Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G)

A major function of tropomyosin (TPM) in nonmuscle cells may be stabilizati on of F-actin by binding longitudinally along the actin filament axis. Howe ver, no clear evidence exists in vitro that TPM can significantly affect th e critical concentration of actin, We previously made a polymerization-defe ctive mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degreesC but will in the presence of phalloidin or beryllium fluorid e, With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degreesC with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac trop omyosin), indicating the importance of the ends in polymerization rescue. I n the presence of TPM, the apparent critical concentration of actin is 5.5 muM, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 muM). Non N-acetylated TPMs did not rescue GG-actin polymerization, The TPMs did not prevent cold-induced depolymerization of GG F-actin, TPM-dependent GG-actin polymerization did not occur at temperat ures below 20 degreesC, Polymerization rescue may depend initially on the c apture of unstable GG-F-actin oligomers by the TPM, resulting in the streng thening of actin monomer-monomer contacts along the filament axis.