Figure 9. The molecular mass of myosin-VIIa interacted with RIM-BP2 in INS-1 832/13 cells is 170 kDa, whereas that directly binds exophilin-8 in HEK293A cells is 260 kDa.
(A) HEK293A cells were transfected with plasmids expressing RIM-BP2, myosin-VIIa, and/or OSF-tagged, wild-type or PA mutant exophilin-8 ABD. Exophilin-8 ABD and the binding proteins were pulled down using Strep-Tactin beads, and were subjected to SDS-PAGE and Coomassie Brilliant Blue staining. (B) HEK293A cells were transfected with plasmids expressing wild-type or PA mutant exophilin-8 ABD, myosin-VIIa, and OSF-tagged RIM-BP2. RIM-BP2 and the binding proteins were pulled down as in (A). (C) INS-1 832/13 cells were transfected with control siRNA duplexes or siRNA duplexes against myosin-VIIa, and the cell extracts were immunoblotted with anti-myosin-VIIa and anti-α-tubulin antibodies. Note that both 260 kDa and major 170 kDa protein bands were downregulated by myosin-VIIa siRNA. (D) INS-1 832–13 cells were transfected with plasmids expressing myosin-VIIa and/or OSF-tagged RIM-BP2. Because the transfection efficiency in these cells are poor compared with that in HEK293A cells, the binding proteins were detected by immunoblotting. The anti-myosin-VIIa used in this figure was that generated by our laboratory (αMyo7), in contrast to the commercially available antibody used in Figures 6D, 8, 9C and 10 (see Materials and methods). (E) Illustration of the cortical F-actin network with secretory granules and the plasma membrane, the exophilin-8–RIM-BP2–170 kDa myosin-VIIa complex found in the present study, and the previously known, exocytic protein interactions (Südhof, 2013). The exact molecular nature of the 170 kDa form of myosin-VIIa and whether it directly interacts with RIM-BP2 are currently unknown.
Figure 9—figure supplement 1. Sucrose density gradient centrifugation analysis of the exophilin-8–RIM-BP2–myosin-VIIa complex.
