The IC138 and IC140 intermediate chains of the I1 axonemal dynein complex bind directly to tubulin.

Dyneins are minus end directed microtubule motors that play a critical role in ciliary and flagellar movement. Ciliary dyneins, also known as axonemal dyneins, are characterized based on their location on the axoneme, either as outer dynein arms or inner dynein arms. The I1 dynein is the best-characterized subspecies of the inner dynein arms; however the interactions between many of the components of the I1 complex and the axoneme are not well defined. In an effort to elucidate the interactions in which the I1 components are involved, we performed zero-length crosslinking on axonemes and studied the crosslinked products formed by the I1 intermediate chains, IC138 and IC140. Our data indicate that IC138 and IC140 bind directly to microtubules. Mass-spectrometry analysis of the crosslinked product identified both α- and ß-tubulin as the IC138 and IC140 binding partners. This was further confirmed by crosslinking experiments carried out on purified I1 fractions bound to Taxol-stabilized microtubules. Furthermore, the interaction between IC140 and tubulin is lost when IC138 is absent. Our studies support previous findings that intermediate chains play critical roles in the assembly, axonemal targeting and regulation of the I1 dynein complex.

Molecular insight into γ-γ tubulin lateral interactions within the γ-tubulin ring complex (γ-TuRC).

γ-Tubulin is essential for the nucleation and organization of mitotic microtubules in dividing cells. It is localized at the microtubule organizing centers and mitotic spindle fibres. The most well accepted hypothesis for the initiation of microtubule polymerization is that α/ß-tubulin dimers add onto a γ-tubulin ring complex (γTuRC), in which adjacent γ-tubulin subunits bind to the underlying non-tubulin components of the γTuRC. This template thus determines the resulting microtubule lattice. In this study we use molecular modelling and molecular dynamics simulations, combined with computational MM-PBSA/MM-GBSA methods, to determine the extent of the lateral atomic interaction between two adjacent γ-tubulins within the γTuRC. To do this we simulated a γ-γ homodimer for 10 ns and calculated the ensemble average of binding free energies of -107.76 kcal/mol by the MM-PBSA method and of -87.12 kcal/mol by the MM-GBSA method. These highly favourable binding free energy values imply robust lateral interactions between adjacent γ-tubulin subunits in addition to their end-interactions longitudinally with other proteins of γTuRC. Although the functional reconstitution of γ-TuRC subunits and their stepwise in vitro assembly from purified components is not yet feasible, we nevertheless wanted to recognize hotspot amino acids responsible for key γ-γ interactions. Our free energy decomposition data from converting a compendium of amino acid residues identified an array of hotspot amino acids. A subset of such mutants can be expressed in vivo in living yeast. Because γTuRC is important for the growth of yeast, we could test whether this subset of the hotspot mutations support growth of yeast. Consistent with our model, γ-tubulin mutants that fall into our identified hotspot do not support yeast growth.

IC138 is a WD-repeat dynein intermediate chain required for light chain assembly and regulation of flagellar bending.

Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation of IC138 phosphorylation controls flagellar bending, we cloned the IC138 gene. IC138 encodes a novel protein with a calculated mass of 111 kDa and is predicted to form seven WD-repeats at the C terminus. IC138 maps near the BOP5 locus, and bop5-1 contains a point mutation resulting in a truncated IC138 lacking the C terminus, including the seventh WD-repeat. bop5-1 cells display wild-type flagellar beat frequency but swim slower than wild-type cells, suggesting that bop5-1 is altered in its ability to control flagellar waveform. Swimming speed is rescued in bop5-1 transformants containing the wild-type IC138, confirming that BOP5 encodes IC138. With the exception of the roadblock-related light chain, LC7b, all the other known components of the I1 complex, including the truncated IC138, are assembled in bop5-1 axonemes. Thus, the bop5-1 motility phenotype reveals a role for IC138 and LC7b in the control of flagellar bending. IC138 is hyperphosphorylated in paralyzed flagellar mutants lacking radial spoke and central pair components, further indicating a role for the radial spokes and central pair apparatus in control of IC138 phosphorylation and regulation of flagellar waveform.