1 Division of Gene Regulation and Expression, School of Life Sciences, MSI/WTB Complex, University of Dundee, Dundee, DD1 5EH, UK
2 Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA
* Present address: Scottish Enterprise Tayside, 45 North Lindsay Street, Dundee, DD1 1HT, UK
Present address: Fred Hutchinson Cancer Research Center, Division of Basic Research, 1100 Fairview Avenue North, Seattle, WA 98109, USA
Author for correspondence (e-mail: m.j.r.stark{at}dundee.ac.uk)
Accepted September 19, 2001
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SUMMARY |
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Key words: Protein phosphatase 1, SDS22, GLC7
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Introduction |
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SDS22 is the budding yeast homologue of Schizosaccharomyces pombe sds22+ (Hisamoto et al., 1995; MacKelvie et al., 1995). S. pombe Sds22 is a nuclear protein that binds directly to the catalytic subunit of PP1 and which largely consists of 11 tandem leucine-rich repeats (Stone et al., 1993), although it lacks the -V/IXF- motif found in many PP1C-interacting proteins. The corresponding gene (sds22+) was isolated as a high-copy suppressor of a conditional mutation (dis2-11) affecting the major fission yeast PP1 catalytic subunit Dis2 (Ohkura and Yanagida, 1991). A temperature-sensitive sds22 allele led to a metaphase-like arrest of fission yeast cells at the restrictive temperature, with high histone H1 kinase activity, a short spindle and condensed chromosomes (Stone et al., 1993). Like fission yeast Sds22, S. cerevisiae Sds22p interacts with PP1C (Glc7p) as established by multiple criteria (Hisamoto et al., 1995; Hong et al., 2000; MacKelvie et al., 1995). Extra copies of SDS22 suppress the temperature-sensitivity of glc7-12, a GLC7 allele that confers a mitotic arrest phenotype (MacKelvie et al., 1995). Thus, work in both S. cerevisiae and S. pombe supports a model whereby Sds22p activates mitosis-specific functions of PP1C, and evidence from the latter yeast suggests that, like other PP1 regulatory subunits, Sds22p might act by modifying the substrate specificity of PP1C (Stone et al., 1993). A human homologue of Sds22p has also been identified (Renouf et al., 1995), but like several of the other nuclear proteins that bind PP1C, human Sds22 appears to act as an inhibitor of PP1C using the specific substrates tested (Dinischiotu et al., 1997).
In this study, we have generated conditional sds22 alleles in order to develop a better understanding of how Sds22p regulates Glc7p function. Surprisingly, we found no evidence of a mitotic arrest phenotype when strains carrying two distinct, temperature-sensitive sds22 alleles were shifted to the restrictive temperature. However, the mutant strains showed clear evidence of chromosome instability, suggestive of a chromosome segregation defect. Strikingly, the sds22 mutations led to a rapid loss of nuclear Glc7p localization under restrictive conditions, showing that Sds22p plays a role in maintaining the normal nuclear localization of Glc7p.
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Materials and Methods |
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Sds22p-myc3 and Sds22-6p-myc3
A triple myc epitope tag was inserted immediately after the last sense codon of SDS22 as follows. First, equimolar amounts of primers 180 and 181, which share 25 bp of complementarity at their 3' ends, were annealed together by heating at 100°C for 5 minutes followed by slow cooling to room temperature. The 3' ends were extended at 72°C by addition of dNTPs and ExpandTM High-Fidelity Polymerase (Roche), forming a DNA fragment encoding a triple myc epitope (myc3). After addition of primers 183 and 184, the 130 bp myc3 fragment was amplified by 30 cycles of 1 minute at 95°C, 1 minute at 50°C and 2 minutes at 72°C such that it was flanked by sequences just preceding and just following the SDS22 stop codon. In a second PCR reaction, the SDS22 open reading frame was similarly amplified using primers SDS22-3 and 182. The two products were recovered using the High Pure PR product purification kit (Roche) and then fused by mixing equimolar amounts, annealing as described above and amplifying the fusion product using primers Sds22-3 and 184. The product was recovered as above and cleaved with NdeI and BclI to generate a 306-bp fragment corresponding to the C-terminal, myc-tagged region of SDS22. This fragment was inserted into psds22-6 cleaved with the same enzymes, generating a myc3-tagged sds22-6 allele. The tagged gene was verified by DNA sequencing and moved as an SpeI-HindIII fragment into YCplac111 (cut with the XbaI-HindIII) to generate YCp3Msds22-6. A wild-type tagged SDS22 construct was then generated by replacing the
800 bp PstI-NdeI interval of YCp3Msds22-6 with the corresponding wild-type sequence, generating YCp3MSDS22. Strains dependent on the tagged wild-type or mutant alleles were generated by transformation of SAY100 followed by selection for loss of pLMY-SDS22 using 5-fluoroorotic acid.
