Cdk-phosphorylation of the Cln3 C-terminus is required for its degradation.

<p>(A) Mutation of Cdk consensus sites stabilizes Cln3. Cycloheximide-chase assay showing levels of Cln3-13Myc, Cln3-5A-13Myc, Cln3-3A-13Myc and Cln3-9A-13Myc after the addition of cycloheximide for the indicated number of minutes (min chx). Cdc28 is shown as a loading control. Diagram of mutant alleles is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002851#pgen.1002851.s002" target="_blank">Figure S2B</a>. (B) Expression of Cln3-9A accelerates progression through G1 phase. Cell cycle profiles of asynchronous wild-type cells and cells expressing Cln3-3A, Cln3-5A, or Cln3-9A. (C) Cdc4 and Grr1 bind to Cdk-phosphorylated Cln3. Myc and GST Western blots showing pull-downs of GST, GST-Cdc4ΔF and GST-Grr1ΔF proteins from <i>grr1Δ cdc4-1</i> cells expressing Cln3-13Myc (wt) or Cln3-9A-13Myc (9A). 2% input (i) and glutathione-sepharose bound proteins (b) are shown. (D) Cdc4 and Grr1 require different regions of the Cln3 C-terminus for targeting. Cycloheximide-chase assays of Cln3-5A-13Myc (top panels) and Cln3-3A-13Myc (bottom panels) in wild-type (wt), <i>grr1Δ</i> or <i>cdc4-1</i> cells. Cells were shifted to 37°C for 2 hours and cycloheximide was added for the indicated number of minutes. Cln3-13Myc and Cdc28 Western blots are shown. (E) Cdk-phosphorylation of Cln3 occurs <i>in cis</i>. Cycloheximide-chase assay of full-length Cln3-13Myc, Cln3ΔN-13Myc (Cln3 lacking amino acids 2-207), and Cln3ΔN-9A-13Myc. Each protein is expressed from the <i>TEF1</i> promoter. For all gels, molecular weight markers are indicated at the left.