REGULATION OF 22S DYNEIN BY A 29-KD LIGHT-CHAIN

Previously, a 29-kD axonemal polypeptide (p29) that copurifies with 22 S dynein has been shown to be phosphorylated in a cAMP- and Ca2+-sensi tive manner, consistent with a role for this molecule in the signal tr ansduction cascade leading to fast forward swimming in Paramecium tetr aurelia (Hamasaki, T., K. Barkalow, J. Richmond, and P, Satir. 1991. P roc. Natl. Acad. Sci. USA. 88:7912-7922). This study demonstrates the nature of the relationship between p29 and 22S dynein. Chaotropic agen ts can be used to separate p29 fractions from 22S dynein. When extract ed p29 is exchanged into physiological buffers, it regains the ability to recombine with 22S dynein with an apparent dissociation constant o f 25 nM; no recombination is seen with 14S dynein or with unrelated co ntrol proteins. p29 from Paramecium will also recombine with Tetrahyme na 22 but not 14S dynein. After chymotryptic digestion of 22S dynein, p29 preferentially binds to a single-headed fragment, homologous to th e ct H chain of Tetrahymena 22S dynein. 22S dynein treated in vitro by Paramecium protein kinase A in the presence of cAMP and ATP to phosph orylate p29 translocates bovine brain microtubules significantly (1.53 x; p < 0.001) faster than before phosphorylation. Similarly, 22S dynei n reconstituted in vitro with thiophosphorylated p29 translocates micr otubules significantly (1.31x; p < 0.001) faster than controls reconst ituted with nonthiophosphorylated p29. p29 is the only moiety thiophos phorylated 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 ph osphorylation state.