Fig. 2.

Exogenous Emi1 protein is destroyed through SCF^βTrCP in CSF extract; nonetheless, Emi1 can accumulate by de novo translation. (A) IVT Emi1 is destroyed in mitotic extract and CSF extract. IVT, radiolabeled Emi1 was incubated in mitotic Δ90 cyclin B extract or CSF extract and processed for autoradiography at the indicated times. (B) Destruction of exogenous Emi1 in CSF extract is conserved and DSGxxS sequence-dependent. IVT [³⁵S]Met Emi1 or Emi1 S95N was added to CSF extract and processed for autoradiography at the indicated times. (C) Accumulation of exogenous Emi1 protein translated in CSF egg extract. myc-Emi1 mRNA (WT or S95N mutant) was added to CSF extract with or without CHX and processed for immunoblot at the indicated times. (D) Exogenous Emi1 destruction in metaphase II eggs is mediated by the SCF^βTrCP ligase. Stage VI oocytes were injected with myc-Emi1, myc-Emi1 S95N, or simultaneously with myc-Emi1 and βTrCPΔF mRNA. Injected oocytes were left at G₂ arrest or matured by progesterone stimulation and processed for immunoblot. (E) Endogenous Emi1 is protected from destruction in CSF extract. Purified MBP-Emi1 protein was added to CSF extract supplemented with CHX and prepared for immunoblot at the indicated times after additions. (F) Endogenous Emi1 is stable in the maturing oocyte. Stage VI oocytes were injected with purified MBP-Emi1 protein and induced to mature by progesterone treatment. Emi1 was detected by immunoblotting lysates from immature oocytes, MI (GVBD) oocytes, and metaphase II eggs.