👤 David P Toczyski

Levels of G1 cyclins fluctuate in response to environmental cues and couple mitotic signaling to cell cycle entry. The G1 cyclin Cln3 is a key regulator of cell size and cell cycle entry in budding yeast. Cln3 degradation is essential for proper cell cycle control; however, the mechanisms that control Cln3 degradation are largely unknown. Here we show that two SCF ubiquitin ligases, SCF(Cdc4) and SCF(Grr1), redundantly target Cln3 for degradation. While the F-box proteins (FBPs) Cdc4 and Grr1 were previously thought to target non-overlapping sets of substrates, we find that Cdc4 and Grr1 each bind to all 3 G1 cyclins in cell extracts, yet only Cln3 is redundantly targeted in vivo, due in part to its nuclear localization. The related cyclin Cln2 is cytoplasmic and exclusively targeted by Grr1. However, Cdc4 can interact with Cdk-phosphorylated Cln2 and target it for degradation when cytoplasmic Cdc4 localization is forced in vivo. These findings suggest that Cdc4 and Grr1 may share additional redundant targets and, consistent with this possibility, grr1Δ cdc4-1 cells demonstrate a CLN3-independent synergistic growth defect. Our findings demonstrate that structurally distinct FBPs are capable of interacting with some of the same substrates; however, in vivo specificity is achieved in part by subcellular localization. Additionally, the FBPs Cdc4 and Grr1 are partially redundant for proliferation and viability, likely sharing additional redundant substrates whose degradation is important for cell cycle progression. Show less

The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. We analyzed the architecture of the APC using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. We found that the largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). We found that the three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Delta complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. We have also found that the IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. We have provided an updated model for the subunit architecture of the APC. Show less