Molecular basis for JIP1–JIP3–kinesin-1 ternary complex formation. (A). Lysates prepared from HEK293T cells expressing TAP-JIP1, V5-KLC, V5-KHC and GFP-JIP3, or GFP alone, were precipitated with SA and analyzed by WB with the indicated antibodies. Input, cell lysate used for the immunoprecipitation assay. SA, proteins bound to streptavidin sepharose. Graph shows quantification of kinesin-1 (KLC) in the TAP-JIP1-bound fraction “SA”. Each value was normalized to the amount of TAP-JIP1 in the corresponding SA fraction. *: p <0.001 compared with TAP-JIP1-WT + GFP. Error bars indicate ± SEM. n = 3. (B). Upper: schematic illustration of the JIP3 constructs. The L residues (431, 438, 452 and 459) were substituted for A in the JIP3-LZ4A mutants. These constructs are GFP- or TAP-tagged at their N-termini. JBD: JNK binding domain; LZ: leucine zipper; CC: coiled-coil. Lower: lysates prepared from HEK293T cells expressing TAP-JIP3, V5-KLC and V5-KHC were precipitated with SA. TAP-JIP1 constructs were used as controls. (C). Lysates of HEK293T cells expressing TAP-JIP1-WT, V5-KLC, V5-KHC, GFP-JIP3 or GFP alone were precipitated with SA and analyzed by WB with the indicated antibodies. (D). Lysates of HEK293T cells expressing TAP-JIP3, V5-KLC, V5-KHC, GFP-JIP1 or GFP alone were precipitated with SA and analyzed by WB with the indicated antibodies. (E). A model of the JIP1–JIP3–kinesin-1 complex. JIP1 binds to the TPR domain of KLC via the C-terminal region. JIP3 also binds to the TPR domain of KLC via the LZ domain. Because JIP1 and JIP3 bind to distinct surfaces within the TPR domain of KLC , JIP3 binding to the JIP1-PTB domain can form a molecular complex that grasps the TPR domain and binds to the TPR domain more stably than JIP1 or JIP3 alone.
Satake et al. BMC Cell Biology 2013 14:12 doi:10.1186/1471-2121-14-12