Recent studies on the conventional motor protein kinesin have identified a putative cargo-binding domain (residues 827-906) within the heavy chain. To identify possible cargo proteins which bind to this kinesin domain, we employed a yeast two-hybrid assay. A human brain cDNA library was screened, using as bait residues 814-963 of human ubiquitous kinesin heavy chain. This screen initially identified synaptosome-associated protein of 25 kDa (SNAP25) as a kinesin-binding protein. Subsequently, synaptosome-associated protein of 23 kDa (SNAP23), the nonneuronal homologue of SNAP25, was also confirmed to interact with kinesin. The sites of interaction, determined from in vivo and in vitro assays, are the N-terminus of SNAP25 (residues 1-84) and the cargo-binding domain of kinesin heavy chain (residues 814-907). Both regions are composed almost entirely of heptad repeats, suggesting the interaction between heavy chain and SNAP25 is that of a coiled-coil. The observation that SNAP23 also binds to residues 814-907 of heavy chain would indicate that the minimal kinesin-binding domain of SNAP23 and SNAP25 is most likely residues 45-84 (SNAP25 numbering), a heptad-repeat region in both proteins. The major binding site for kinesin light chain in kinesin heavy chain was mapped to residues 789-813 at the C-terminal end of the heavy chain stalk domain. Weak binding of light chain was also detected at the N-terminus of the heavy chain tail domain (residues 814-854). In support of separate binding sites on heavy chain for light chain and SNAPs, a complex of heavy and light chains was observed to interact with SNAP25 and SNAP23.
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