Figure 2.

Cdc4p and Grr1p are intrinsically unstable and their levels do not change during the cell cycle. (A) The half-life of Cdc4p-myc (JMG107; Upper two panels) and Grr1p-myc (JMG101; Lower two panels) was determined in cells arrested in G₁ by α-factor, in S-phase by hydroxyurea (HU), and in mitosis by nocodazole (Noc). CX was added at time 0, and aliquots were removed at times indicated (minutes) and analyzed by immunoblotting for the presence of the F-box proteins Cdc4p-myc or Grr1p-myc (upper panel of each pair) or Cdc34p (lower panel of each pair). Where indicated Cln2p-HA was used as an internal control (top panel). An extract prepared from exponentially growing cells is shown in the first lane. (B) Cells expressing Cln2p-HA and either Cdc4p-myc (JMG107; Upper two panels) or Grr1p-myc (JMG108; Lower two panels) were released from an α-factor block (time 0); aliquots were removed at the times indicated (minutes) and analyzed by immunoblotting for the levels of Cdc4p-myc or Grr1p-myc, Cln2p-HA, and Cdc34p as indicated. BE marks the time of bud emergence as determined microscopically. Note that the levels of the F-box proteins Cdc4p-myc and Grr1p-myc remain constant throughout the cell cycle.