A Novel Role of the N Terminus of Budding Yeast Histone H3 Variant Cse4 in Ubiquitin-Mediated Proteolysis

Abstract

Regulating levels of centromeric histone H3 (CenH3) variant is crucial for genome stability. Interaction of Psh1, an E3 ligase, with the C terminus of Cse4 has been shown to contribute to its proteolysis. Here, we demonstrate a role for ubiquitination of the N terminus of Cse4 in regulating Cse4 proteolysis for faithful chromosome segregation and a role for Doa1 in ubiquitination of Cse4.C ENTROMERIC histone H3 (CenH3), an evolutionarily conserved histone H3 variant, is essential for chromosome segregation (Stoler et al. 1995;Meluh et al. 1998;Blower and Karpen 2001;Maddox et al. 2012). Mis-localization and overexpression of CenH3 has been observed in cancers and associated with aneuploidy in Drosophila melanogaster (Tomonaga et al. 2003;Heun et al. 2006;Moreno-Moreno et al. 2006). Studies from budding yeast and fruit flies have shown that proteolysis of CenH3 plays an important role in preventing its mis-localization MorenoMoreno et al. 2006 MorenoMoreno et al. , 2011. Psh1, an E3 ligase, interacts with Cse4, the budding yeast CenH3, and mediates its ubiquitination for proteolysis (Hewawasam et al. 2010;Ranjitkar et al. 2010). Despite ubiquitination of Cse4 by Psh1, Cse4 is only partially stabilized when overexpressed in psh1Δ strains (Hewawasam et al. 2010;Ranjitkar et al. 2010), suggesting that additional factors regulate Cse4 protein stability.In this study, we investigated the role of Cse4 domains in directing Cse4 proteolysis and used a genome-wide screen to identify pathways that regulate Cse4 stability. Since subcellular levels of Cse4 are stringently regulated, strains overexpressing CSE4 or cse4 mutants have been used to identify proteolytic pathways and factors such as Psh1 that mediate its degradation (Hewawasam et al. 2010;Ranjitkar et al. 2010). We initiated our studies with a mutant cse416KR (16KR) and 16KR fusion mutants in which lysines (K) are mutated to arginines (R) (Figure 1, A-C; Supporting Information, Table S1 and File S1). Overexpression of 16KR leads to defects in chromosome segregation, and this correlates with enrichment in chromatin and increased protein stability Au et al. 2008) . Our results show that the stability of CK (fusion of Cse4 N terminus amino acids 1-135 and 16KR C terminus) is low (t 1/2 = 26 6 6 min) and is comparable to that of Cse4 (t 1/2 = 34 6 3 min), indicating that the lack of ubiquitination of the C terminus due to K-to-R mutations does not increase protein stability. In contrast, the stability of KC (a fusion of 16KR N terminus and Cse4 C terminus) is high (t 1/2 . 120 min) and similar to 16KR (Figure 1, B and C), showing that K-to-R mutations in the N terminus of Cse4 increase protein stability. Consistent with these results are previous observations showing that Cse4 lacking the N terminus (Δ129) is fivefold more stable than full-length Cse4 (Morey et al. 2004). Increased Cse4 stability correlates with chromosome loss, as KC and 16KR strains exhibit fourfold higher chromosome loss than strains expressing CK or wild-type Cse4 ( Figure 1G). Based on ...

Ectopic Centromere Nucleation by CENP-A in Fission Yeast

“…Together, these findings suggest that the N terminus of CENP-A Cnp1 contributes to the exclusion of CENP-A Cnp1 from noncentromeric chromatin by targeting mislocalized CENP-A Cnp1 for degradation via the ubiquitin-dependent pathway. This is consistent with a study showing that, in budding yeast, the N terminus of Cse4 mediates polyubiquitination of the protein (Au et al 2013), although it remains unknown whether deleting the domain results in assembly of Cse4 at ectopic loci.…”

“…Together, these findings suggest that the N terminus of CENP-A Cnp1 contributes to the exclusion of CENP-A Cnp1 from noncentromeric chromatin by targeting mislocalized CENP-A Cnp1 for degradation via the ubiquitin-dependent pathway. This is consistent with a study showing that, in budding yeast, the N terminus of Cse4 mediates polyubiquitination of the protein (Au et al 2013), although it remains unknown whether deleting the domain results in assembly of Cse4 at ectopic loci.Here we also provide the first evidence that increasing the level of either histone H3 or H4 in cells overexpressing CENP-A cnp1 is able to inhibit the assembly of CENP-A cnp1 chromatin at ectopic loci. In the case of H4, its overexpression can rescue the growth and chromosome segregation defects caused by the overexpression of CENP-A cnp1 .…”

The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A

“…S3; Ranjitkar et al 2010). As expected, these species were substantially decreased but not eliminated in psh1Δ cells, consistent with additional Psh1-independent pathways contributing to CENP-A Cse4 ubiquitylation (Collins et al 2004;Au et al 2013). Strikingly, CENP-A Cse4 purified from psh1ΔC-13Myc cells also displayed a significant decrease in CENP-A Cse4 ubiquitylation (Figs.…”

“…5C) were used as the substrate instead of octamers in ubiquitylation assays in vitro. We speculate that the slight ubiquitylation of CENP-A Cse4 nucleosomes by Psh1 reflects (Au et al 2013). To ensure that the decrease in activity was not due to direct inhibition of Psh1 by DNA, we added the DNA used to assemble the mononucleosomes to the ubiquitylation assays containing CENP-A Cse4 octamers.…”

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…Cse4 can also incorporate into euchromatin, especially at sites of high histone turnover (Collins et al 2004;Lefrancois et al 2009;Krassovsky et al 2012). Cse4 does not stably incorporate into euchromatin because its protein levels are tightly controlled by proteolysis via the Psh1 E3 ubiquitin ligase and additional mechanisms (Collins et al 2004;Hewawasam et al 2010;Ranjitkar et al 2010;Au et al 2013). In the absence of proteolysis, Cse4 levels increase and its overexpression in these cells leads to mislocalization throughout euchromatin and subsequent lethality.…”