Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport.
In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end-directed movement. The latter was selectively blocked in the rigor-mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo.
D000255:Adenosine Triphosphate / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000502:physiology; D048429:Cell Size; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D006056:Golgi Apparatus / Q000378:metabolism; D007425:Intracellular Membranes / Q000502:physiology; D016547:Kinesins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D015388:Organelles / Q000502:physiology; D017354:Point Mutation / Q000502:physiology*
D017931:DNA Primers; D018076:DNA, Complementary; D000255:Adenosine Triphosphate; D016547:Kinesins