Figure 6 .

Nab3 localization and acetylation is altered in esa1 cells. (A) Nab3 localization is aberrant in the esa1 mutant. Top: At a permissive temperature (28°), Nab3 staining in wild type (LPY4909) and esa1 (LPY4911) cells appears as punctate nuclear foci interspersed with diffuse nuclear staining. Sir2 localization demarcates the nucleolus in a crescent shape (inset, green) and is normal. At a restrictive temperature (37°), Nab3 staining is diffuse in the esa1 mutant but appears normal in the wild-type strain. No Sir2 foci are observed in the esa1 mutant. Bottom: WT and esa1 strains used above were grown at permissive and elevated temperatures and used for immunoblots to detect total Nab3 levels using anti-Nab3. Anti-PGK1 (phosphoglycerate kinase) was used as a loading control. (B) Nab3 is acetylated in vivo in an ESA1-dependent manner. To detect posttranslational acetylation of Nab3, a WT (LPY15000) and esa1 (LPY15004) strain containing a chromosomal FLAG-tagged version of Nab3 were grown at an elevated temperature (37°) and used in an anti-FLAG immunoprecipitation followed by an immunoblot with anti-acetyl lysine. Decreased levels of Nab3 acetylation are observed in the esa1 mutant. Quantification of films from independent experiments shows a 48% decrease in Nab3 acetylation in esa1 compared with wild-type. An untagged WT strain (LPY5) is used as a negative control. Nab3-FLAG levels are not themselves altered in the esa1 mutant, as demonstrated by control immunoblotting of immunoprecipitations and inputs with anti-FLAG.