Protein A-tagged constructs
A derivative of strain AYS927 in which the genomic copy of SDS22 was tagged at the 3' end with protein A was generated as described (Rayner and Munro, 1998), using primers SDS22-PrA-5' and SDS22-PrA-3' with pZZ-His5 as template. Following verification by PCR and detection of the tagged protein by western blot analysis, haploid strains in which protein A-tagged Sds22p (Sds22p-PrA) was the sole source of Sds22p function were generated by tetrad analysis.
To generate an integrative plasmid encoding protein A-tagged Glc7p, the HA-tagged GLC7 construct from YCpHA-GLC7 was excised as a HindIII-MscI fragment and cloned between the HindIII and SmaI sites of YIplac204. A small SpeI-ClaI fragment at the 5' end of GLC7 (carrying the HA epitope tag) was removed and replaced with a larger SpeI-ClaI fragment carrying a protein A tag and TEV protease cleavage site, generating YIplac204-PrA-GLC7. This fragment was made by PCR using primers PrA-GLC7-5' and PrA-GLC7-3' with pZZ-His5 as template, and was cleaved by SpeI and ClaI prior to subcloning. The sequence of this fragment was verified by DNA sequencing of the relevant region of the final construct. To generate a strain (LKY150) solely dependent on PrA-Glc7p for Glc7p function, YIplac204-PrA-GLC7was integrated into SBY-SSa at the trp1-1 locus followed by selection on 5-FOA to remove YCp-GLC7(URA3). LKY118 was similarly generated usingYIplac204-HA-GLC7. A control strain expressing unfused protein A (from the NOP1 promoter) was generated by transformation of AY925 with pNOPPATA-1L.
Analysis of Glc7p-Sds22p complexes
To identify proteins complexed with Sds22p, cultures (1.5 l) of strain SAY1228 were grown in YPD medium at 26°C until they reached 107 cells/ml. The cells were harvested by centrifugation and then washed twice with water and finally with 10 ml ice-cold extraction buffer (50 mM Hepes-KOH (pH 7.5), 150 mM KCl, 0.1% Triton X-100, 0.1 mM EDTA, 10% (v/v) glycerol). The cell pellet was resuspended in an equal volume of extraction buffer containing 1x complete protease inhibitors (Roche) and supplemented with an equal volume of acid-washed glass beads (0.4 mm diameter). After 20 cycles of vortexing (30 seconds) and cooling on ice (30 seconds) the cell debris was removed by centrifugation for 10 minutes at 3500 rpm in a Jouan CR/312 centrifuge. The cell pellet was subject to a further 10 cycles of disruption after addition of the same volume of extraction buffer with protease inhibitors and the two resulting supernatants were pooled and spun at 20,000 g for 20 minutes. Protein A-tagged Sds22p (Sds22-PrA) was recovered from the extract by elution over a column containing 0.75 ml IgG-Sepharose (Amersham Pharmacia Biotech) which had been pre-washed with 20 ml extraction buffer. After washing the column with 10 bed volumes of extraction buffer, the column material was resuspended in 3 ml of extraction buffer without Triton X-100 and glycerol but containing 30 units TEV protease (Life Technologies) and incubated for 1 hour at room temperature. The released material was recovered by elution with extraction buffer (lacking Triton X-100 and glycerol) and examined by SDS-PAGE, with silver staining to visualize the protein bands. For identification of protein bands by mass spectrometry, appropriate fractions of eluate were desalted using CentriPlus (Millipore), concentrated using Microcone protein concentrators (Millipore), alkylated with 4-vinylpyridine and separated by SDS-PAGE using 4-12% Bis-Tris gels (Novex). Gels were stained using Coomassie Brilliant Blue and mass fingerprint analysis carried out as described (Morrice and Powis, 1998). Proteins interacting with PrA-Glc7p were similarly identified using strain LKY150. In each case, strain AY925 transformed with pNOPPATA-1L (expressing the unfused protein A tag) (Lau et al., 2000) was used to control for nonspecific protein binding.
