Cytoskeletal-based vesicular trafficking of the bile salt transporter, Ntcp
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Intracellular trafficking and protein sorting regulate the abundance and therefore activity of transporters present at the plasma membrane. The transporter, Na+/taurocholate cotransporting polypeptide (ntcp), is known to exist in an intracellular pool of vesicles from which it traffics to and from the cell surface according to cellular demands. The PI3-kinase pathway, as well as microtubule and microfilament cytoskeletons, have been implicated in this trafficking. However, details of this process have been unclear and the regulation and intracellular localization of ntcp are not well understood. Utilizing in vitro and whole cell immunofluorescence microscopy assays, we demonstrate that ntcp is present on intracellular vesicles that bind to microtubules and move bidirectionally with equal propensity toward their plus and minus-ends, using kinesin-1 and dynein, respectively. PI(3,4,5)P3, which activates PKCzeta, enhances motility of ntcp-containing vesicles and overcomes the partial inhibition produced by a PI3K inhibitor, LY294002. Inhibition of PKCzeta with a specific pseudosubstrate, blocks the motility of ntcp-containing vesicles but not late endocytic vesicles, suggesting a specific requirement of PKCzeta activity for this motility. Ntcp-containing vesicles colocalize with markers for early and recycling endosomes but not late endosomes. They frequently undergo fission, providing a mechanism for their depletion from late endosomes. We reconstituted vesicular fission in vitro and analyzed the segregation kinetics of ntcp and other endocytic markers. The data provide direct evidence that protein segregation occurs by cytoskeletal-based pulling of vesicles, requires activity of PI3K, and suggests a mechanism of protein segregation by generation of 'pure' vesicles. We also established an in vitro actin filament-based motility assay and demonstrated the involvement of myosins IIa and Vb in movement of these vesicles.
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