Blocking membrane traffic triggers a checkpoint arrest. (A) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. Cleavage of Mcd1-6×HA was assayed by Western blotting. (B) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. DNA staining was used to determine the percentage of cells with multiple nuclei. (C) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 45 min after release. Levels of Clb2 were assayed by Western blotting. (D and E) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. The percentage of cells with short or long spindles was determined. Error bars represent SEMs for three biological replicates. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Normal signaling to Mih1 and Swe1 fails to occur when membrane traffic is blocked. (A and B) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 30 min after release. The behavior of Mih1 and Swe1 were assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Blocking membrane traffic triggers rapid signaling to Mih1. (A and B) Cells were released from a G1 arrest and were shifted to the restrictive temperature (34°C) at 70 min after release, when Mih1 dephosphorylation was being initiated. Mih1 phosphorylation was assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

The response to arrest of membrane traffic is not a consequence of indirect effects on actin. (A) Wild-type cells were released from a G1 arrest at room temperature. A sample was taken at 70 min after release (t = 0), and the cells were divided into two aliquots. Latrunculin A (LatA) was added to one aliquot, and solvent (DMSO) was added to the other as a control. Mih1 phosphorylation was assayed by Western blotting. (B) Cells were grown to log phase in YPD media and then shifted to the restrictive temperature (34°C) for 5 min. Cells were then fixed and stained with FITC-phalloidin. Bar, 5 µm. Numbers shown next to the Western blot indicate molecular mass in kilodaltons.

Overexpression of Zds1 drives cells through the checkpoint arrest. (A–C) Cells were grown in YEP with 2% raffinose and arrested in G1 with α-factor at 25°C. Cells were released from the arrest into YEP with 2% raffinose at 25°C. When 10% of cells had undergone bud emergence, they were shifted to the restrictive temperature (34°C) to induce the checkpoint arrest. After 30 min, galactose was added to induce expression of ZDS1. Cleavage of Mcd1-3×HA and phosphorylation of Mih1 and Swe1 were assayed by Western blotting. The asterisks denote background bands that appear with some batches of purified anti-Mih1 antibody. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Pkc1 associates with PP2A^Cdc55 and is required for Mih1 dephosphorylation. (A) Anti-HA antibodies were used to immunoprecipitate PP2A^Cdc55-3×HA from wild-type, zds1Δ zds2Δ, and untagged control cells. Coprecipitation of Pkc1 was assayed by Western blotting with anti-Pkc1 antibodies. Crude extract samples were electrophoresed longer than the immunoprecipitated samples to resolve phosphorylation forms. (B) Cells were inoculated into YPD media at low density and grown at 30°C until they reached an OD of ∼1.7. Bar, 10 µm. (C) Cells were released from a G1 arrest and shifted to the restrictive temperature (34°C) at 75 min after release. Mih1 phosphorylation and Clb2 levels were assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Pkc1 signals to Mih1 via PP2A^Cdc55-Zds1/2. (A) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 30°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. (B) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 25 or 34°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. (C) Cells were grown to log phase in YEP media containing 2% glycerol and 2% ethanol. Cells were shifted to 34°C for 60 min to induce a checkpoint arrest, and galactose was then added. Mih1 phosphorylation was assayed by Western blotting. (D) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol. Galactose was added, and cells were shifted to 30°C at t = 0. Zds1 phosphorylation was assayed by Western blotting. The asterisks denote background bands that appear with some batches of purified anti-Mih1 antibody. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Rho1 signals to Mih1 via Pkc1. (A) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 35 min after release. Mih1 phosphorylation was assayed by Western blotting. The arrowhead marks a hyperphosphorylated form that appears in the rho1-2 cells. At 34°C, rho1-2 cells grow slowly but are viable. (B) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 45 min after release. Swe1 phosphorylation was assayed by Western blotting. (C) Cells were released from a G1 arrest and shifted to a semirestrictive temperature (34°C) at 45 min after release. Clb2 levels were assayed by Western blotting. (D) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol. Galactose was added, and the cells were shifted to 34°C at t = 0. Mih1 phosphorylation was assayed by Western blotting. The pkc1-21 allele was used because it was found to cause rapid inactivation of Pkc1. (E) Cells were grown to log phase in YEP with 2% glycerol and 2% ethanol. The cells were shifted to 34°C for 60 min to induce a checkpoint arrest, and galactose was then added. Mih1 phosphorylation was assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

Signaling to Pkc1 is dependent on membrane traffic. (A) Cells were released from a G1 arrest at room temperature. The behavior of Pkc1, Cln2-3×HA, and Clb2 were assayed by Western blotting. All blots are from the same samples, so timing of events may be directly compared. (B) Wild-type and sec6-4 cells were released from a G1 arrest. A sample was taken at 70 min (t = 0), and the cells were then shifted to the restrictive temperature (34°C). Pkc1 phosphorylation was assayed by Western blotting. Numbers shown next to the Western blots indicate molecular mass in kilodaltons.

A model for signals that link mitotic entry to membrane growth. (A) Dependency relationships in the Rho1–Pkc1 signaling axis. (B) Known binding interactions in the Rho1–Pkc1 signaling axis. (C) A hypothetical model for generation of a signal that is proportional to membrane growth. Rho1 is activated at the site of membrane growth by a guanine nucleotide exchange factor (GEF). As more Rho1-bearing vesicles are delivered to the site of growth, the amount of active Rho1 increases. Downstream components of the signaling axis read the signal and flip a switch to initiate mitosis when the signal reaches a threshold level.