Cdc123 and Checkpoint Forkhead Associated with RING Proteins Control the Cell Cycle by Controlling eIF2γ Abundance

Abstract

Eukaryotic initiation factor 2 (eIF2) is a central regulator of translational initiation in times of growth and times of stress. Here we discovered three new conserved regulators of eIF2 in Saccharomyces cerevisiae. cdc123, homolog of mammalian D123, is a new cell division cycle mutant with a G 2 delay at permissive temperature and a terminal, mating-proficient G 1 arrest point. Cdc123 protein is regulated by nutrient availability. CHF1 and CHF2, homologs of mammalian checkpoint forkhead associated with RING genes, are required for G 2 delay and G 1 arrest of cdc123-4 and promote G 1 delay when overexpressed. Cell cycle delaying activity and the natural instability of Chf1 and Chf2 depend on the integrity of both domains and association with Cdc123. Genetic analysis maps the Chf1 forkhead associated domainbinding site to the conserved Thr-274 of Cdc123, suggesting that mammalian D123 is a key target of Chfr. Gcd11, the ␥ subunit of eIF2, is an additional Cdc123-interacting protein that is an essential target of the Cdc123 cell cycle promoting and Chf cell cycle arresting activity whose abundance is regulated by Cdc123, Chf1, and Chf2. Loss of cdc123 activity promotes Chf1 and Chf2 accumulation and Gcd11 depletion, accounting for the essentiality of Cdc123. The data establish the Cdc123-Chf-Gcd11 axis as an essential pathway for nutritional control of START that runs parallel to the Tor-Gcn2-Sui2 system of translational control.

Yeast Chfr homologs retard cell cycle at G1and G2/M via Ubc4 and Ubc13/Mms2-dependent ubiquitination

“…To address E2 utilization by Chf proteins in vivo, we exploited the slow cell division phenotype of Chf2 overexpression 44 to identify genes encoding factors essential for Chf2-dependent cell cycle delays. Because the slow cell division phenotypes of GAL1-driven Chf proteins depend on intact RING domains, we hypothesized that overexpression of the E3 ligases causes depletion or dysregulation of one or more cell cycle regulators by ubiquitination in a manner that depends on a specific E2 Ub conjugating enzyme.…”

“…46,47 Chf1 and Chf2 interact with the positive cell cycle regulator Cdc123 and are required for both the G 1 and G 2 cell cycle phenotypes of cdc123-4. 44 At the permissive temperature of cdc123-4, most cells are delayed in G 2 /M and the mitotic accumulation of cdc123-4 cells is lost in cdc123-4 chf1 chf2 mutants. 44 Additionally, chf1 chf2 mutants fail to assemble a septin ring properly.…”

“…44 At the permissive temperature of cdc123-4, most cells are delayed in G 2 /M and the mitotic accumulation of cdc123-4 cells is lost in cdc123-4 chf1 chf2 mutants. 44 Additionally, chf1 chf2 mutants fail to assemble a septin ring properly. 45 However, two lines of evidence indicate that Chf proteins function in G 1 in addition to G 2 /M.…”

“…Chf1 and Chf2 from S. cerevisiae, 44 alternately termed Dma1 and Dma2, 45 possess Chfr and Rnf8-like domain structures consisting of variable N-terminal regions, central FHA domains and C-terminal RING domains. Consistent with the requirement of Chfr to impose a checkpoint late in the cell cycle, 24 S. pombe dma1 plays an important role in cytokinesis.…”

“…Second, overexpression of Chf1 or Chf2 retards the cell cycle in a RING and FHA-dependent manner and produces an excess of cells in G 1 phase. 44 Here we report a detailed biochemical and genetic analysis of the functions of the Chfr and Rnf8 related proteins, Chf1 and Chf2. We provide genetic evidence that Ubc4 and Ubc13/Mms2 are required for distinct cell cycle-delaying activities of Chf2, early and late in the cell cycle respectively.…”