F-actin-like ATPase activity in a polymerization-defective mutant yeast actin (V266G/L267G).
Polymerization increases a low level G-actin ATPase activity yielding ADP-P(i) F-actin and then ADP F-actin following release of P(i). By monitoring P(i) release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG. In this mutant, two hydrophobic residues at the tip of a proposed hydrophobic plug between actin subdomains 3 and 4, Val(266) and Leu(267), were mutated to Gly. Although GG-actin does not polymerize by itself in vitro, GG cells are viable. We show that GG-actin ATPase activity increases under normal polymerization conditions, although stable filaments do not form. A plot of P(i) release rate versus actin concentration yields an apparent critical concentration, like that seen for actin polymerization, of approximately 8 microm for Mg(2+) GG-actin and 11 microm for Ca(2+) GG-actin. In contrast to WT-actin, P(i) release from GG-actin is cold-sensitive, reflecting the temperature sensitivity associated with mutations that decrease hydrophobicity in this region. Thus, under polymerization conditions, GG-actin exhibits a continuous F-actin-like ATPase activity resulting from the temperature-sensitive formation of unstable cycling F-actin oligomers. Tropomyosin limits the extent and rate of this activity and restores polymerization by capturing and stabilizing these oligomers rather than enhancing filament nucleation.
Yao
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Department of Biochemistry, University of Iowa College of Medicine, Iowa City, IA 52242, USA.
Rubenstein
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PA
D000199:Actins / Q000235:genetics* / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D011108:Polymers; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae; D013696:Temperature; D014335:Tropomyosin / Q000378:metabolism
D000199:Actins; D010710:Phosphates; D011108:Polymers; D014335:Tropomyosin; D000244:Adenosine Diphosphate; D000251:Adenosine Triphosphatases; D008274:Magnesium