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 ...