Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein.
Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.
Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202, USA.
B L
BL
Goode
D G
DG
Drubin
P
P
Lappalainen
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000737:chemistry / Q000502:physiology*; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins; D008840:Microfilament Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology*; D008969:Molecular Sequence Data; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
D051339:Actin Depolymerizing Factors; D000199:Actins; D005656:Fungal Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D011505:Protein-Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485190:Twf1 protein, mouse; C113980:TWF1 protein, S cerevisiae; C485188:TWF1 protein, human