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Cell-cycle-dependent localisation of Ulp1, a Schizosaccharomyces pombe Pmt3 (SUMO)-specific protease

Deborah L. Taylor, Jenny C. Y. Ho, Alejandro Oliver and Felicity Z. Watts*

Genome Damage and Stability Centre, School of Biological Sciences, University of Sussex, Falmer, Brighton, BN1 9QG, UK



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  		Fig. 1. Ulp1 processes the Pmt3 precursor. (A) Recombinant 6xHis-tagged Ulp1 expressed in E. coli. Extracts were made from cells transformed with empty pRSETB (lane 1) or pRSETB-Ulp1 (lane 2), separated by SDS PAGE (10% polyacrylamide) and western blotted using anti-His antisera. (B) Recombinant Ulp1 was tested for Pmt3-processing activity as described in Materials and Methods. Lanes 1-10, 35S-labelled Pmt3 or Pmt3-GG produced in the TnT system. Lane 1, full-length Pmt3 with no additions; lane 2, Pmt3-GG with no additions. Lanes 3-9 full-length Pmt3 incubated with 0.72 µg of recombinant Ulp1. Lane 3, no inhibitors; lane 4, 1:1000 mammalian protease inhibitor cocktail; lane 5, 1:1000 yeast protease inhibitor cocktail; lane 6, 5 mM benzamidine; lane 7, 10 mM iodoacetamide; lane 8, 10 mM NEM; lane 9, 1 mM PMSF; lane 10, 2 µl elution fraction from E. coli BL21 + pRSETB. Lanes 11-13, full-length Pmt3 (600 ng) or Pmt3-GG (600 ng) purified from E. coli BL21 + pET15b-Pmt3 or E. coli BL21 + pET15b-Pmt3-GG respectively. Lane 11, full-length Pmt3, no additions; lane 12, Pmt3-GG no additions; lane 13 full-length Pmt3 incubated with 0.72 µg Ulp1. All samples were incubated at 30°C for 30 minutes.

 


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  		Fig. 2. Ulp1 can remove Pmt3 from conjugates in S. pombe cell extracts. Recombinant Ulp1 was tested for deconjugating activity using wild-type (sp.011) S. pombe cell extract corresponding to 0.3 OD600 units of cells per assay. Assays were analysed by SDS PAGE (12.5% polyacrylamide in (A) and 10% polyacrylamide in (B)) and western blotted with anti-Pmt3 antisera. (A) Lanes 1 and 2, no additions; lanes 3-6, 3.7 µg Ulp1. Lane 1, 0 minutes; lanes 2,6, 40 minutes at 30°C; lane 3, 5 minutes at 30°C; lane 4, 10 minutes at 30°C, lane 5, 20 minutes at 30°C. (B) Lanes 1,2, no additions; lanes 3-6, 3.7 µg Ulp1; lane 4, 10 mM iodoacetamide; lane 5, 10 mM NEM; lane 6, 0.5 mM PMSF. Lanes 7-9, 3.7 µg Ulp1 preincubated for 10 minutes on ice in the presence of 100 mM iodoacetamide (lane 7), 100 mM NEM (lane 8) or 5 mM PMSF (lane 9) before addition of cell extract. Lanes 2-9, incubated at 30°C for 30 minutes.

 


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  		Fig. 3. Disruption of the ulp1 gene. (A) ulp1 disruption strategy. A 1.1 kb BamHI-SalI fragment within the ulp1 coding sequence was replaced with the ura4 gene. (B) Microscopic analysis of ulp1-null cells. Wild-type, sp.011, (a) and ulp1.d, sp.651, (b) cells were fixed in 70% ethanol, stained with DAPI and analysed using a Zeiss Axioskop 2 microscope. (C-E) Response of ulp1-null cells to UV and ionising radiation and the DNA synthesis inhibitor HU. Wildtype (sp.011) and ulp1.d (sp.651) were exposed to a range of doses of UV (C), ionising radiation (D) or 20 mM HU and samples taken over 10 hours (E). (F) Epistasis analysis. Wild-type (sp.011), ulpl.d (sp.651), rad17.d (sp.310) and ulp1.d,rad17.d (sp.666) cells were subject to UV radiation.

 


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  		Fig. 4. Effect of over-expressing Pmt3 and Pmt3-GG in wild-type and ulp1.d cells. (A,B) Total cell extracts from wild-type (sp.011) (lanes 1-3) and ulp1.d (sp.651) cells (lanes 4-6) transformed with pREP41HA (lanes 1,4), pREP41HA-Pmt3 (lanes 2,5) or pREP41HA-Pmt3-GG (lanes 3,6) and grown for 18 hours in selective medium lacking thiamine were analysed by western blotting with anti-Pmt3 antisera (A) or anti-HA antisera (B). Equal amounts of protein were loaded in each lane as determined by staining with Coomassie Brilliant Blue (data not shown). (a) Pmt3-GG, (b) full-length Pmt3, (c) HA-Pmt3-GG, (d) HA-Pmt3.

(C,D) Wild-type (sp.011) and ulp1.d (sp.651) cells transformed with pREP41HA, pREP41HA-Pmt3 or pREP41HA-Pmt3-GG were grown for 12 hours to midlog phase in selective medium lacking thiamine. Cells were then diluted (t=0) to 106 cells/ml in fresh thiamine-free medium and counted every 2 hours.

(E) Transformed cells were grown for 18 hours to 4x106 cells/ml in selective media lacking thiamine and then subjected to UV radiation as indicated.

 


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  		Fig. 5. Ulp1 protein levels vary in response to increased temperature. Strain sp.611 (ulp1-13myc) was grown overnight to mid-log phase at 30°C (A) or at 25°C (B). TCA extracts were analysed by SDS PAGE and western blotting using anti-myc antisera (upper panels) or anti-tubulin antisera (lower panels). (A) Effect of ionising radiation and HU. Lane 1, no treatment; lane 2, 20 mM HU for 3 hours; lane 3 cells exposed to 500 Gy ionising radiation and incubated for a further 1.5 hours at 30°C. (B) Effect of temperature. Lane 1, no treatment; lane 2, 3 hours at 30°C; lane 3, 3 hours at 35.5°C.

 


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  		Fig. 6. Ulp1 protein is located at the nuclear periphery in a cell-cycle-dependent manner. S. pombe strain sp.611 containing an integrated myc epitope-tagged version of ulp1 was used for DAPI staining and immunofluorescence with anti-myc antisera (TRITC) (A,B) and anti-tubulin antisera (FITC) (B). (A) Field of cells from an asynchronously growing culture. Cells, from the top to the bottom of the field, are in late M/G1, S, early anaphase and G2. (B) Composite of cells taken from an asynchronous culture, arranged in sequence according to cell cycle stage as determined by anti-tubulin staining. Merge is an overlay of DAPI and anti-myc antisera images. A number of examples of each stage are shown to give the range of intensity of Ulp1 staining.

 

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© The Company of Biologists Ltd 2002