F-Box Protein Specificity for G1 Cyclins Is Dictated by Subcellular Localization

Abstract

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, SCFCdc4 and SCFGrr1, 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.

Hst3 is turned over by a replication stress-responsive SCF Cdc4 phospho-degron

“…GST-Cdc4 fusion is expressed at lower levels than GST alone and therefore is not seen in the input with this exposure. The asterisk denotes a background band previously seen with these constructs (17). this notion, we find evidence for phosphorylation of many of the sites identified in degrons 1 and 2 even in the absence of RAD53.…”

“…We used GST-tagged Cdc4 lacking its F-box motif (GST-Cdc4ΔF) (17), which cannot interact with the other subunits of the SCF Cdc4 complex and therefore allows easier observation of the interaction with its substrates. As shown in Fig.…”

“…The Cdc4-binding experiment was performed as described in ref. 17. Cells were grown in C-URA medium with galactose to induce Hst3 (wild-type or d1m/d2m) for 9 h at 23°C.…”

“…The F-box protein is the substrate recognition module that confers substrate specificity to SCF ligases, with different F-box proteins recognizing different sets of substrates. The essential F-box protein Cdc4 regulates many cell-cycle-regulated proteins after their phosphorylation, including Sic1, Eco1, Cln3, and many others (14)(15)(16)(17)(18)(19).…”

Multiple Pho85-Dependent Mechanisms Control G1 Cyclin Abundance in Response to Nutrient Stress

“…Intriguingly, a closer look at the sequence surrounding site T520 phosphorylated by Pho80-Pho85 reveals a row of three S/TP sites that fulfill the requirement of a 2-to 3-aminoacid spacing characteristic of SCF Cdc4 -binding diphosphodegrons: WP-514 SPL 517 TPT 520 TPSLM (the S/TP amino acids are highlighted by boldface and underlining). Indeed, these three sites were previously shown to be required for Cdc4-dependent degradation (5). However, the phosphorylation of T520 by Pho85 introduces a negative charge in position ϩ3.…”