The heavy chain of conventional kinesin interacts with the SNARE proteins SNAP25 and SNAP23.
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.
Diefenbach
Russell J
RJ
Centre For Virus Research, Westmead Millennium Institute, Westmead Hospital and The University of Sydney, NSW 2145, Australia. russell_diefenbach@wmi.usyd.edu.au
Diefenbach
Eve
E
Douglas
Mark W
MW
Cunningham
Anthony L
AL
D000595:Amino Acid Sequence; D001923:Brain Chemistry / Q000235:genetics; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000032:analysis / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D050600:SNARE Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D050825:Synaptosomal-Associated Protein 25; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D020409:Molecular Motor Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D021122:Protein Subunits; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D011993:Recombinant Fusion Proteins; C100493:SNAP23 protein, human; C494208:SNAP25 protein, human; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D033921:Vesicular Transport Proteins; D016547:Kinesins