The behaviour of Sds22p-PrA on gel filtration of yeast cell extract (prepared as above from strain SAY1228) was examined using a Superose-200 column calibrated with the following markers (Sigma): apoferritin, -amylase, alcohol dehydrogenase, bovine serum albumin (BSA). SAY1228 also expressed HA-Glc7p as the sole source of PP1C. Sds22p-PrA was located by western blot analysis and its apparent Stokes radius calculated using a plot of (log KAV)1/2 against published values for the Stokes radii of the marker proteins. The sedimentation of Sds22p-PrA in a 10-40% (v/v) glycerol gradient centrifuged at 60,000 rpm in a Beckman SW60 rotor for 8 hours at 4°C was examined using a similar sample of cell extract from LKY168, with thyroglobulin, catalase, aldolase and BSA as markers (Amersham Pharmacia Biotech). The molecular size of Sds22p-PrA was calculated from its Stokes radius and sedimentation coefficient as previously described (Siegel and Monty, 1966).
Immunoprecipitation of Sds22p-Glc7p complexes and western blot analysis
Cultures of yeast cells (200 ml) were grown at 26°C to a density of 1x107 cells/ml in YPD, harvested by centrifugation and washed in an equal volume of water. Cells were collected in a 15 ml centrifuge tube and the pellet supplemented with an equal volume of lysis buffer containing 50 mM Tris-HCL (pH 7.5), 100 mM NaCl, 10 mM MgCl2, 5 mM EDTA, 1 mM DTT, 1% (v/v) Triton X-100, 1x complete protease inhibitors (Roche). Acid-washed glass beads (0.4 mm diameter; 0.7 g per ml) were added and the cells lysed by 20 cycles of vortexing for 30 seconds followed by 30 seconds on ice. Extracts were centrifuged for 5 minutes at 21,000 g to pellet cell debris and the supernatant removed. The glass beads were washed with one pellet volume of lysis buffer and the supernatants pooled. Protein concentrations were determined using the Bio-Rad protein assay. To make Sds22p-myc3 immunoprecipitates, Protein G-Sepharose beads (Amersham Pharmacia Biotech) were first equilibrated in lysis buffer and 80 µl of a 50% suspension used to pre-clear lysates (containing 2.5 mg protein) at 4°C for 2 hours on a rotary mixer. Purified 9E10 monoclonal antibody (1 µg) was added to the pre-cleared lysate and incubated at 4°C for 2 hours with gentle mixing. The antibody-lysate mix was divided into two equal parts and 20 µl 50% Protein G-Sepharose bead suspension added to each. One was incubated at 4°C for 2 hours while the other was incubated at 30°C, both with gentle mixing as above. The beads were recovered by centrifugation, washed three times in lysis buffer and then resuspended in 30 µl 2x SDS-PAGE sample buffer and boiled for 2 minutes. Recovered proteins were separated by SDS-PAGE on 10% polyacrylamide gels and Sds22p-myc3 and HA-tagged Glc7p visualized by western blotting with ECL detection using either 9E10 with sheep anti-mouse IgG-HRP conjugate (Amersham Pharmacia Biotech) or mouse anti-HA HRP conjugate (Roche), respectively. Similar procedures were used to compare Sds22p-myc3 and HA-tagged Glc7p levels in total cell extracts using anti-calmodulin antibodies (Stirling et al., 1994) to confirm equivalent protein loading.
Immunofluorescence microscopy
Cells were prepared for immunofluorescence microscopy as described (Ayscough and Drubin, 1998) using the general immunofluorescence protocol, but omitting the methanol and acetone fixing step. High-affinity 3F10 (anti-HA) and 9E10 (anti-myc) antibodies were used at dilutions of 1:100 while both sheep anti-mouse IgG-Cy3 conjugate (Sigma) and goat anti-rat IgG-FITC conjugate (Cappel) secondary antibodies were used at 1:200 dilutions. To visualize DNA, the mounting medium contained 1 mg/ml 4', 6-diamidino-2-phenylindole dihydrochloride (DAPI). Images were acquired using a Deltavision Restoration microscope (Applied Precision Inc., USA) fitted with a Nikon PlanApo 100x (1.4NA) objective and a Photometrics series 350 cooled CCD camera, taking a Z series encompassing the whole cell. Images were deconvolved and processed using the Deltavision Softworx application on a Silicon Graphics Octane Workstation (Silicon Graphics Inc., USA) and a single optical section presented.
