Eddy R J, Han J, Sauterer R A, Condeelis J S
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Biochim Biophys Acta. 1996 Dec 12;1314(3):247-59. doi: 10.1016/s0167-4889(96)00108-5.
Stimulation of starved Dictyostelium amoebae with the chemoattractant cAMP produces a rapid increase in actin nucleation activity at 5 seconds which is cotemporal with an increase in actin assembly and a decrease in Ca(2+)-insensitive capping activity [1]. Further characterization of this capping activity, called aginactin, led to the isolation of an Hsc70 [2]. Here, we demonstrate that purified aginactin contains both Hsc70 and the heterodimeric barbed-end capping protein, cap32/34. Immunoprecipitation of cap32/34 from purified aginactin removes all capping activity while immunoprecipitation of Hsc70 does not, indicating that the capping activity of aginactin is an intrinsic property of cap32/34. Gel filtration and immunoprecipitation assays fail to demonstrate the existence of a stable, high affinity complex between Hsc70 and cap32/34 in either lysate supernatants or aginactin pools but indicate the presence of a transient, ATP-sensitive interaction in cell lysates. Reconstitution experiments with purified Hsc70 and cap32/34 demonstrate that Hsc70 neither stimulates nor inhibits the capping activity of native cap32/34. Furthermore, we measured a Kd of approx. 0.8 nM for the binding of cap32/34 to barbed ends of actin filaments in the absence or presence of Hsc70, in agreement with Kd values measured for purified capping protein from other sources. We conclude, therefore, that cap32/34 is responsible for the capping activity called aginactin and that Hsc70 is not a regulatory cofactor for cap32/34 in Dictyostelium but may function as a chaperone during assembly of the cap32/34 heterodimer.
用趋化因子cAMP刺激饥饿的盘基网柄菌变形虫，5秒时肌动蛋白成核活性迅速增加，这与肌动蛋白组装增加和钙不敏感封端活性降低同时发生[1]。对这种称为aginactin的封端活性的进一步表征导致分离出一种热休克蛋白70（Hsc70）[2]。在这里，我们证明纯化的aginactin同时含有Hsc70和异源二聚体的肌动蛋白丝尖端封端蛋白cap32/34。从纯化的aginactin中免疫沉淀cap32/34会消除所有封端活性，而免疫沉淀Hsc70则不会，这表明aginactin的封端活性是cap32/34的固有特性。凝胶过滤和免疫沉淀分析未能证明在裂解物上清液或aginactin组分中Hsc70和cap32/34之间存在稳定的高亲和力复合物，但表明在细胞裂解物中存在短暂的、ATP敏感的相互作用。用纯化的Hsc70和cap32/34进行的重组实验表明，Hsc70既不刺激也不抑制天然cap32/34的封端活性。此外，我们测量了在不存在或存在Hsc70的情况下cap32/34与肌动蛋白丝尖端结合的解离常数（Kd）约为0.8 nM，这与从其他来源纯化的封端蛋白测得的Kd值一致。因此，我们得出结论，cap32/34负责称为aginactin的封端活性，并且Hsc70不是盘基网柄菌中cap32/34的调节辅因子，但可能在cap32/34异源二聚体组装过程中作为伴侣蛋白发挥作用。
