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

Abstract

Restricting the localization of the histone H3 variant CENP-A (Cse4 in yeast, CID in flies) to centromeres is essential for faithful chromosome segregation. Mislocalization of CENP-A leads to chromosomal instability (CIN) in yeast, fly and human cells. Overexpression and mislocalization of CENP-A has been observed in many cancers and this correlates with increased invasiveness and poor prognosis. Yet genes that regulate CENP-A levels and localization under physiological conditions have not been defined. In this study we used a genome-wide genetic screen to identify essential genes required for Cse4 homeostasis to prevent its mislocalization for chromosomal stability. We show that two Skp, Cullin, Fbox (SCF) ubiquitin ligases with the evolutionarily conserved F-box proteins Met30 and Cdc4 interact and cooperatively regulate proteolysis of endogenous Cse4 and prevent its mislocalization for faithful chromosome segregation under physiological conditions. The interaction of Met30 with Cdc4 is independent of the D domain, which is essential for their homodimerization and ubiquitination of other substrates. The requirement for both Cdc4 and Met30 for ubiquitination is specifc for Cse4; and a common substrate for Cdc4 and Met30 has not previously been described. Met30 is necessary for the interaction between Cdc4 and Cse4, and defects in this interaction lead to stabilization and mislocalization of Cse4, which in turn contributes to CIN. We provide the first direct link between Cse4 mislocalization to defects in kinetochore structure and show that SCF-mediated proteolysis of

Reduced Gene Dosage of Histone H4 Prevents CENP-A Mislocalization inSaccharomyces cerevisiae

“…The HHT1/hhf1-20 psh1Δ strain showed rapid degradation of Cse4 when compared to the HHT1/HHF1 psh1Δ and HHT1/hhf1-10 psh1∆ strains ( Figure S5). The rapid degradation of overexpressed Cse4 in the HHT1/hhf1-20 psh1Δ strain is consistent with previous studies for a correlation between higher protein stability and GALCSE4 SDL (HEWAWASAM et al 2010;RANJITKAR et al 2010;CIFTCI-YILMAZ et al 2018;AU et al 2020;EISENSTATT et al 2020) and suggests that defective Cse4-H4 interaction contributes to the lack of GALCSE4 SDL in psh1∆ strains.…”

“…Our ChIP-qPCR data showed reduced levels of Cse4 at peri-centromeric and non-centromeric regions in psh1Δ hhf1Δ and psh1Δ hhf2Δ strains when compared to the psh1Δ strain. The mislocalization of overexpressed Cse4 to non-centromeric regions contributes to highly stable Cse4 in psh1Δ, slx5Δ, doa1Δ, hir2Δ, cdc4-1, and cdc7-4 strains (HEWAWASAM et al 2010;RANJITKAR et al 2010;AU et al 2013;OHKUNI et al 2016;CIFTCI-YILMAZ et al 2018;AU et al 2020;EISENSTATT et al 2020). We reasoned that reduced mislocalization of Cse4 to non-centromeric regions in psh1Δ hhf2∆ strains may contribute to faster degradation of Cse4 in these strains.…”

“…Previous studies have shown that mislocalization of Cse4 to non-centromeric regions contributes to the GALCSE4 SDL in psh1Δ, slx5Δ, doa1Δ, hir2Δ, cdc4-1, and cdc7-4 strains (HEWAWASAM et al 2010;RANJITKAR et al 2010;AU et al 2013;OHKUNI et al 2016;CIFTCI-YILMAZ et al 2018;AU et al 2020;EISENSTATT et al 2020). We sought to define mechanisms that prevent lethality due to mislocalization of overexpressed Cse4.…”

“…To determine if the SDL suppression by reduced H4 gene dosage is limited to the psh1Δ GALCSE4 strain, we deleted HHF1 or HHF2 in deletion or mutant strains encoding Slx5, Doa1, Hir2, Cdc4, and Cdc7 as deletion or mutation of these factors show SDL with GALCSE4 and mislocalization of transiently overexpressed Cse4 (AU et al 2013;OHKUNI et al 2016;CIFTCI-YILMAZ et al 2018;AU et al 2020;EISENSTATT et al 2020). Growth on galactose media revealed that the SDL of doa1Δ, slx5Δ, cdc4-1, and cdc7-4 GALCSE4 strains is suppressed when either HHF1 or HHF2 is deleted ( Figures 2E and 2F and Table 2), while the SDL of hir2Δ GALCSE4 is suppressed only when HHF2 is deleted ( Figure 2E and Table 2).…”

“…In budding yeast, post-translational modifications (PTMs) of Cse4, such as ubiquitination, sumoylation, and isomerization, are important for regulating steady-state levels of Cse4 and preventing its mislocalization to non-centromeric regions, thereby maintaining chromosome stability (COLLINS et al 2004;HEWAWASAM et al 2010;RANJITKAR et al 2010;OHKUNI et al 2014;OHKUNI et al 2016;CHENG et al 2017;AU et al 2020). Ubiquitin-mediated proteolysis of Cse4 by E3 ubiquitin ligases such as Psh1 (HEWAWASAM et al 2010;RANJITKAR et al 2010), SUMO-targeted ubiquitin ligase (STUbL) Slx5 (OHKUNI et al 2016), SCF Met30/Cdc4 (AU et al 2020), SCF Rcy1 (CHENG et al 2016), and Ubr1 (CHENG et al 2017) and the proline isomerase Fpr3 (OHKUNI et al 2014) regulate the cellular levels of Cse4.…”