Nuclear localization of Glc7p
To monitor the localization of Glc7p in wild-type and sds22 mutant strains, a functional GFP-Glc7p construct (Bloecher and Tatchell, 2000) was integrated into the ura3 locus of strains KT2070 (SDS22/SDS22), KT2067 (SDS22/sds22-6) and KT2066 (sds22-6/sds22-6). The sds22-6 and sds22::TRP1 alleles were introduced into KT2066 and KT2067 after backcrossing SAY304 seven times to KT1357. For imaging, cells were grown to mid logarithmic phase in synthetic complete medium, collected by centrifugation and then placed on a microscope slide over a thin agarose slab and under a cover slip, as described elsewhere (Waddle et al., 1996). Cells were observed through a 100x N.A. 1.25 Olympus objective equipped with a Bioptechs objective heater (Butler, PA) and images were collected with a 12-bit Princeton Instruments Micro Max CCD camera, capturing images at 30 minute intervals using 2 second exposures at 6% full intensity. All images for a given strain were normalized in the same way but the normalization was different for each strain. To calculate the ratio of nuclear/cytoplasmic fluorescence at each time point, a minimum of 102 cells in which the focal plane went through the nucleus were quantitated using the IPLab Spectrum software. Individual cells were imaged only once to avoid photobleaching.
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Results |
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Conditional sds22 alleles
To gain greater insight into the role of Sds22p, we next generated temperature-sensitive sds22 alleles by random PCR-mediated mutagenesis. Of several such alleles isolated we chose to characterize two in particular. sds22-5 was found to carry four point mutations leading to four amino acid replacements in the protein (T25A, Q28R, L73P, F285S), while sds22-6 encodes two missense mutations (I54T, I193T) together with a silent mutation in codon 51. The L73P and F285S mutations in sds22-5 and the I193T mutation in sds22-6 represent nonconservative changes to key hydrophobic residues located in leucine-rich repeats (LRRs) 1 and 10 (sds22-5) and in LRR 6 (sds22-6). Both alleles were found to be recessive (not shown). Strains dependent on either plasmid-borne or integrated copies of sds22-5 were unable to grow at 32°C or higher, while the equivalent sds22-6 strains grew up to 35°C. In the presence of nocodazole, strains containing either the sds22-5 or sds22-6 allele arrested with a large-budded morphology at the same concentration required to provoke an equivalent arrest in the SDS22 control strain, and this arrest was maintained for 3 hours when the nocodazole-arrested cultures were shifted to 37°C (not shown). Thus, continued Sds22p function is not required for maintenance of a mitotic checkpoint arrest. Although in W303 strains high-copy GLC7 is unable to compensate for complete loss of SDS22 function (not shown), the restrictive temperature of each sds22 mutant was raised significantly by high-copy GLC7 (Fig. 3; and data not shown), suggesting that elevated levels of Glc7p can in part compensate for loss of Sds22p function. High-copy SDS22 was similarly able to raise the restrictive temperature of the glc7 alleles glc7-5 and glc7-12 (Fig. 3). Taken together, these genetic data support a model whereby Sds22p and Glc7p function in an interdependent manner.
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Discussion |
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Localization of Sds22p
As previously found for its human and fission yeast homologues, S. cerevisiae Sds22p is a nuclear protein despite the absence of any clearly defined monopartite (Kalderon et al., 1984) or bipartite (Robbins et al., 1991) nuclear localization sequence (NLS). Although a 39 kDa polypeptide such as Sds22p could potentially gain entry to the nucleus without the aid of a specific import signal, we think this is unlikely in the case of Sds22p, which is largely composed of LRRs and is therefore expected to be non-globular and rather asymmetric (Kobe and Deisenhofer, 1994). In this case, Sds22p may either contain a novel signal for nuclear import, or alternatively it could be imported as a complex with another protein such as Glc7p. Although Glc7p has an excellent candidate monopartite NLS (RKKK) at its extreme C-terminus, recent work suggests that this region is not essential either for Glc7p nuclear localization or function (Hong et al., 2000). Furthermore, mutant Sds22 proteins in fission yeast that failed to bind PP1C could nonetheless localize to the nucleus, while other variants which were excluded from the nucleus could still bind PP1C (Stone et al., 1993), strengthening the idea that PP1C binding and nuclear import are not obligatorily linked. Additional examples of nuclear LRR proteins that also apparently lack a classical NLS include Drosophila LRR47 (Buchanan et al., 1998) and the human splicing factor U2A (Sillekens et al., 1989), although other LRR proteins such as CIITA (a transactivator of human MHC class II genes) (Hake et al., 2000), contain potential NLSs as well as LRRs. Mutations in the LRR region of CIITA specifically affect its nuclear localization (Hake et al., 2000). It is therefore conceivable that LRRs constitute a novel determinant of nuclear import, although this could be due to their role as mediators of protein-protein interaction rather than because they contain an intrinsic nuclear import signal. In fission yeast Sds22, mutational analysis revealed that C-terminal truncation or point mutations in LRRs 5 or 9 led to nuclear exclusion of the mutant proteins and loss of function, although mutations in some of the other LRRs failed to affect nuclear localization (Stone et al., 1993).
