Reconstitution of the human cytoplasmic dynein complex.

ABSTRACT: Cytoplasmic dynein is the major motor protein responsible for microtubule minus-end-directed movements in most eukaryotic cells. It transports a variety of cargoes and has numerous functions during spindle assembly and chromosome segregation. It is a large complex of about 1.4 MDa composed of six different subunits, interacting with a multitude of different partners. Most biochemical studies have been performed either with the native mammalian cytoplasmic dynein complex purified from tissue or, more recently, with recombinant dynein fragments from budding yeast and Dictyostelium. Hardly any information exists about the properties of human dynein. Moreover, experiments with an entire human dynein complex prepared from recombinant subunits with a well-defined composition are lacking. Here, we reconstitute a complete cytoplasmic dynein complex using recombinant human subunits and characterize its biochemical and motile properties. Using analytical gel filtration, sedimentation-velocity ultracentrifugation, and negative-stain electron microscopy, we demonstrate that the smaller subunits of the complex have an important structural function for complex integrity. Fluorescence microscopy experiments reveal that while engaged in collective microtubule transport, the recombinant human cytoplasmic dynein complex is an active, microtubule minus-end-directed motor, as expected. However, in contrast to recombinant dynein of nonmetazoans, individual reconstituted human dynein complexes did not show robust processive motility, suggesting a more intricate mechanism of processivity regulation for the human dynein complex. In the future, the comparison of reconstituted dynein complexes from different species promises to provide molecular insight into the mechanisms regulating the various functions of these large molecular machines.

SUBMITTER: Trokter M 

PROVIDER: S-EPMC3529016 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature