IDENTIFICATION AND MOLECULAR CHARACTERIZATION OF THE P24 DYNACTIN LIGHT-CHAIN

Intracellular transport along microtubules uses the motor proteins cyt oplasmic dynein and kinesin. Cytoplasmic dynein is responsible for mov ement to the minus ends of microtubules and the evidence indicates tha t dynein interacts with another protein complex, dynactin. In order to better understand how these proteins function, we have sought to iden tify and clone the subunit polypeptides of these two complexes, in par ticular their light chains. Dynactin is made up of eight subunits of s imilar to 24,000 to 160,000 Da. In order to clone the p24 subunit, the components of purified dynactin were resolved by SDS polyacrylamide g el electrophoresis. The amino acid sequence of a tryptic peptide from the 24,000-M-r region of the gel was obtained and a candidate polypept ide identified by a screen of the databases. This polypeptide has a pr edicted molecular weight of 20,822 Da. Using an antibody to a differen t region of this protein, we demonstrate that it copurifies with micro tubules and elutes from the microtubule pellet with characteristics si milar to those of the dynactin complex and distinct from those of cyto plasmic dynein. This polypeptide co-sediments with dynactin on sucrose density gradients and it also co-immunoprecipitates with dynactin, bu t not with kinesin or cytoplasmic dynein. Together these results demon strate that this polypeptide is the p24 subunit of dynactin. Analysis of the predicted amino acid sequence of p24 shows that it is a unique protein that has no significant similarity to known enzymes or other p roteins. Structural analysis indicates that most of this protein will form an or-helix and that portions of the molecule may participate in the formation of coiled-coils. Since stoichiometric analysis of dynact in indicates that there is one molecule of p24 per dynactin complex, t hese characteristics suggest that this polypeptide may be involved in protein-protein interactions, perhaps in the assembly of the dynactin complex. Cell Motil. Cytoskeleton 41.154-167. (C) 1998 Wiley-Liss, Inc .