Bentley et al. use a novel assay to identify the kinesin motor proteins that bind to different types of endosomes.

Membrane trafficking is controlled by a large number of proteins that bind to specific vesicles and organelles. But defining which proteins bind to which membranes within a crowded cell—by fluorescence colocalization, for example—is often difficult. Bentley et al. therefore developed an assay that can directly test whether a protein binds to a specific vesicle population in vivo.

The researchers fused candidate proteins to an FRB tag that, in the presence of a rapamycin-like drug called AP21967, binds to an FKBP-tagged version of the dynein motor adaptor molecule Bicaudal D2. If a candidate protein binds to a specific vesicle population, dynein will be recruited to these organelles in the presence of AP21967 and subsequently transport them to the minus ends of microtubules, which, in most cell types, are clustered around a microtubule-organizing center (MTOC) in the middle of the cell.

Bentley et al. verified their assay by showing that an FRB-tagged version of the small GTPase Rab5 could misdirect early endosomes to microtubule minus ends, whereas FRB-Rab7 caused late endosomes to cluster around the MTOC. The researchers then systematically analyzed the endosome-binding properties of Kinesin-3 family members and found that KIF13A and KIF13B bound to early endosomes, whereas KIF1A and KIF1Bβ associated with late endosomes and lysosomes. Bentley et al. think that the approach should be applicable to many other families of membrane trafficking proteins as well.


, et al
J. Cell Biol.

Author notes

Text by Ben Short