First published online 6 January 2004
doi: 10.1242/jcs.00880
Journal of Cell Science 117, 545-557 (2004)
Published by The Company of Biologists 2004
Swm1p subunit of the APC/cyclosome is required for activation of the daughter-specific gene expression program mediated by Ace2p during growth at high temperature in Saccharomyces cerevisiae
Sandra Ufano1,2,
M. Evangelina Pablo1,
Arturo Calzada1,3,
Francisco del Rey1 and
Carlos R. Vázquez de Aldana1,*
1 Instituto de Microbiología-Bioquímica, Departamento de Microbiología y Genética, CSIC /Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain
2 Centro Nacional de Biotecnología, CSIC/UAM, Cantoblanco, 28049, Madrid, Spain
3 Centro de Investigación del Cáncer, CSIC / Universidad de Salamanca, Campus Miguel de Unamuno, 37007, Salamanca, Spain
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Fig. 1. Growth of wild-type and swm1 mutants. Growth of wild-type (YPA24) and mutant (YPA207) cells at 38°C in solid medium. Cells were streaked out onto YEPD or YEPD plates supplemented with 1 M sorbitol (YEPD +S) and incubated for 48 hours at 38°C.
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Fig. 2. Microscopic appearance of wild-type and swm1 mutant cells during growth at 38°C. Diploid wild-type (YPA24) or swm1 mutant (YPA207) cells were grown for 8 hours at the restrictive temperature on YEPD medium, washed and stained with Calcofluor. Photographs of differential interference contrast microscopy or Calcofluor-stained cells are shown. Arrowheads indicate the position of thickened septa.
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Fig. 3. Electron microscopic appearance of wild-type and swm1 mutant cells incubated for 12 hours at 38°C. (A,C) Wild-type strain YPA24; (B,D-F) swm1 strain YPA207. Cells grown in YEPD supplemented with 1 M sorbitol are shown at low magnification in (A,B), whereas higher magnifications of the septal region are shown in (C-F). The structure of the septum is clearly visible in wild-type cells, in which the chitin-rich primary septum (ps) is surrounded by the secondary septum (ss), composed mainly of glucans and mannoproteins. The chitin ring (cr) synthesized by Chs3p is visible at both sides of the primary septum. In mutant cells (D-F), the same structure is visible but the region corresponding to secondary septum (ss) is thickened. Scale bars: 1 µm (A,B), 0.5 µm (C-F).
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Fig. 4. (A) Chitinase activity in culture supernatants. Wild-type (YPA24, black circles) and mutant cells (YPA207, white circles) cells were grown overnight in YEPD medium at 28°C and then diluted into fresh YEPD medium that was transferred to 38°C. At the indicated times, aliquots were taken, cells were removed by centrifugation and chitinase activity was measured using the fluorogenic substrate 4-methylumbelliferyl-ß-D-N,N',N''-triacetylchitotrioside. Activity is expressed in nmoles 4-methylumbelliferone (4-MU) released per minute and per millilitre of culture. (B) Expression of CTS1 in wild-type and mutant cells. RNA was purified from wild-type (strain TD28) and swm1 (strain LS40) cells at the indicated times (hours) after transfer to 38°C. RNA blots were hybridized sequentially with radioactively labelled probes for CTS1 and ACT1. (C) Analysis of CTS1 mRNA stability. RNA was purified from swm1 cells (strain LS40) transformed with vector (left, swm1) or pSU50 (right, swm1+PTPI-CTS1) containing the CTS1 ORF under the control of the TPI promoter (PTPI-CTS1 allele) at the indicated times (hours) after transfer to 38°C. RNA blots were hybridized with radioactively labelled probes for CTS1 and ACT1. Arrows indicate the position of the transcript corresponding to the heterologous promoter (upper) or the chromosomal locus (lower). (D) Expression of genes involved in cell separation in swm1 cells. RNA was purified from wild-type (strain TD28) and swm1 cells (strain LS40) at the indicated times (hours) after transfer to 38°C. RNA blots were hybridized sequentially with radioactively labelled probes for CTS1, DSE1, DSE2, SCW11, EGT2 and ACE2. The ACT1 gene was used to test for equal RNA loading in all lanes.
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Fig. 5. Localization of Ace2p in wild-type and swm1 mutants. (A) Diploid wild-type (YPA24) cells carrying the Ace2-YFP or Ace2-G128EYFP fusion proteins were grown at 38°C for 4 hours and stained with DAPI. (Left) Differential interference contrast microscopy (DIC); (middle) YFP fluorescence; (right) DAPI fluorescence. (B) Diploid wild-type (YPA24) or swm1 (YPA207) cells were transformed with plasmid pACE2-YFP and the localization of the protein was determined by fluorescence microscopy during growth at 28°C or after 2 hours or 8 hours of incubation at 38°C. (Left) Differential interference contrast microscopy; (right) YFP fluorescence. (C) Localization of Ace2-G128Ep in wild-type and swm1 mutants. Diploid wild-type (YPA24) or swm1 (YPA207) cells were transformed with plasmid pACE2-G128E-YFP and the localization of the protein was determined by fluorescence microscopy during growth at 28°C or after 4 hours of incubation at 38°C. (Left) Differential interference contrast microscopy; (right) YFP fluorescence.
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Fig. 6. Cells with the swm1 mutation have a defect in exit from mitosis. Cultures of isogenic wild-type (W9317) or swm1 mutant (SEP3) cells containing an epitope-tagged version of the CLB2 gene (CLB2HA) were arrested in mitosis with nocodazole (2.5 hours) before transfer to the restrictive temperature for the swm1 mutation (38°C, 1.5 hours). Cultures were then released from nocodazole arrest and incubated at 38°C. Samples were taken at the indicated intervals after the release (minutes) and processed for FACS analysis (A), Clb2 and Pgk1 western analysis (B) or Clb2-associated H1 kinase activity assays (C). In each panel asynchronous cultures (A) are also shown for reference.
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© The Company of Biologists Ltd 2004
