Article |
Address correspondence to Clarence S.M. Chan, Section of Molecular Genetics and Microbiology, ESB 226, The University of Texas at Austin, Austin, TX 78712. Tel.: (512) 471-6860. Fax: (512) 471-7088. E-mail: clarence_chan{at}mail.utexas.edu
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Abstract |
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Key Words: chromosome segregation; Ipl1; Sli15; Dam1; kinetochore
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Introduction |
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One protein that appears to be required for Ipl1 function is Sli15, which binds directly to Ipl1 (Kim et al., 1999). Mutations in SLI15 exacerbate the Ts- phenotype of ipl1-2 cells, and Ts- sli15-3 mutant cells have cytological phenotypes very similar to those of ipl1-2 cells. Both proteins also colocalize to spindle microtubules and spindle poles. These observations suggest that Sli15 may be a positive regulator or major physiological target of Ipl1. Recently, a sequence motif known as the IN box (Adams et al., 2000; Kaitna et al., 2000) was found to be present at the COOH termini of both Sli15 and the metazoan "chromosomal passenger" inner centromere protein (INCENP), which localizes along chromosomes, at kinetochores, and at the spindle midzone in a cell cycle stagespecific manner (Cooke et al., 1987). Interestingly, INCENP proteins from Caenorhabditis elegans, Xenopus, and humans bind to aurora-B (AIRK2) and these two proteins colocalize in human cells (Adams et al., 2000; Kaitna et al., 2000). Aurora-B and INCENP, like their counterparts in yeast, are required for chromosome segregation (Mackay et al., 1998; Kaitna et al., 2000; Giet and Glover, 2001).
Dam1 and Duo1 are components of an essential protein complex required for both mitotic spindle integrity and kinetochore function. These two proteins associate with each other and colocalize to spindle poles, spindle microtubules, and kinetochores (Hofmann et al., 1998; Jones et al., 1999; Cheeseman et al., 2001). Although Dam1 by itself can bind microtubules directly in vitro, Dam1 and Duo1 require each other for their localization to the spindle microtubules. Interestingly, in addition to missegregating chromosomes severely, some dam1 mutant cells exhibit premature anaphase events, such as spindle elongation while arrested in metaphase, as well as genetic interactions with a subset of kinetochore components. Similar abnormalities have been observed in some ipl1 mutant cells (Biggins et al., 1999), and they point toward a potential functional connection between Ipl1 and Dam1 in the correct functioning of the kinetochores.
Here we report a molecular characterization of Ipl1 and Sli15 and demonstrate a novel interaction with Dam1. Our results show that, like Dam1, Ipl1 and Sli15 are microtubule-binding proteins associated with yeast kinetochores. Both Sli15 and Dam1 are likely physiological targets of Ipl1, and Sli15 also stimulates the kinase activity of Ipl1 and facilitates its association with the mitotic spindle. The microtubule-binding and Ipl1-stimulating activities of Sli15 reside in different regions of Sli15.
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Results |
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Sli15 is a phosphoprotein that is under-phosphorylated in ipl1-2 mutant cells
We have previously shown that multiple forms of Sli15 differing in electrophoretic mobility exist in vivo (Kim et al., 1999). Three to four forms that could be resolved by SDS-PAGE were detected in most experiments. The slower-migrating forms represented phosphorylated Sli15, since they could be converted to the fastest migrating form by phosphatase treatment (unpublished data). To find out whether the in vivo phosphorylation of Sli15 is dependent on Ipl1 function, we examined the phosphorylation state of a functional version of Sli15HA in wild-type and ipl1-2 mutant cells. Strikingly, the abundance of the two slowest-migrating forms of Sli15HA was greatly reduced in ipl1-2 cells even at the permissive growth temperature of 26°C (Fig. 3 C, lanes 1 and 2). These two forms of Sli15HA almost totally disappeared in ipl1-2 cells after a 2.5-h incubation at the restrictive growth temperature of 37°C (Fig. 3 C, lanes 3 and 4). These results are consistent with our previous observation that despite a normal growth rate and cell cycle distribution, ipl1-2 cells exhibit an 10-fold increase in the frequency of chromosome gain and thus have reduced Ipl1 function at the permissive temperature (Chan and Botstein, 1993). The disappearance of two hyperphosphorylated forms of Sli15 in ipl1-2 cells is also consistent with the observation that Ipl1 could phosphorylate both the middle and COOH-terminal regions of Sli15 in vitro (Fig. 3 A). Together, the in vitro and in vivo results indicate Sli15 most likely functions as a physiological substrate of Ipl1.
