ABSTRACT
Capping protein (CP) is a ubiquitously expressed, heterodimeric actin binding protein that is essential for normal actin dynamics in cells. The existing methods for purifying native CP from tissues and recombinant CP from bacteria are time-consuming processes that involve numerous conventional chromatographic steps and functional assays to achieve a homogeneous preparation of the protein. Here, we report the rapid purification of Acanthamoeba CP from amoeba extracts and recombinant mouse CP from E. coli extracts using as an affinity matrix GST-fusion proteins containing the CP binding site from Acanthamoeba CARMIL and mouse CARMIL-1, respectively. This improved method for CP purification should facilitate the in vitro analysis of CP structure, function, and regulation.
Subject(s)
Actin Capping Proteins/isolation & purification , Carrier Proteins/chemistry , Protozoan Proteins/isolation & purification , Acanthamoeba/chemistry , Actin Capping Proteins/genetics , Actin Capping Proteins/metabolism , Animals , Binding Sites , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Extracts/chemistry , Electrophoresis, Polyacrylamide Gel , Escherichia coli/genetics , Escherichia coli/metabolism , Immunoblotting , Mice , Microfilament Proteins , Protein Structure, Tertiary , Protozoan Proteins/genetics , Protozoan Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Sepharose/chemistryABSTRACT
Capping protein (CP) is an integral component of Arp2/3-nucleated actin networks that drive amoeboid motility. Increasing the concentration of capping protein, which caps barbed ends of actin filaments and prevents elongation, increases the rate of actin-based motility in vivo and in vitro. We studied the synergy between CP and Arp2/3 using an in vitro actin-based motility system reconstituted from purified proteins. We find that capping protein increases the rate of motility by promoting more frequent filament nucleation by the Arp2/3 complex and not by increasing the rate of filament elongation as previously suggested. One consequence of this coupling between capping and nucleation is that, while the rate of motility depends strongly on the concentration of CP and Arp2/3, the net rate of actin assembly is insensitive to changes in either factor. By reorganizing their architecture, dendritic actin networks harness the same assembly kinetics to drive different rates of motility.