Fig. 4
From: Coupling fission and exit of RAB6 vesicles at Golgi hotspots through kinesin-myosin interactions
Definition of the RAB6-BD of KIF20A. RAB6 partially recruits KIF20A on the Golgi complex. RAB6 is required for KIF20A-Myosin II interaction. Relative binding affinities between RAB6, KIF20A, and Myosin II. a–c KIF20A-RAB6-binding domain (KIF20A-RBD) is a dimer composed of parallel helices that form a right-handed coiled-coil stabilized by an inter-helical cysteine bridge and two RAB6 molecules bind on opposite sides of the dimer b. The RAB6:KIF20A interface makes hydrophobic and polar contacts between the molecules b, c, in particular K629 and S631, are buried in the interface and involved in direct interactions at the center of the interface. d Yeast two-hybrid interactions between the KIF20A-RBD-529-665 fragment or KIF20A-RBD-529-665-K626W-S631W fragment with RAB6, Myosin II-RBD, and KIF20A-RBD. The Saccharomyces cerevisiae reporter strain L40 was co-transformed with a plasmid encoding fusion proteins to detect interactions between the KIF20A-RBD-529-665 fragment or KIF20A-RBD-529-665-K626W-S631W fragment and RAB6-Q72L, KIF20A-RBD, Myosin II-RBD, RAB1Q70L, and Lamin A. Growth on medium lacking histidine (-W -L -H) indicates an interaction between the encoded proteins. e Golgi association of the KIF20A-RBD-529-665 fragment or KIF20A-RBD-529-665-K626W-S631W fragments. Staining for endogenous Giantin (red) and overexpressed myc-tagged KIF20A-RBD-529-665 fragment or KIF20A-RBD-529-665-K626W-S631W fragment. f Staining of endogenous Giantin (green) and KIF20A (red) in HeLa cells 3 days after transfection with specific RAB6 siRNAs. Quantification of Golgi-associated KIF20A fluorescence intensity in cells treated as described above (mean ± SEM, n = 34-37 cells). **P < 10−3 (Student’s t test). Bar, 10 µm. g RAB6 is required for KIF20A/Myosin II interaction. GFP-KIF20A or GFP expressing HeLa cell extracts treated for 3 days with control or RAB6-specific siRNAs were immunoprecipitated using the GFP-trap system. Myosin II bound to GFP-KIF20A was revealed by western blot analysis using anti-Myosin II antibody. Input represents a 5% load of the total cell extracts used in all conditions. h 96-well plates were coated with recombinant GST-RAB6 or GST-Myosin II-1148-1652 and incubated with increasing amounts of recombinant 6 × His-KIF20A-RBD-529-665 or 6 × His-RAB6, as indicated (solid-phase assay). After washes, KIF20A-RBD-529-665 or RAB6 proteins bound to RAB6 or Myosin II-1148-1652 were detected (arbitrary units) using anti-6 × His antibodies and a chromogenic substrate (mean ± SEM, n = 3 experiments). No binding to GST-GFP alone was detected. MW molecular weight in kDa
