RhoBTB3 is part of a CUL3–ubiquitin ligase complex on the Golgi that targets Cyclin E. (A) Colocalization of transfected GFP-RhoBTB3 and FLAG-CUL3 on the Golgi of HeLa cells, detected with anti-GCC185 antibodies. Bar, 20 µm. Insets, enlarged Golgi regions. Bar, 2 µm. (B) GFP-RhoBTB3 binds 3XFLAG-CUL3. Total cell extract inputs (2%) are shown at left; anti-GFP bound material is shown at right, detected by immunoblot. (C) Interaction of RhoBTB3 with FLAG-CUL3 and HA-RBX1 in membranes from HEK293T cells. Cells were transfected as indicated at the top, immunoprecipitated with anti-FLAG antibody, and bound proteins detected by immunoblot with antibodies to the proteins indicated at right. Molecular mass marker mobility is shown at the left in kilodaltons. C, cytosol; M, membrane. (D) Schematic diagram of a canonical CUL3–ubiquitin ligase complex. The N-terminal domain (NTD) of CUL3 interacts with the RING E3 ubiquitin ligase RBX1; the C-terminal domain (CTD) binds a BTB domain–containing substrate adaptor (i.e., RhoBTB3). (E) Interaction of FLAG-RhoBTB3 with HA-RBX1 after 72 h of control or CUL3 siRNA depletion in HEK293T cells. Inputs are at left (5%); anti-FLAG precipitates in duplicate are at right, immunoblotted with anti-FLAG (top) or anti-HA (bottom). Quantitation of coprecipitated RBX1 is shown in relative values at the bottom of the anti-HA panel. (E) CUL3 depletion disrupts the Golgi but does not release RhoBTB3 from the Golgi. Bar, 5 µm. (F) Interaction of RhoBTB3 with Cyclin E on membranes. Membrane extracts from cells cotransfected with indicated constructs were immunoprecipitated and analyzed as in C. Molecular mass marker mobility is shown at the left in kilodaltons.