Transferable Domain in the G₁ Cyclin Cln2 Sufficient To Switch Degradation of Sic1 from the E3 Ubiquitin Ligase SCF^Cdc4 to SCF^Grr1

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.

DNA Damage Response-mediated Degradation of Ho Endonuclease via the Ubiquitin System Involves Its Nuclear Export

“…However, despite the fact that deletion of its PEST sequence stabilizes Ho (6), we found that this does not abolish binding to Ufo1. This suggests that there are other phosphorylated residues on Ho that participate in binding to Ufo1 and is consistent with the finding that PEST sequences alone, when fused to a heterologous protein, are not enough to destabilize it (12,43). Indeed our data show that Ho with a single mutation in its PEST sequence, threonine 225 to alanine, although resistant to degradation, still binds Ufo1 with an affinity not visibly different from that shown by wild type Ho.…”

BSCTV C2 Attenuates the Degradation of SAMDC1 to Suppress DNA Methylation-Mediated Gene Silencing in Arabidopsis

“…The PEST sequence has been reported to be located in some proteins with fast turnover rates and be essential for the interaction between SCF complex and target proteins, which consequently leads to ubiquitination/26S proteasome-mediated degradation (Kiernan et al, 2001;Berset et al, 2002;Blondel et al, 2005;Pal et al, 2007). Human SAMDC was reported to have a short half-life and is regulated at multiple levels, such as the transcriptional, translational, and posttranslational levels (Shantz et al, 1992;Stanley et al, 1994;Hanfrey et al, 2003;Yerlikaya and Stanley, 2004).…”

“…SAMDC is synthesized as an inactive proenzyme that generates two subunits, termed a and b, by an autocatalytic cleavage reaction. SAMDCs in many organisms are highly conserved, sharing a defined autocleavage recognition site and a PEST region enriched in Pro (P), Glu (E), Ser (S), and Thr (T) that is correlated with the protein's rapid turnover (Salama et al, 1994;Rechsteiner and Rogers, 1996;Berset et al, 2002;Blondel et al, 2005;Pal et al, 2007). SAMDC is expressed throughout the cell, and its expression is regulated at multiple levels, such as the transcriptional, translational, and posttranslational levels (Shantz et al, 1992;Stanley et al, 1994;Hanfrey et al, 2003;Yerlikaya and Stanley, 2004).…”

Direct Binding to Rsp5 Mediates Ubiquitin-independent Sorting of Sna3 via the Multivesicular Body Pathway

“…Note that the low endogenous expression of Doa4 is difficult to detect. (D) Western blot analysis of extracts prepared from pep4⌬ doa4 C571S SNA3-HA cells (MMY64) that had been transformed either with empty low-copy vector (pRS414) or pRS414 encoding Myc-Ub (pUB223; Berset et al, 2002) and incubated with 250 M CuSO 4 to induce overexpression of Myc-Ub. Unmodified full-length Sna3-HA migrates at ϳ25 kDa.…”