The F-box Protein Rcy1 Is Involved in the Degradation of Histone H3 Variant Cse4 and Genome Maintenance

Abstract

Cse4, a histone H3-like centromeric protein, plays critical functions in chromosome segregation. Cse4 level is tightly regulated, but the underlying mechanism remains poorly understood. We employed a toxicity-based screen to look for the degradation components involved in Cse4 regulation. Here, we show that the F-box containing protein Rcy1 is required for efficient Cse4 turnover as Cse4 degradation is compromised in yeast cells lacking RCY1. Excessive Cse4 accumulation in rcy1⌬ cells leads to growth retardation. Furthermore, the deletion of RCY1 is tied to enhanced chromosome instability and temperature-sensitive cell growth. Our results reveal the involvement of Rcy1 in chromosome regulation and another regulatory pathway controlling the Cse4 level and activity.Chromosome segregation is key to equally divide the duplicated DNA to daughter cells. Following DNA replication, sister chromatids are joined at a highly condensed and constricted chromosome region called centromere (1,2). A distinct feature of the specialized centromeric chromatin is the incorporation of an essential histone H3 variant CENP-A (called Cse4 in yeast Saccharomyces cerevisiae), instead of histone H3 in nucleosomes (1,3,4).As altered localization and expression of CENP-A and its homologues have been linked to genome instability and cancers (1,5,6), CENP-A activity must be tightly regulated. Human CENP-A and yeast Cse4 have been found to be subject to proteasome-mediated degradation (1,7,8). Proteasome substrates are selected by a ubiquitin ligase E3, which collaborates with a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2) to covalently attach ubiquitin molecules onto substrates (9, 10). Ubiquitin-marked substrates are then delivered to and degraded by the proteasome. The key to understanding the role of proteolysis in CENP-A regulation is to identify E3 enzymes involved.Two groups previously isolated Psh1 as a yeast Cse4-binding protein via the affinity purification-mass spectrometry coupled approach and further demonstrated that Psh1 acts as an E3 ligase for Cse4 ubiquitylation and degradation (11,12). The mechanism underlying Psh1-mediated Cse4 turnover remains to be elucidated. Because Cse4 degradation is partially impaired in psh1⌬ cells, other E3s likely also promote Cse4 turnover (11-15).We have previously developed a synthetic dosage lethality screen to uncover physiological targets of several proteolytic pathways (16 -18). The synthetic lethality screen can be easily adapted to isolate relevant components involved in the degradation of a given substrate. Because Cse4 overexpression leads to toxicity in psh1⌬ cells (11, 12), we looked for the degradation mutants that grow slower upon Cse4 overexpression. Yeast cells lacking RCY1 are found to be sensitive to Cse4 expression and exhibit compromised Cse4 degradation. RCY1 encodes a F-box containing protein, which is a substrate recognition component of the Skp1-Cdc53-F box (SCF) 2 E3 complex (19). Rcy1 is one of the eight F-box proteins in yeast, but little is ...

Posttranslational mechanisms controlling centromere function and assembly

“…So far, four E3 ubiquitin ligases have been reported to mediate CENP-A Cse4 ubiquitylation. These include Psh1, Rcy1, Slx5 and Ubr1 [17–19,20 • ,21 • ,22,23]. CENP-A Cse4 is protected from degradation at the centromere, while CENP-A Cse4 in chromosome arms is degraded.…”

Skp, Cullin, F-box (SCF)-Met30 and SCF-Cdc4-Mediated Proteolysis of CENP-A Prevents Mislocalization of CENP-A for Chromosomal Stability in Budding Yeast

“…Ubiquitination of substrates for proteasomemediated degradation is catalyzed by three classes of enzymes, namely the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase [20][21][22]. Studies with budding yeast have identified the non-essential E3 ubiquitin ligase Psh1, Sumo-targeted ubiquitin ligases (STUbLs) Slx5, Slx8 and the Skp-Cullin-F-box (SCF)-Rcy1 in ubiquitin-mediated proteolysis of overexpressed Cse4 [23][24][25][26][27][28]. Both the N-terminus and the CENP-A targeting domain (CATD) in the C-terminus of Cse4 are required for Psh1-mediated proteolysis of overexpressed Cse4 [19,24,25].…”

Posttranslational modifications of CENP-A: marks of distinction

“…Ubiquitylation has been reported to prevent the ectopic localization of Cse4 by triggering its degradation, and the E3 ubiquitin ligases Psh1, Rcy1, Slx5, and Ubr1 have been shown to ubiquitylate Cse4 (Fig. 5) (Au et al 2013; Cheng et al 2017; Cheng et al 2016; Collins et al 2004; Hewawasam et al 2010; Ohkuni et al 2016; Ranjitkar et al 2010). While Psh1 ubiquitylates Lys-4, Lys-131, Lys-155, Lys-163 and Lys-172 (Hewawasam et al 2010), Slx5-mediated proteolysis of Cse4 is directed by sumoylation at Lys-65 (Ohkuni et al 2018) (Fig.…”