bindings with both structural and contractile myofibrillar elements. It has been demonstrated that this giant protein plays a major role in striated muscle cell passive tension and contractile filament alignment. The in vitro interaction of titin with a new partner (tropomyosin) reported here is reinforced by our recent in vitro motility study using reconstituted Ca-regulated thin filaments, myosin and a native 800-kDa titin fragment. In the presence of the tropomyosin-troponin complex, the actin filament movement onto coated S1 is improved by the titin fragment. Here, we found that two purified native titin fragments of 150 and 800 kDa, covering respectively the N1-line and the N2-line/PEVK region in the I-band and known to contain actin-binding sites, directly bind tropomyosin in the absence of actin. We have also shown that binding of the 800-kDa fragment with filamentous actin inhibited the subsequent interaction of tropomyosin with actin, as judged by cosedimentation. However, this was not the case if the complex of actin and tropomyosin was formed before the addition of the 800-kDa fragment. We thus conclude that a sequential arrangement of contacts exists between parts of the titin I-band region, tropomyosin and actin in the thin filament.
Raynaud
Fabrice
F
UMR 5539, cc107, University of Montpellier 2 place Eugéne Bataillon, 34090 Montpellier, France. raynaudf@univ-montp2.fr
Astier
Catherine
C
Benyamin
Yves
Y
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D064211:Connectin; D046911:Macromolecular Substances; D009038:Motion; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000737:chemistry; D009363:Neoplasm Proteins / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D014335:Tropomyosin / Q000378:metabolism*
D064211:Connectin; D046911:Macromolecular Substances; D009124:Muscle Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D014335:Tropomyosin; C081763:sarcoma-associated antigen S1; D011494:Protein Kinases; D002118:Calcium