Molecular characterization and structure-function analysis of the intermediate chain of cytoplasmic dynein.

摘要

Cytoplasmic dynein is a ubiquitous microtubule-based motor implicated in mitotic spindle function as well as in the retrograde transport of organelles. The heavy chain of the multi-subunit molecule is thought to contain the microtubule binding and the catalytic sites, while associated intermediate and light chain subunits have been hypothesized to confer functional specificity or regulate dynein binding and activity. The fact that Dictyostelium discoideum is amenable to both molecular genetic and biochemical approaches makes it an attractive system in which to investigate the role of the component subunits in dynein function.;In order to examine the role of the IC in dynein function, we analyzed the phenotypic consequences of expressing regions of the protein in wildtype Dictyostelium cells. Myc-tagged, full-length IC, the divergent N-terminal domain, and the highly conserved C-terminal domain were expressed in AX3 cells under control of an inducible promoter. The expressed, full-length IC successfully competes with endogenous IC for binding to the dynein HC. The N-terminal domain does not associate with the HC, but the C-terminal domain binds at an estimated 1:1 molar ratio with the endogenous IC. A dominant-negative growth defect is observed in cells induced to express high levels of the C-terminal domain. These cells have a 4-fold higher mitotic index than uninduced cells, with 43% of mitotic cells blocked in prometaphase. We propose that high levels of overexpression of the C-terminal IC domain renders dynein functionally inactive because it binds the dynein HC, but lacks the proposed "cargo-binding" domain. These results indicate that IC function is required for an essential role of cytoplasmic dynein in the production of a functional mitotic spindle in Dictyostelium.;The intermediate chain (IC) of Dictyostelium dynein was cloned and characterized. The deduced amino acid sequence of the Dictyostelium IC is most similar (71%) to the rat IC in the C-terminal two-thirds of the molecule. Southern analysis suggests the presence of a single IC gene in the Dictyostelium genome. Northern blot analysis of poly A RNA indicates that a single transcript is expressed in both vegetative and developed cells. However, multiple isoelectric species of the IC are expressed late in development, suggesting that the IC is post-translationally modified. Phosphorylation of the IC in vegetative and developed cells accounts for at least some of the observed heterogeneity of IC isoforms.