Identification and molecular characterization of the p24 dynactin light chain.
Intracellular transport along microtubules uses the motor proteins cytoplasmic dynein and kinesin. Cytoplasmic dynein is responsible for movement to the minus ends of microtubules and the evidence indicates that dynein interacts with another protein complex, dynactin. In order to better understand how these proteins function, we have sought to identify and clone the subunit polypeptides of these two complexes, in particular their light chains. Dynactin is made up of eight subunits of approximately 24,000 to 160,000 Da. In order to clone the p24 subunit, the components of purified dynactin were resolved by SDS polyacrylamide gel electrophoresis. The amino acid sequence of a tryptic peptide from the 24,000-Mr region of the gel was obtained and a candidate polypeptide identified by a screen of the databases. This polypeptide has a predicted molecular weight of 20,822 Da. Using an antibody to a different region of this protein, we demonstrate that it copurifies with microtubules and elutes from the microtubule pellet with characteristics similar to those of the dynactin complex and distinct from those of cytoplasmic dynein. This polypeptide co-sediments with dynactin on sucrose density gradients and it also co-immunoprecipitates with dynactin, but not with kinesin or cytoplasmic dynein. Together these results demonstrate 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 proteins. Structural analysis indicates that most of this protein will form an alpha-helix and that portions of the molecule may participate in the formation of coiled-coils. Since stoichiometric analysis of dynactin indicates that there is one molecule of p24 per dynactin complex, these characteristics suggest that this polypeptide may be involved in protein-protein interactions, perhaps in the assembly of the dynactin complex.
Pfister
K K
KK
Department of Cell Biology, University of Virginia Health Science Center, Charlottesville 22908-0439, USA. kkp9w@virginia.edu
Benashski
S E
SE
Dillman
J F
JF
Patel-King
R S
RS
King
S M
SM
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002499:Centrifugation, Density Gradient; D017124:Conserved Sequence; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D020224:Expressed Sequence Tags; D006160:Guanosine Triphosphate / Q000378:metabolism; D016547:Kinesins / Q000378:metabolism; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D012965:Sodium Chloride / Q000378:metabolism
D000906:Antibodies; D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D012965:Sodium Chloride; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004398:Dyneins; D016547:Kinesins