Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1)

Mol Cell Biol. 2002 Jul;22(13):4463-76. doi: 10.1128/MCB.22.13.4463-4476.2002.

Abstract

Degradation of Saccharomyces cerevisiae G(1) 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 (SCF(Grr1)). 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 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-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 SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Motifs
  • CDC28 Protein Kinase, S cerevisiae / genetics
  • CDC28 Protein Kinase, S cerevisiae / metabolism
  • Carrier Proteins*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism*
  • Cyclin-Dependent Kinase Inhibitor Proteins
  • Cyclins / genetics
  • Cyclins / metabolism*
  • F-Box Proteins*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • G1 Phase
  • Peptide Synthases / genetics
  • Peptide Synthases / metabolism
  • Phosphorylation
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • SKP Cullin F-Box Protein Ligases
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases*

Substances

  • CDC4 protein, S cerevisiae
  • CLN2 protein, S cerevisiae
  • Carrier Proteins
  • Cell Cycle Proteins
  • Cyclin-Dependent Kinase Inhibitor Proteins
  • Cyclins
  • F-Box Proteins
  • Fungal Proteins
  • Recombinant Fusion Proteins
  • SIC1 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin
  • GRR1 protein, S cerevisiae
  • SKP Cullin F-Box Protein Ligases
  • Ubiquitin-Protein Ligases
  • CDC28 Protein Kinase, S cerevisiae
  • Peptide Synthases