Figure 3.
Biochemical Activation and Subcellular Localization Are Independent Layers of Yen1 Regulation
(A) Nuclear localization of Yen1 throughout the cell cycle. Yen1-myc18 at different stages of the cell cycle was analyzed by immunofluorescence. Spindle morphology and DNA were visualized using anti-tubulin antibodies and DAPI. The contours of the cells, determined from differential interference contrast (DIC) images, are depicted by a dotted line.
(B) As in (A), but with Yen1ON.
(C) Subcellular localization of Yen1 phosphomutants. Nuclear enrichment (as opposed to diffuse pan-cellular staining) of the indicated Yen1 mutants was analyzed as in (A) and quantified (200 cells per condition). The schematic indicates the mutations at the Cdk sites in each allele. Protein domains are colored as in Figure 1A.
(D) Analysis of the biochemical activities of the Yen1 mutants used in (C). Myc9-tagged proteins were immunoprecipitated from S phase-arrested cells, and their nuclease activities were assayed.
(E) Suppression of the mus81Δ phenotype depends on both the biochemical activation and nuclear localization of Yen1. mus81Δ strains carrying the indicated YEN1 alleles were analyzed as in Figure 2B.