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Michio Tomishige and Ronald D. Vale
KIF1A/UNC-104 transports synaptic vesicle precursors in nerve axons. Although they contain several putative coiled-coil regions, mouse KIF1A and C. elegans UNC-104 have been shown to be monomeric in solution. The mechanism of vesicle transport is unknown, although it was proposed that either many monomers are involved in the transport (Pierce et al., Biochemistry, 38:5412, 1999) or that a single monomer moves a vesicle through a biased diffusion mechanism (Okada and Hirokawa, Science, 283:1152, 1999). Another possibility is that KIF1A/UNC-104 transiently dimerizes and moves vesicles processively using a hand-over-hand mechanism like conventional kinesin. To test this possibility, we engineered a stable dimer of C. elegans UNC-104 by attaching a leucine zipper coiled-coil (from GCN4) after the UNC-104 neck region. This UNC-104 dimer showed unidirectional processive movement along axonemes with a similar or greater run length than conventional kinesin, but at a four-fold higher velocity of 100 µm/min. This fast unidirectional motion is similar to that observed for UNC-104-GFP in C. elegans axons (Zhou, J. Neurosci., 21:3749, 2001). Optical trapping assay showed that the step size of the UNC-104 dimer was 8 nm and its stall force was 7 pN, which are the same as conventional kinesin. In contrast, monomeric UNC-104, without the coiled-coil region, did not show processive movement. At 100 fold higher motor density (multiple motor binding condition), monomer UNC-104 coated beads moved in an optical trap, but the velocity (36 µm/min) and the stall force (4 pN) were smaller than dimeric UNC-104. These results suggest that UNC-104 has structural and kinetic properties that allow it to move processively like conventional kinesin, provided that it is dimerized. We propose that UNC-104 may dimerize in a regulated manner in vivo and that the dimerized UNC-104 propels processive motion of vesicle cargo.
ASCB 2001
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