Ipl1 and Sli15 bind to microtubules in vitro
Because Ipl1 and Sli15 both localize along the length of the mitotic spindle (Biggins et al., 1999; Kim et al., 1999), we tested whether either of these proteins can bind to microtubules directly by examining the ability of GST and GST fusion proteins (that were purified from E. coli) to cosediment with taxol-stabilized microtubules in vitro. GST did not sediment with microtubules (Fig. 4 A). In contrast, whereas only a small fraction of GSTIpl1 sedimented in the absence of microtubules, the bulk of GSTIpl1 did cosediment with microtubules in a microtubule concentrationdependent manner (Fig. 4 B). The estimated dissociation constant of the GSTIpl1microtubule interaction is 0.5 µM, which represents a slightly lower affinity than that between tau and microtubules (Goode and Feinstein, 1994).
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Ipl1Sli15 can associate with Dam1Duo1 in vivo
To identify additional proteins that associate with Sli15 (and possibly Ipl1) in vivo, we performed a two-hybrid screen (James et al., 1996) with full-length Sli15 as the bait. From this screen, we identified a prey plasmid (pCC1428) that encoded a truncated form of Duo1 lacking the first nine residues (Duo110247). Because Duo1 is known to bind to Dam1 (Hofmann et al., 1998; Cheeseman et al., 2001), we tested whether Dam1 also could interact with Sli15 in the two-hybrid assay. Our results indicated that Sli15 interacted with full-length Dam1 even more strongly than with full-length Duo1 (unpublished data).
To confirm the in vivo association of Dam1Duo1 with Sli15 and possibly Ipl1, we purified GST, GSTDam1, or GSTDuo1 (which were expressed under the control of the GAL1/10 promoter) from yeast cells that also expressed functional HAIpl1 or Sli15Myc. Our results showed that HAIpl1 and Sli15Myc could be copurified with GSTDam1 (Fig. 6 A, lanes 6 and 11), whereas HAIpl1 but not Sli15Myc could be copurified with GSTDuo1 (Fig. 6 A, lanes 5 and 12). The association of HAIpl1 with GSTDam1 appeared to be stronger than that with GSTDuo1 since repeated rounds of washing led to the dissociation of most HAIpl1 from GSTDuo1 but not from GSTDam1. These results are consistent with the two-hybrid results described above, which suggested that Sli15 interacted more strongly with Dam1 than with Duo1. One possible interpretation of these results is that Dam1 may mediate the association between Duo1 and Ipl1Sli15.
