Analysis of dynactin subcomplexes reveals a novel actin-related protein associated with the Arp1 minifilament pointed end

The multisubunit protein, dynactin, is a critical component of the cytoplas mic dynein motor machinery. Dynactin contains two distinct structural domai ns: a projecting sidearm that interacts with dynein and an actin-like minif ilament backbone that is thought to bind cargo. Here, we use biochemical, u ltrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynact in with recombinant dynamitin yields two assemblies: the actin-related prot ein, Arp1, minifilament and the p150(Glued) sidearm. Both contain dynamitin . Treatment of dynactin with the chaotropic salt, potassium iodide, complet ely depolymerizes the Arp1 minifilament to reveal multiple protein complexe s that contain the remaining dynactin subunits. The shoulder/sidearm comple x contains p150(Glued), dynamitin, and p24 subunits and is ultrastructurall y similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assemb ly that may link the shoulder/sidearm complex to the Arp1 minifilament. Poi nted-end complex contains p62, p27, and p25 subunits, plus a novel actin-re lated protein, Arp11. p62, p27, and p25 contain predicted cargo-binding mot ifs, while the Arp11 sequence suggests a pointed-end capping activity. Thes e isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.