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Regulation of the Subcellular Localization of Cyclic AMP-Dependent Protein Kinase in Response to Physiological Stresses and Sexual Differentiation in the Fission Yeast Schizosaccharomyces pombe

Yasuhiro Matsuo, Brittney McInnis, Stevan Marcus
Yasuhiro Matsuo
Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487
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Brittney McInnis
Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487
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Stevan Marcus
Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487
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  • For correspondence: smarcus@bama.ua.edu
DOI: 10.1128/EC.00168-08
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  • FIG. 1.
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    FIG. 1.

    Subcellular localization of S. pombe PKA regulatory and catalytic subunits. (A) Immunoblot analysis of GFP fusion protein expression in cell lysates prepared from SP870 (Control), YMSM105 (cgs1-GFP), and YMSM101 (pka1-GFP) strains cultured overnight to log phase in YEAU medium. (B) Fluorescence photomicrographs of SP870 (Control), cgs1-GFP (Cgs1-GFP), and pka1-GFP (Pka1-GFP) S. pombe strains cultured in YEAU medium to mid-log phase. (C) Fluorescence photomicrographs of cgs1-GFP (Cgs1-GFP) and pka1-GFP (Pka1-GFP) cells were cultured in glucose-limited YEAU medium (YEAU-Glycerol) to mid-log phase.

  • FIG. 2.
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    FIG. 2.

    cAMP is required for nuclear localization of Cgs1-GFP and Pka1-GFP. (A) Fluorescence photomicrographs of cyr1Δ cgs1-GFP (left panels) and cyr1Δ pka1-GFP (right panels) cells cultured in YEAU (YEAU-glucose) or YEAU-glycerol to mid-log phase. (B) Fluorescence photomicrographs of cyr1Δ cgs1-GFP and cyr1Δ pka1-GFP cells cultured in YEAU with 100 mM cAMP for 1.5 h and YEAU with 5 mM cAMP for 0.5 h, respectively. (C) Fluorescence photomicrographs of pka1Δ cgs1-GFP cells cultured in YEAU to mid-log phase.

  • FIG. 3.
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    FIG. 3.

    Cgs1p contributes to regulation of subcellular localization of Pka1p. Fluorescence photomicrographs of cgs1Δ pka1-GFP (A) and cyr1Δ cgs1Δ pka1-GFP (B) strains cultured in YEAU (YEAU-Glucose) (left panels) or YEAU-Glycerol (right panels) to mid-log phase are shown.

  • FIG. 4.
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    FIG. 4.

    KCl-induced nuclear-cytoplasmic redistribution of Cgs1-GFP and Pka1-GFP. (A) Fluorescence photomicrographs of cgs1-GFP (Cgs1-GFP) and pka1-GFP (Pka1-GFP) cells cultured to mid-log phase in YEAU containing 1.2 M KCl (YEAU+KCl). cgs1-GFP (B) and pka1-GFP (C) strains were cultured in YEAU to 2 × 106 cells/ml and then diluted with YEAU containing 3 M KCl for a final concentration of 1.2 M KCl. The cultures were then examined by fluorescence microscopy at the indicated time points. (D) Fluorescence photomicrograph of cgs1Δ pka1-GFP cells cultured to mid-log phase in YEAU containing 1.2 M KCl.

  • FIG. 5.
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    FIG. 5.

    Sorbitol-induced nuclear-cytoplasmic redistribution of Cgs1p. (A) Fluorescence photomicrographs of cgs1-GFP (Cgs1-GFP) and pka1-GFP (Pka1-GFP) cells cultured to mid-log phase in YEAU containing 2 M sorbitol. (B) The cgs1-GFP strain was cultured in YEAU to 2 × 106 cells/ml and then diluted with an equal volume of YEAU containing 4 M sorbitol for a final concentration of 2 M sorbitol. The culture was then examined by fluorescence microscopy at the indicated time points.

  • FIG. 6.
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    FIG. 6.

    Nuclear-cytoplasmic redistribution of Cgs1 and Pka1 is induced during stationary phase. SP870 wild-type (A to C), cgs1-GFP (A), pka1-GFP (B), and cgs1Δ pka1-GFP (C) S. pombe strains were cultured in YEAU and examined by fluorescence microscopy at late log phase (6 × 107 cells/ml) and at 3-h intervals upon reaching stationary-phase density (about 108 cells/ml). (A) Cells imaged with 3-s digital camera exposures. (B and C) Cells imaged with 4-s camera exposures.

  • FIG. 7.
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    FIG. 7.

    Subcellular localization of Cgs1-GFP and Pka1-GFP during S. pombe sexual differentiation. Fluorescence photomicrographs of cgs1-GFP (left panels) and pka1-GFP (right panels) nitrogen-starved cells (Ai), zygotes (Aii to Aiv), and zygotic asci (Bi to Biii). (Ai) Cells were cultured in EMM, washed three times with EMM-nitrogen, resuspended in EMM-nitrogen at 2 × 106 cells/ml to induce nitrogen starvation, and incubated for 18 h at 28°C prior to photomicroscopy. (Aii to Aiv and B) Same conditions as for panel Ai, except the cells were resuspended at 2 × 107 cells/ml to induce mating (Aii to Aiv) and sporulation (B).

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Regulation of the Subcellular Localization of Cyclic AMP-Dependent Protein Kinase in Response to Physiological Stresses and Sexual Differentiation in the Fission Yeast Schizosaccharomyces pombe
Yasuhiro Matsuo, Brittney McInnis, Stevan Marcus
Eukaryotic Cell Sep 2008, 7 (9) 1450-1459; DOI: 10.1128/EC.00168-08

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Regulation of the Subcellular Localization of Cyclic AMP-Dependent Protein Kinase in Response to Physiological Stresses and Sexual Differentiation in the Fission Yeast Schizosaccharomyces pombe
Yasuhiro Matsuo, Brittney McInnis, Stevan Marcus
Eukaryotic Cell Sep 2008, 7 (9) 1450-1459; DOI: 10.1128/EC.00168-08
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KEYWORDS

Cyclic AMP-Dependent Protein Kinases
Gene Expression Regulation, Enzymologic
Gene Expression Regulation, Fungal
Protein Subunits
Schizosaccharomyces
Schizosaccharomyces pombe Proteins

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