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Dam1 binding primarily involves the middle region of Sli15
To determine whether different regions of Sli15 are responsible for its binding to Dam1Duo1 and Ipl1, we repeated the two-hybrid assays with truncated versions of Sli15. Our results showed that Sli15-M, but not Sli15-N and Sli15-C, interacted with Dam1 and Duo1 in this assay (Fig. 1; unpublished data). To confirm the in vivo association of Sli15-M with Dam1 or Duo1, we affinity-purified GST, GSTSli15, GSTSli15-N, GSTSli15-M, and GSTSli15-C from yeast. Duo1 did not copurify with any GST fusion proteins (unpublished data). For reasons that we do not understand, Dam1 also did not copurify with GSTSli15 (Fig. 6 C, lane 7). However, Dam1 was readily copurified with GSTSli15-M and, to a lesser degree, with GSTSli15-C, but not with GST or GSTSli15-N (Fig. 6 C, lanes 610). Interestingly, the Dam1 species that copurified with GSTSli15-C had a slower electrophoretic mobility than the Dam1 species that copurified with GSTSli15-M. Because reduced electrophoretic mobility of Dam1 is caused by its hyperphosphorylation (see below) and since Dam1 is known to bind directly to Ipl1 and Sli15 (Fig. 6 B), these results suggest that Dam1 associates directly with the middle region of Sli15 (Sli15-M) and indirectly with the COOH-terminal region of Sli15 (Sli15-C) through Ipl1.
Because Sli15-M binds to Dam1 and is localized to the mitotic spindle, we tested whether the localization of Ipl1Sli15 and Dam1 to the mitotic spindle is interdependent. Our results showed that GFPSli15 and GFPIpl1, unlike Dam1 and Duo1 (Cheeseman et al., 2001), were properly localized to the mitotic spindle in dam1-1, dam1-9, and dam1-11 cells at 25°C as well as 37°C (unpublished data). Similarly, Dam1 and Duo1 were properly localized in ipl1-2 and sli15-3 cells at both temperatures (unpublished data). Thus, the localization of Ipl1Sli15 and Dam1 to the mitotic spindle does not appear to be interdependent.
Dam1, but not Duo1, is an in vitro substrate of Ipl1
Because Dam1 and Duo1 can associate with Ipl1Sli15, this raised the possibility that Dam1 or Duo1 may be substrates of Ipl1. Therefore, we performed in vitro kinase assays with His6Dam1, GSTDam1-C (containing the COOH-terminal residues 175343 of Dam1), GSTSli15, and GSTIpl1 purified from E. coli, as well as GSTDuo1 that was purified from yeast. As shown in Fig. 7 A (lanes 68), GSTDuo1 was not phosphorylated by GSTIpl1 even in the presence of GSTSli15. In contrast, His6Dam1 and GSTDam1-C were readily phosphorylated by GSTIpl1 (Fig. 7 A, lanes 2 and 3). Furthermore, the addition of GSTSli15 to the kinase reaction greatly stimulated the ability of GSTIpl1 to phosphorylate these two proteins (Fig. 7 A, lanes 4 and 5). GSTDam1-C could be phosphorylated almost quantitatively, resulting in the conversion of the majority of GSTDam1-C to an electrophoretically slowermigrating form (unpublished data). Thus, Dam1 is a very good in vitro substrate of Ipl1Sli15. Because the amount of GSTSli15 used in these experiments was much less than that of His6Dam1 or GSTDam1-C, the relatively low level of Sli15 phosphorylation (Fig. 7 A, lanes 4 and 5) did not necessarily mean that Dam1 was a much better in vitro substrate than Sli15.
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Dam1 is a phosphoprotein that is underphosphorylated in ipl1 and sli15 mutant cells
Previous studies using anti-Dam1 antibodies indicated that Dam1 exists as multiple forms that differ in electrophoretic mobility (Cheeseman et al., 2001). On a 10% polyacrylamide gel, as many as five different forms of Dam1 were visible (Fig. 7 C, lane 1). The slower-migrating forms were caused by phosphorylation since phosphatase treatment of immunoprecipitated Dam1 led to the disappearance of the slower-migrating forms (Fig. 7 B). The abundance of the different forms of Dam1 does not change over the cell cycle (unpublished data).
