Outer arm dynein regulation through a motor-light chain-tubulin ternary complex

Stephen M. King and Ramila S. Patel-King

Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3305, U.S.A..

The LC1 light chain is leucine-rich repeat protein with a C-terminal helical domain that protrudes from the main protein axis; two copies are bound to the motor domain of the outer arm dynein γ heavy chain (HC). Crosslinking studies revealed a direct interaction between LC1 and an axonemal component of ~45 kDa. Blot overlay and direct binding assays indicate that this protein is tubulin suggesting that the γ HC is permanently tethered to axonemal doublet microtubules by both its N-terminal domain and via LC1 bound to the motor domain. Based on the location of a large hydrophobic patch within LC1 that likely mediates association with the γ HC, the C-terminal helix which contains two Arg residues is predicted to insert within the motor; backbone dynamics studies indicate that the orientation of this helix is controlled by M182 and D185. As no LC1 mutant currently exists, we expressed a series of myc-tagged mutant forms in a wildtype background to test whether LC1 is involved in dynein regulation. Insertion of the myc tag alone had essentially no effect on either swimming velocity or flagellar beat frequency. In contrast, alteration of M182 and D185 to either Gly or Pro caused substantial decreases in swimming velocity (from ~115 μm/s to <40 μm/s). Large decreases in swimming velocity were also observed when R189 and R196 in the terminal helix were altered to Ala or Asp/Glu. Intriguingly, these mutations (with the apparent exception of D185P) had only minor effects on flagellar beat frequency. These results suggest a novel dynein regulatory mechanism mediated by the direct interaction of LC1 with an outer arm motor unit and doublet microtubules.

e-mail address of presenting author: king@neuron.uchc.edu