title: Importance of a Lys113-Glu195 intermonomer ionic bond in F-actin stabilization and regulation by yeast formins Bni1p and Bnr1p.
abstract: Proper actin cytoskeletal function requires actin's ability to generate a stable filament and requires that this reaction be regulated by actin-binding proteins via allosteric effects on the actin. A proposed ionic interaction in the actin filament interior between Lys(113) of one monomer and Glu(195) of a monomer in the apposing strand potentially fosters cross-strand stabilization and allosteric communication between the filament interior and exterior. We interrupted the potential interaction by creating either K113E or E195K actin. By combining the two, we also reversed the interaction with a K113E/E195K (E/K) mutant. In all cases, we isolated viable cells expressing only the mutant actin. Either single mutant cell displays significantly decreased growth in YPD medium. This deficit is rescued in the double mutant. All three mutants display abnormal phalloidin cytoskeletal staining. K113E actin exhibits a critical concentration of polymerization 4 times higher than WT actin, nucleates more poorly, and forms shorter filaments. Restoration of the ionic bond, E/K, eliminates most of these problems. E195K actin behaves much more like WT actin, indicating accommodation of the neighboring lysines. Both Bni1 and Bnr1 formin FH1-FH2 fragment accelerate polymerization of WT, E/K, and to a lesser extent E195K actin. Bni1p FH1-FH2 dramatically inhibits K113E actin polymerization, consistent with barbed end capping. However, Bnr1p FH1-FH2 restores K113E actin polymerization, forming single filaments. In summary, the proposed ionic interaction plays an important role in filament stabilization and in the propagation of allosteric changes affecting formin regulation in an isoform-specific fashion.
authors: Department of Biochemistry, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242, USA.
authors: Kuo-Kuang
authors: KK
authors: Wen
authors: Melissa
authors: M
authors: McKane
authors: Peter A
authors: PA
authors: Rubenstein
mesh_terms: D000199:Actins / Q000737:chemistry*; D000495:Allosteric Site; D002942:Circular Dichroism; D003598:Cytoskeletal Proteins / Q000737:chemistry*; D003599:Cytoskeleton / Q000737:chemistry; D015966:Gene Expression Regulation, Fungal; D018698:Glutamic Acid / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D008840:Microfilament Proteins / Q000737:chemistry*; D008928:Mitochondria / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*
chemical_list: D000199:Actins; C106463:BNR1 protein, S cerevisiae; C105191:Bni1 protein, S cerevisiae; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D008239:Lysine
keywords: Actin; Contractile Protein; Cytoskeleton; Formin; Mutagenesis Site-specific; Polymerization; Yeast
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