Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain.
Conventional kinesin transports membranes along microtubules in vivo, but the majority of cellular kinesin is unattached to cargo. The motility of non-cargo-bound, soluble kinesin may be repressed by an interaction between the amino-terminal motor and carboxy-terminal cargo-binding tail domains, but neither bead nor microtubule-gliding assays have shown such inhibition. Here we use a single-molecule assay that measures the motility of kinesin unattached to a surface. We show that full-length kinesin binds microtubules and moves about ten times less frequently and exhibits discontinuous motion compared with a truncated kinesin lacking a tail. Mutation of either the stalk hinge or neck coiled-coil domain activates motility of full-length kinesin, indicating that these regions are important for tail-mediated repression. Our results suggest that the motility of soluble kinesin in the cell is inhibited and that the motor becomes activated by cargo binding.
Friedman
D S
DS
Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.
Vale
R D
RD
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D008870:Microtubules / Q000502:physiology*; D008969:Molecular Sequence Data; D009068:Movement; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D011994:Recombinant Proteins; D016547:Kinesins