Because Ipl1 can phosphorylate Dam1 in vitro (Fig. 7 A), we sought to determine whether Dam1 is an in vivo target of Ipl1. As shown in Fig. 7 C (lanes 1 and 4), Dam1 existed as multiple phosphorylated forms in wild-type cells incubated at either 25°C or 37°C. In contrast, the abundance of the three slowest-migrating forms of Dam1 was greatly reduced in ipl1-2 and sli15-3 mutant cells incubated at the permissive temperature of 25°C (Fig. 7 C, lanes 2 and 3), and these phosphorylated forms of Dam1 were absent in mutant cells that had been incubated at 37°C for 3 h (lanes 5 and 6). These results are very similar to those observed for Sli15HA (Fig. 3 C). The in vitro and in vivo results together indicate that Ipl1 is required for Dam1 phosphorylation in yeast, most likely because Dam1 functions as a physiological target of Ipl1. The disappearance of three hyperphosphorylated forms of Dam1 in ipl12 cells suggests that there are at least three Ipl1 phosphorylation sites within Dam1, or that phosphorylation of Dam1 by Ipl1 is required for its subsequent phosphorylation by other kinases. At least one of the Ipl1 phosphorylation sites most likely resides within the COOH-terminal 169 residues of Dam1 (Dam1-C; Fig. 7 A). Furthermore, these results support the idea that Sli15 functions in vivo as a positive regulator of Ipl1.
Genetic links between IPLSLI15, DUO1-DAM1, and GLC7
In addition to the physical and biochemical interactions observed between Ipl1Sli15 and Duo1-Dam1, we sought to determine whether there was any genetic evidence to support the importance of such interactions in vivo. Crosses were conducted between ipl1 or sli15 mutants and duo1 or dam1 mutants. A synthetic lethal interaction was observed at 25°C (a permissive temperature for all single mutant cells) in dam1-1 ipl1-2 and dam1-1 sli15-3 double mutants. In contrast, no genetic interactions were observed in crosses between ipl1-2 or sli15-3 and duo1-2, dam1-9, or dam1-11 mutants.
Previous studies have indicated that the Ipl1 protein kinase activity is opposed by the action of the Glc7 type-1 protein phosphatase (Francisco et al., 1994; Tung et al., 1995). In fact, ipl1 mutant cells cannot tolerate a large increase in the dosage of GLC7 (Francisco et al., 1994). Wild-type cells carrying a multicopy GLC7 plasmid did not show a major growth defect when compared with cells carrying an empty vector (unpublished data). In contrast, duo1-2 and dam1-9 mutants carrying the GLC7 plasmid showed a significant lowering of the restrictive growth temperature when compared with mutant cells carrying the empty vector (Fig. 8). Thus, increased Glc7 type-1 protein phosphatase activity exacerbates the phenotype of not only ipl1 but also dam1 and duo1 mutant cells. Together, these genetic interactions highlight the in vivo importance of Dam1 as a physiological target for Ipl1Sli15.
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Discussion |
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In addition to its stimulatory function, Sli15 also facilitates the association of Ipl1 with the mitotic spindle, as Ipl1 becomes more concentrated on the mitotic spindle in cells with elevated levels of Sli15. Although Ipl1 can bind to microtubules in vitro, very little, if any, Ipl1 is found on cytoplasmic microtubules in vivo (Biggins et al., 1999; Kim et al., 1999). Thus, Ipl1 may be prevented from acting in the cytoplasm through its nuclear import. Sli15, which contains a putative nuclear localization signal in its middle region, potentially may target Ipl1 to the nucleus. The finding that the middle region of Sli15 also binds directly to microtubules in vitro suggests that once inside the nucleus, Ipl1 and Sli15 may bind cooperatively to microtubules. Together, the targeting and stimulatory functions of Sli15 should ensure that only correctly targeted Ipl1 can phosphorylate its mitotic spindleassociated physiological substrates. This is clearly true in the case of Dam1, which is hypophosphorylated in ipl1-2 as well as sli15-3 mutant cells. Interestingly, Dam1 can bind to Ipl1 and Sli15 directly in vitro. Thus, Sli15 may enhance the association of Ipl1 with at least one of its substrates. This may represent yet another means by which Sli15 may facilitate the ability of Ipl1 to phosphorylate its physiological substrates.
