Regulation of 22S dynein by a 29-kD light chain.
Previously, a 29-kD axonemal polypeptide (p29) that copurifies with 22S dynein has been shown to be phosphorylated in a cAMP- and Ca(2+)-sensitive manner, consistent with a role for this molecule in the signal transduction cascade leading to fast forward swimming in Paramecium tetraurelia (Hamasaki, T., K. Barkalow, J. Richmond, and P. Satir. 1991. Proc. Natl. Acad. Sci. USA. 88:7912-7922). This study demonstrates the nature of the relationship between p29 and 22S dynein. Chaotropic agents can be used to separate p29 fractions from 22S dynein. When extracted p29 is exchanged into physiological buffers, it regains the ability to recombine with 22S dynein with an apparent dissociation constant of 25 nM; no recombination is seen with 14S dynein or with unrelated control proteins. p29 from Paramecium will also recombine with Tetrahymena 22 but not 14S dynein. After chymotryptic digestion of 22S dynein, p29 preferentially binds to a single-headed fragment, homologous to the alpha H chain of Tetrahymena 22S dynein. 22S dynein treated in vitro by Paramecium protein kinase A in the presence of cAMP and ATP to phosphorylate p29 translocates bovine brain microtubules significantly (1.53x; p < 0.001) faster than before phosphorylation. Similarly, 22S dynein reconstituted in vitro with thiophosphorylated p29 translocates microtubules significantly (1.31x; p < 0.001) faster than controls reconstituted with nonthiophosphorylated p29. p29 is the only moiety thiophosphorylated in the reconstituted dynein. We conclude that p29 functions as a 22S dynein regulatory light chain in that it alone is sufficient to control the rate of microtubule translocation by changes in its phosphorylation state.
Barkalow
K
K
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
Hamasaki
T
T
Satir
P
P
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D004398:Dyneins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010247:Paramecium / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D015398:Signal Transduction; D013768:Tetrahymena / Q000378:metabolism*
D000242:Cyclic AMP; D004398:Dyneins; D002118:Calcium