Transferable Domain in the G1 Cyclin Cln2 Sufficient To Switch Degradation of Sic1 from the E3 Ubiquitin Ligase SCFCdc4 to SCFGrr1

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Abstract
Degradation of Saccharomyces cerevisiae G1 cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCFGrr1). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (ΔN-Sic1), a substrate of SCFCdc4, results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to ΔN-Sic1 switches degradation of Sic1 from SCFCdc4 to SCFGrr1. ΔN-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCFCdc4 target for SCFGrr1-mediated degradation by the ubiquitin-proteasome pathway.