A dynactin subunit with a highly conserved cysteine-rich motif interacts directly with Arp1

Dynactin is a multisubunit complex and a required cofactor for most, or all , of the cellular processes powered by the microtubule-based motor cytoplas mic dynein. Using a dynein affinity column, the previously uncharacterized p62 subunit of dynactin was isolated and microsequenced. Two peptide sequen ces were used to clone human cDNAs encoding p62. Sequence analysis of the p redicted human polypeptide of 53 kDa revealed a highly conserved pattern of eleven cysteine residues, eight of which fit the consensus sequence for a Zn2+-binding RING domain. We have characterized p62 as an integral componen t of 20 S dynactin by biochemical and immunocytochemical methods. Affinity chromatography experiments demonstrate that pea binds directly to the Arp1 subunit of dynactin, Immunocytochemistry with antibodies to p62 demonstrate s that this polypeptide has a punctate cytoplasmic distribution as well as centrosomal distribution typical of dynactin, In transfected cells, overexp ression of p62 did not disrupt microtubule organization or the integrity of the Golgi but did cause both cytosolic and nuclear distribution of the pro tein, suggesting that this polypeptide may be targeted to the nucleus at ve ry high expression levels.