Sds22p function
The cellular role of Sds22p is clearly a critical question and at least two types of model can be proposed for how the Sds22p-Glc7p complex might influence PP1C activity. One possibility is that Sds22p activates Glc7p function towards key nuclear PP1C substrates that are required for chromosome stability and other functions. This is supported by the reciprocal, high-copy suppression by each gene of recessive mutations in the other, and by the finding that sds22-6 mimics glc7 mutations in partially suppressing ipl1-2 temperature-sensitivity (Hsu et al., 2000). This suggests that Sds22p and Glc7p function together in a positive sense to create a nuclear PP1 activity. Furthermore, although fission yeast Sds22 immunoprecipitates lacked the phosphorylase phosphatase activity shown by PP1C alone, they contained a histone H1 phosphatase activity (Stone et al., 1993), supporting a model whereby the Sds22-PP1C complex can be active as a phosphatase, at least against certain substrates. However, when Sds22p function is lost, the normally uniform nuclear localization of Glc7p is dramatically changed such that the overall level of nuclear PP1 is reduced and the remaining PP1 becomes localized in a small number of foci. Since the interaction of Glc7p with the mutant sds22-6 polypeptide is itself temperature-sensitive, this suggests that Sds22p binding is required to maintain proper nuclear localization of Glc7p. A second possibility is therefore that Sds22p plays a chaperone-like role for nuclear Glc7p, preventing aggregation of the free PP1C subunit and/or helping to retain it in the nucleus, but not necessarily directing it towards specific substrates. This model would predict that the sds22 mutations might affect a wider range of nuclear PP1C functions.
In addition to the dramatic effect of sds22-6 on nuclear localization of Glc7p, both sds22 alleles described here confer a profound chromosome loss phenotype at higher growth temperatures. If reflected uniformly across all 16 chromosomes, the high rates of loss seen in sds22-6 at 32°C would suggest that only 75% of cells would inherit a complete genome at each cell division. High-copy GLC7 also promotes chromosome instability (Francisco et al., 1994) and exacerbates the chromosome loss defect in the sds22 mutants despite partially suppressing their growth defect (M.W.P. and M.J.R.S., unpublished). Thus the lethality of the sds22 mutants is unlikely to result from chromosome loss per se. A number of glc7 alleles including glc7-10 (Sassoon et al., 1999), glc7-129 (Bloecher and Tatchell, 1999) and glc7-12 (MacKelvie et al., 1995) A. Engles and M.J.R.S., unpublished) confer a mitotic arrest phenotype due to mitotic checkpoint activation. Such mutants show an in vitro defect in microtubule binding by kinetochores (Sassoon et al., 1999) (I. Sassoon et al., unpublished) that if representative of the in vivo situation would be sufficient to account for the observed checkpoint activation This is in contrast to the budding yeast sds22 mutants described in this work that do not arrest in mitosis despite showing severe chromosome instability at higher growth temperatures. Conversely, the conditional fission yeast sds22 mutant described by Stone et al. (Stone et al., 1993) arrested homogeneously in mitosis, although it is not known whether this synchronous arrest is checkpoint-dependent. If Sds22p is required generally for nuclear PP1C function then it is perhaps surprising that our sds22 mutants do not also activate the checkpoint. However, Glc7p most likely has multiple nuclear roles that do not all result in checkpoint activation. For example, some glc7 mutants that clearly suppress the temperature-sensitivity of ipl1-2 and raise the phosphorylation level of histone H3 on ser-10 (glc7-127), dont by themselves activate the mitotic checkpoint. Conversely, glc7-129 mutants lacking the Mad/Bub checkpoint are still delayed at the end of the cell cycle, pointing to an additional defect late in mitosis or during cytokinesis. We have also observed synthetic lethality between glc7-129 and several genes encoding microtubule motors (A.B. and K.T., unpublished) that point to a cell cycle role separate from microtubule binding at the kinetochore. Perhaps either the sds22 mutants show chromosome loss for some reason other than faulty kinetochore regulation that does not lead to checkpoint activation or, alternatively, the checkpoint-dependent arrest expected to ensue from the type of kinetochore defect seen in the above glc7 mutants is masked due to defects in multiple Glc7p nuclear functions.
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ACKNOWLEDGMENTS |
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