![Figure 5. Fus2p phosphorylation regulates localization. (A) The N-terminal domain of Fus2p is sufficient to localize GFP to the nucleus in mitotic cells and the cytoplasm in α factor–arrested cells. MY9181 containing full-length PGAL1-FUS2∷GFP104 (pMR5469) or the truncation mutant PGAL1-fus21-104-GFP (pMR5774) were pregrown in selective media with galactose; α factor was added 2 h before imaging. (B) Mutations at S84 affect the nuclear/cytoplasmic distribution of NTD. MY9181 containing PGAL1-fus21-104-GFP (pMR5774) with the indicated point mutations (Table II) were grown as in A. For each strain, 18 ≤ n ≤ 37. Asterisks indicate mutants significantly different from the wild-type control (P ≤ 0.0001 by one-way analysis of variance and Dunnett's multiple comparison). (C) NTD is phosphorylated in a Fus3p-dependent manner during pheromone signaling. MY9181 (wild type [WT]) or MY10273 (fus3 cln3) containing PGAL1 vector (YCpIF5), PGAL1-fus21-104-GFP (pMR5774), or pMR5774 with the indicated S84 and S100 mutations were grown in selective medium containing galactose and treated with α factor as indicated. Samples were run on standard gels (top) or gels containing 25 µM Phos-tag (bottom) to resolve phosphoisoforms. Proteins were detected by Western blot using α-GFP. SS, SA, and AA refer to the specific amino acids at residues 84 and 100. (D) Fus3p phosphorylates Fus2p in vitro. Flag-tagged Fus3p or kinase-dead (KD) Fus3pK42R was immunoprecipitated from α factor–treated yeast cultures and used to phosphorylate 6xHN-tagged Fus21–328 purified from Escherichia coli. (E) Mutations at S84 affect the nuclear/cytoplasmic distribution of full-length Fus2p-GFP. MY10174 containing PGAL1-FUS2∷GFP104 (pMR5469) or derivatives containing the indicated point mutations were grown and examined as in A. (F) Quantification of the experiment shown in E. For each strain/condition, 20 ≤ n ≤ 26. (G) S84E is insensitive to Fus3p for export. MY10174 or MY10273 containing either wild-type PGAL1-FUS2∷GFP104 (pMR5469) or the S84E mutant were grown, treated with α factor, and examined as in A. (H) Quantification of the experiment shown in G. For each strain, 22 ≤ n ≤ 25 cells. (B, F, and H) Box and whisker plots of the ratio of GFP fluorescence in the nucleus relative to an equal area of the cytoplasm are shown. Rectangles show the two inner quartiles separated by the median. Error bars indicate the entire range, and circles indicate outliers. Bars, 1 µm.](https://cdn.rupress.org/rup/content_public/journal/jcb/184/3/10.1083_jcb.200809066/7/jcb_200809066r_gs_fig5.jpeg?Expires=1767782509&Signature=xlB7~MyEHSwlXrWQ2PI4QnjwUxtj~kpV2JJJ~CEjrXqyb8GeW2YwsVmoyI980kRfNXQ7JLojeEiBcLOzeskW00o6wN4m6XZjI1CbWH8hLnONxRmWYPhmSCKwKgtYADaUgc~Hjg9OUOCfynivpOozADeRDt9XmTJS90d4~Z-VPruG9Z1vN47lmmW3w-KV-Y4yq10pSxmCrYz9E-s9MJVZw9ukk8Ke7PPR~ceoWor8IeaDFLNSUQgcH~mxHcf7~ICJQDMCbmDfMt9T1~FEyrU7ZruawthYT6NEtsUlCUx~lCw-DHGXosePGYHymfXOdrpq2atJLIsUDv0CqjxE6mUd7A__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Fus2p phosphorylation regulates localization. (A) The N-terminal domain of Fus2p is sufficient to localize GFP to the nucleus in mitotic cells and the cytoplasm in α factor–arrested cells. MY9181 containing full-length PGAL1-FUS2∷GFP104 (pMR5469) or the truncation mutant PGAL1-fus21-104-GFP (pMR5774) were pregrown in selective media with galactose; α factor was added 2 h before imaging. (B) Mutations at S84 affect the nuclear/cytoplasmic distribution of NTD. MY9181 containing PGAL1-fus21-104-GFP (pMR5774) with the indicated point mutations (Table II) were grown as in A. For each strain, 18 ≤ n ≤ 37. Asterisks indicate mutants significantly different from the wild-type control (P ≤ 0.0001 by one-way analysis of variance and Dunnett's multiple comparison). (C) NTD is phosphorylated in a Fus3p-dependent manner during pheromone signaling. MY9181 (wild type [WT]) or MY10273 (fus3 cln3) containing PGAL1 vector (YCpIF5), PGAL1-fus21-104-GFP (pMR5774), or pMR5774 with the indicated S84 and S100 mutations were grown in selective medium containing galactose and treated with α factor as indicated. Samples were run on standard gels (top) or gels containing 25 µM Phos-tag (bottom) to resolve phosphoisoforms. Proteins were detected by Western blot using α-GFP. SS, SA, and AA refer to the specific amino acids at residues 84 and 100. (D) Fus3p phosphorylates Fus2p in vitro. Flag-tagged Fus3p or kinase-dead (KD) Fus3pK42R was immunoprecipitated from α factor–treated yeast cultures and used to phosphorylate 6xHN-tagged Fus21–328 purified from Escherichia coli. (E) Mutations at S84 affect the nuclear/cytoplasmic distribution of full-length Fus2p-GFP. MY10174 containing PGAL1-FUS2∷GFP104 (pMR5469) or derivatives containing the indicated point mutations were grown and examined as in A. (F) Quantification of the experiment shown in E. For each strain/condition, 20 ≤ n ≤ 26. (G) S84E is insensitive to Fus3p for export. MY10174 or MY10273 containing either wild-type PGAL1-FUS2∷GFP104 (pMR5469) or the S84E mutant were grown, treated with α factor, and examined as in A. (H) Quantification of the experiment shown in G. For each strain, 22 ≤ n ≤ 25 cells. (B, F, and H) Box and whisker plots of the ratio of GFP fluorescence in the nucleus relative to an equal area of the cytoplasm are shown. Rectangles show the two inner quartiles separated by the median. Error bars indicate the entire range, and circles indicate outliers. Bars, 1 µm.