Ipl1Sli15 and their homologues in other eukaryotes
Our finding that the COOH-terminal IN boxcontaining region of Sli15 binds to Ipl1 is consistent with the recent report that the IN boxcontaining COOH-terminal region of INCENP (ICP-1) from C. elegans is sufficient for its binding to aurora-B (AIRK2) (Kaitna et al., 2000). The observation that this region of Sli15 functions as a stimulator of Ipl1 suggests that the IN box in INCENP also may stimulate AIRK. This idea is further supported by our recent finding that a fusion protein containing essentially only the IN box of Sli15 can stimulate the kinase activity of Ipl1 (unpublished results). Our finding that Sli15 facilitates the association of Ipl1 with the mitotic spindle is also consistent with the recent reports that the mitotic spindle association of aurora-B (AIRK2) is dependent on INCENP in humans and C. elegans (Adams et al., 2000; Kaitna et al., 2000). The middle region of Sli15 can bind to microtubules directly and is localized to the mitotic spindle. The equivalent region of chicken INCENP also localizes to microtubules in mammalian cells (Mackay et al., 1993, 1998). Because the sequence homology between Sli15 and metazoan INCENP is largely restricted to the COOH-terminal IN box domain, it remains to be determined whether a less conserved sequence motif is responsible for the binding of Sli15 to microtubules.
Sli15 and Dam1 as physiological targets of Ipl1
Two lines of evidence suggest that Sli15 and Dam1 are physiological targets of Ipl1. First, they are very good in vitro substrates of Ipl1. In fact, Sli15 is by far the best in vitro substrate of Ipl1 that we have identified. It is a better substrate than the previously identified in vitro (and possibly in vivo) substrate Ndc10 (Biggins et al., 1999) and the physiological target histone H3 (Hsu et al., 2000). Second, both Sli15 and Dam1, like histone H3, are hypophosphorylated in ipl1-2 cells even at a permissive growth temperature. Even though the significance of the phosphorylation of these two proteins by Ipl1 is not yet known, the binding or phosphorylation of Dam1 by Ipl1Sli15 is likely to be biologically important since certain combinations of ipl1, sli15, and dam1 mutations show synthetic lethal genetic interactions (Kim et al., 1999; this study).
Although it may not be apparent in Fig. 7 C, the abundance of at least one slower-migrating and presumably phosphorylated form of Dam1 remains unchanged in ipl1-2 and sli15-3 cells, thus indicating that Dam1 may be the target of one additional protein kinase. Dam1 is known to interact genetically with the Mps1 protein kinase (Jones et al., 1999). However, the electrophoretic mobility of Dam1 appears unaffected in mps1-1 cells (unpublished results), thus it is not yet possible to conclude whether Dam1 is a physiological target for Mps1. Nevertheless, this observation argues that Ipl1 protein kinase activity is not greatly altered in mps1-1 cells.
Ipl1, Sli15, and Dam1 as microtubule-binding proteins that associate with centromeres
For chromosome segregation to occur, spindle microtubules from opposite poles must attach to the kinetochores of sister chromatids. Microtubule-binding proteins that are found at kinetochores potentially may play important roles in the poorly understood process of kinetochoremicrotubule attachment. The finding that Ipl1, Sli15, and Dam1 associate with both microtubules and kinetochores suggests that these proteins may connect kinetochores to spindle microtubules (this study; Hofmann et al., 1998; Cheeseman et al., 2001). The phosphorylation of Sli15 and Dam1 by Ipl1 may modulate their ability to associate with each other, the kinetochores, or microtubules, thus affecting kinetochoremicrotubule attachments or the movement of kinetochores along kinetochore microtubules. Consistent with the idea that Ipl1 function is required for normal kinetochoremicrotubule attachment or movement, Biggins et al. (1999) have shown that Ipl1 phosphorylates the kinetochore protein Ndc10 and inhibits the binding of kinetochores to microtubules in vitro. We have also shown that the kinetochore protein Nuf2 (Janke et al., 2001; Wigge and Kilmartin, 2001) is found along the length of mitotic spindles much more frequently in ipl1 and sli15 mutant cells than in wild-type cells (Kim et al., 1999). This pattern of Nuf2 localization suggests that kinetochores are attached to spindle microtubules in ipl1 and sli15 cells but these kinetochores do not move efficiently toward the opposite spindle poles. Interestingly, recent live-cell analysis of chromosome dynamics in strains carrying mutant kinetochore proteins revealed that ipl1 and dam1 mutants are similar in that sister chromatids show close association with a single pole, presumably reflecting monopolar microtubule attachment (He et al., 2001).
