Kuhn Jeffrey R, Pollard Thomas D
Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520, USA.
J Biol Chem. 2007 Sep 21;282(38):28014-24. doi: 10.1074/jbc.M705287200. Epub 2007 Jul 26.
We investigated how heterodimeric capping proteins bind to and dissociate from the barbed ends of actin filaments by observing single muscle actin filaments by total internal reflection fluorescence microscopy. The barbed end rate constants for mouse capping protein (CP) association of 2.6 x 10(6) M(-1) s(-1) and dissociation of 0.0003 s(-1) agree with published values measured in bulk assays. The polyphosphoinositides (PPIs), phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)), PI(4,5)P(2), and PI(3,4,5)P(3), prevent CP from binding to barbed ends, but three different assays showed that none of these lipids dissociate CP from filaments at concentrations that block CP binding to barbed ends. The affinity of fission yeast CP for barbed ends is a thousandfold less than mouse CP, because of a slower association rate constant (1.1 x 10(5) M(-1) s(-1)) and a faster dissociation rate constant (0.004 s(-1)). PPIs do not inhibit binding of fission yeast CP to filament ends. Comparison of homology models revealed that fission yeast CP lacks a large patch of basic residues along the actin-binding surface on mouse CP. PPIs binding to this site might interfere sterically with capping, but this site would be inaccessible when CP is bound to the end of a filament.
我们通过全内反射荧光显微镜观察单个肌肉肌动蛋白丝，研究了异二聚体封端蛋白如何与肌动蛋白丝的尖端结合以及从其解离。小鼠封端蛋白（CP）结合的尖端速率常数为2.6×10⁶ M⁻¹ s⁻¹，解离速率常数为0.0003 s⁻¹，与在大量实验中测得的已发表值一致。多磷酸肌醇（PPI），磷脂酰肌醇3,4 - 二磷酸（PI(3,4)P₂）、PI(4,5)P₂和PI(3,4,5)P₃，可阻止CP与尖端结合，但三种不同的实验表明，在阻止CP与尖端结合的浓度下，这些脂质均不能使CP从丝上解离。裂殖酵母CP对尖端的亲和力比小鼠CP低一千倍，这是因为其结合速率常数较慢（1.1×10⁵ M⁻¹ s⁻¹）且解离速率常数较快（0.004 s⁻¹）。PPI不抑制裂殖酵母CP与丝末端的结合。同源模型比较显示，裂殖酵母CP在小鼠CP的肌动蛋白结合表面缺少一大片碱性残基。PPI结合到该位点可能会在空间上干扰封端，但当CP与丝末端结合时，该位点将无法接近。
