Screening of novel dominant negative mutant actins using glycine targeted scanning identifies G146V actin that cooperatively inhibits cofilin binding.
A number of studies suggested that the structure of actin filaments changes by interaction with actin-binding proteins such as cofilin and myosin, and that the conformational changes of the actin subunits within a filament are cooperative. To understand the functions of these cooperative conformational changes induced by actin-binding proteins, we sought to obtain dominant negative mutant actins impaired in cooperative conformational changes. A series of mutant actin genes in which glycine residues in actin were systematically substituted by valine residues were constructed, and were expressed individually in yeast cells that carry a wild-type endogenous actin gene. Six dominant negative actin mutations were identified on the basis of growth inhibition. Among them, G146V mutation was chosen for further biochemical analysis because the Gly146 residue is located at the strategic hinge position connecting the large and small domains of an actin molecule. We found that G146V actin filaments hardly bind cofilin, consistent with a previous suggestion that cofilin binding causes conformational changes of actin around Gly146 (Galkin et al. [3]). Notably, copolymer that consists of 1:10 mixture of the mutant and wild-type actin molecules showed significantly reduced affinity for cofilin, suggesting that G146V mutant actin affects the conformation of neighboring wild-type actin within a filament, and inhibits cofilin binding.
Noguchi
Taro Q P
TQ
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Toya
Ryo
R
Ueno
Hironori
H
Tokuraku
Kiyotaka
K
Uyeda
Taro Q P
TQ
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051339:Actin Depolymerizing Factors / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D000199:Actins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D004252:DNA Mutational Analysis; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism; D005998:Glycine / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
C465210:Act1 protein, S cerevisiae; D051339:Actin Depolymerizing Factors; D000199:Actins; D029701:Saccharomyces cerevisiae Proteins; D005998:Glycine