The three panels are labeled A, B, and C, each depicting different biochemical analyses of K I F 3 and K A P 3 subtypes. Panel A shows schematic diagrams of major constructs used for biochemical analysis, tagged by 6 His or G S T. These constructs include A C, A C T, A T for K I F 3 A, and B C T, B T for K I F 3 B. Panel B illustrates a pull-down assay evaluating the interaction among K I F 3 A, K I F 3 B, and K A P 3. The assay results show the presence of G S T-A C T and His-B C T at equal levels in lane 8. The comparable levels of His-B T in lanes 12 and 18 indicate an indirect interaction between K I F 3 A and K I F 3 B mediated by K A P 3, and suggest that direct A B dimer formation requires the coiled-coil region. Panel C presents analytical S E C analysis of reconstructed K I F 3 and K I F 3 and K A P 3 complexes, with different constructs labeled as A A, A B, B B, A A K, A B K, and B B K. The S E C analysis shows the retention volume on the horizontal axis and U V 280 on the vertical axis, with distinct peaks for each construct combination. All data is approximate.
Biochemical analyses indicated the existence of different KIF3/KAP3 subtypes in vitro. (A) Schematic diagrams showing the major constructs used for biochemical analysis, which are tagged by 6×His or GST for different uses. AC, KIF3A 376–706, CC1–CC2–CC3-tail construct; ACT, KIF3A 481–701, CC2–CC3-tail construct; AT, KIF3A 600–701, tail construct; BCT, KIF3B 475–747, CC2–CC3-tail construct; BT, KIF3B 592–747, tail construct. (B) Pull-down assay evaluating the interaction among KIF3A, KIF3B, and KAP3. In the GST-AT group, because His-BCT and GST-AT have similar molecular weights, western blotting was probed with anti-6×His and anti-GST antibodies. The observation that GST-ACT and His-BCT are present at equal levels in lane eight suggests the formation of a dimer. The comparable levels of His-BT (20 kDa) in lanes 12 and 18 are consistent with an indirect interaction between KIF3A and KIF3B mediated by KAP3 (AT–KAP3–BT) and further suggest that direct AB dimer formation requires the coiled-coil region. Note that KAP3 band intensities are equivalent after normalization to the GST immunoblot signal in lanes 16–18. B, beads of glutathione agarose. (C) Analytical SEC analysis of reconstructed KIF3 and KIF3/KAP3 complexes. His-tagged KIF3A constructs were used for all the complex purification. AAK: ACT/ACT/KAP3. Source data are available for this figure: SourceData F4.