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Abstract
Conserved Asp-11 of actin is a part of the nucleotide binding pocket, and its mutation to Gln is dominant lethal in yeast, whereas the mutation to Asn in human α-actin dominantly causes congenital myopathy. To elucidate the molecular mechanism of those dominant negative effects, we prepared Dictyostelium versions of D11N and D11Q mutant actins and characterized them in vitro. D11N and D11Q actins underwent salt-dependent reversible polymerization, although the resultant polymerization products contained small anomalous structures in addition to filaments of normal appearance. Both monomeric and polymeric D11Q actin released bound nucleotides more rapidly than the wild type, and intriguingly, both monomeric and polymeric D11Q actins hardly bound cofilin. The deficiency in cofilin binding can be explained by rapid exchange of bound nucleotide with ATP in solution, because cofilin does not bind ATP-bound actin. Copolymers of D11Q and wild type actins bound cofilin, but cofilin-induced depolymerization of the copolymers was slower than that of wild type filaments, which may presumably be the primary reason why this mutant actin is dominantly toxic in vivo. Purified D11N actin was unstable, which made its quantitative biochemical characterization difficult. However, monomeric D11N actin released nucleotides even faster than D11Q, and we speculate that D11N actin also exerts its toxic effects in vivo through a defective interaction with cofilin. We have recently found that two other dominant negative actin mutants are also defective in cofilin binding, and we propose that the defective cofilin binder is a major class of dominant negative actin mutants.
Highlights
Mutation of Asp-11 is dominant negative in yeast and human actins
Our results demonstrated that D11N/D11Q actin polymers are abnormal in a number of ways, including rapid release of bound nucleotides and defective cofilin binding and cofilin-mediated depolymerization, the former presumably causing the latter
Because cofilin is essential for yeast viability [17, 18] and for proper muscle development [19] and functions [20, 21], defective cofilin-induced depolymerization of WT and D11Q actin copolymers suggested that defective cofilin binding is the primary reason why D11N/D11Q actins are dominantly toxic in yeast and human
Summary
Mutation of Asp-11 is dominant negative in yeast and human actins. Results: Mutant actins exchange bound nucleotides rapidly, cannot bind cofilin, and cofilin-induced depolymerization of mutant and wild type copolymers is slow. Actin plays a number of important roles in eukaryotic cells, including amoeboid movement, cytokinesis, adhesion, intracellular transport, endocytosis/exocytosis, as well as nuclear roles in transcription regulation To perform these functions, actin filaments need to be polymerized and depolymerized in a spatially and temporally regulated manner. Native final residue of actin, to separate the actin and thymosin moieties [13] This expression system was used to characterize dominant negative actin mutations [14] previously identified in Drosophila indirect flight muscle [2], as well as in our own genetic screens using yeast cells [3]. Our results demonstrated that D11N/D11Q actin polymers are abnormal in a number of ways, including rapid release of bound nucleotides and defective cofilin binding and cofilin-mediated depolymerization, the former presumably causing the latter. Because cofilin is essential for yeast viability [17, 18] and for proper muscle development [19] and functions [20, 21], defective cofilin-induced depolymerization of WT and D11Q actin copolymers suggested that defective cofilin binding is the primary reason why D11N/D11Q actins are dominantly toxic in yeast and human
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