RhoBTB3–CUL3 represents an alternative pathway for Cyclin E ubiquitylation. (A) Fbw7 depletion in HEK293T cells assayed by immunoblot. Cyclin E1 increases after 72 h of Fbw7 siRNA treatment; Golgin 97 was used as a loading control. (B) Characterization of anti-FLAG immunopurified complexes containing Myc-RhoBTB3, FLAG-CUL3, and HA-RBX1 proteins in HEK293T cells treated either with control or Fbw7 siRNA. (C) In vitro–reconstituted ubiquitylation of Cyclin E using complexes shown in B. Top panel represents a long exposure of the same gel shown below (blotted using anti–Cyclin E). (D) Immunoblot detection of Cyclin E1 levels in HeLa cells treated with control (first lane) or Fbw7-siRNA (second and third lanes). After 24 h, cells were transfected either with Myc-empty vector (second lane) or Myc-RhoBTB3 (third lane); after 72 h total, extracts were analyzed as indicated. Overexpression of RhoBTB3 was detected using anti-Myc antibody; Golgin 97 was a loading control. Graph below shows mean Cyclin E levels from at least two independent experiments; error bars represent SEM. Molecular mass marker mobility is shown at left (A, B, and D) or right (C) in kilodaltons. (E and F) Model for RhoBTB3-mediated ubiquitylation of Cyclin E on the Golgi. Under physiological conditions (E), Golgi-associated RhoBTB3 binds to free Cyclin E and presents it to a CUL3–ubiquitin ligase for modification. Cyclin E can also be ubiquitylated by an SCF–Fbw7 pathway. Upon RhoBTB3 depletion (F), Cyclin E accumulates and Golgi fragmentation is observed.