The fate of the Kinesin-1 motor in neurite tips and microtubule posttranslational modifications influence Kinesin-1 motor protein trafficking.

The mechanisms contributing to polarized trafficking are poorly understood but are of great interest. Long-distance intracellular travel is driven by the kinesin and dynein motor proteins that ferry their cargoes along microtubule tracks. Kinesin-1 is a heterotetramer consisting of two kinesin heavy chains (KHC) and two kinesin light chains (KLC). Using neuronal cells as a polarized cellular model, we show JNK-interacting protein 1, JIP1, which is a cargo of Kinesin-1, is localized to a subset of neurites. The mechanism of polarized trafficking of JIP1 involves the Kinesin-1 motor domain preferentially recognizing microtubules containing certain posttranslational modifications. Genetic, biochemical, and pharmacological approaches were taken to dissect the mechanism of polarized trafficking of JIP1. Mutations to eliminate certain posttranslational modifications in Tetrahymena strains showed that alpha-tubulin lysine-40 acetylation influences Kinesin-1 motor domain interaction with the microtubules. In vivo studies utilizing pharmacological treatments that increase microtubule acetylation caused a redirection of Kinesin-1 transport of JIP1 to nearly all neurite tips. Collectively, these results suggest that microtubule acetylation promotes Kinesin-1 binding and motor-protein trafficking. The fate of the molecular motor in the neurite tips following successful cargo delivery is not understood. Here we dissect several possibilities of the Kinesin-1 motor fate. First we show Kinesin-1 is not degraded in the neurite tips nor brought back to the cell body by minus-end motors. Utilizing photoactivatable, GFP-tagged Kinesin-1 motor subunits; Kinesin-1-specific cargoes; and freely diffusible protein, we monitored the dynamics of the motor leaving the neurite tips in vivo. For the first time, a sub-population of the Kinesin-1 motor has been monitored in vivo, and our data suggest that Kinesin-1 is not degraded in the neurite tip; rather, Kinesin-1 returns to the cell body in an ATP--independent manner. The regulation of fast axonal transport and the mechanisms that exist to selectively enrich membrane proteins in neuronal subdomains are poorly understood. A putative GSK3 consensus site has been identified on a KLC2 splice variant on the C-terminus. Our results indicate that GSK3 inhibition does not influence Kinesin-1 delivery of cargo to tips of primary hippocampal neurons or neuronal-like-cells.