In addition to serving as a physical link between kinetochores and microtubules, Ipl1, Sli15, and Dam1 may play a role in monitoring the attachment of kinetochores to microtubules or their function may be regulated by this attachment. In this regard, it is interesting to note that one human homologue of Ipl1 (aurora-A or AIRK1) is known to bind to Cdc20, a key activator of the anaphase-promoting complex, which is the target for the kinetochore attachment checkpoint control (Farruggio et al., 1999).
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Materials and methods |
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Chromatin immunoprecipitation
Chromatin immunoprecipitation was performed as previously described (Saitoh et al., 1997), with some minor modifications. Approximately 4 x 108 yeast cells growing exponentially at 30°C in SC medium lacking leucine or uracil were fixed at room temperature with 1% formaldehyde for 15 min and then with 125 mM glycine for 5 min. Cells were harvested and washed three times with TBS (20 mM Tris-HCl [pH 7.6], 200 mM NaCl), and then resuspended in 400 µl of lysis buffer D (50 mM Hepes-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100 [vol/vol], 0.1% SDS [wt/vol]) that contained protease inhibitors (complete mini from Roche Diagnostics GmbH). Cell lysis and fragmentation of chromosomes were achieved by sonicating the samples for 14 rounds of 20 s each, using a Tekmar sonic disruptor with microtip and set at 60% duty cycle. This yielded chromatin fragments with average lengths of 300700 bp. Cell debris was removed by centrifuging in a microcentrifuge at 15,000 rpm for 5 min and then for 15 min at 4°C. The volume of the supernatant (whole cell extracts [WCE]) was adjusted to 450 µl with lysis buffer D. 400 µl of each WCE was used for immunoprecipitation and the rest was stored at -20°C as untreated WCE. WCE was incubated for 4 h at 4°C with 4 µg of antibodies (9E10 in the case of anti-Myc; 16B12 in the case of anti-HA; both from BAbCO). 60 µl of protein ASepharose CL-4B beads (Amersham Pharmacia Biotech) was added and incubated for 1 h at 4°C. Beads were harvested by centrifugation, followed by five rounds of washing with 1.5 ml of lysis buffer D. Washed beads were resuspended in 200 µl of TE (10 mM Tris-HCl [pH 7.0], 1 mM EDTA). Immunoprecipitated sample and untreated WCE (see above) were adjusted to give final concentrations of 0.25% SDS (wt/vol) and 250 µg/ml proteinase K (Promega). These samples were incubated at 37°C for at least 8 h. Cross-linking was reversed by incubating the samples at 65°C for 6 h, followed by phenol/chloroform extraction. DNA was precipitated and resuspended in 50 µl of TE. 1 µl of each sample was used as template for 30 cycles of PCR reactions with Taq DNA polymerase (Roche Diagnostics GmbH). CEN3 and CEN16 primers were described by Meluh and Koshland (1997). CEN16 proximal primers had the following sequences (5'-ACACCATGGTAGCGGTTCTA-3' and 5'- GGTAGAAGCCTTTGTACCAT-3').
Purification and analysis of proteins
GST or GST fusion proteins were purified from yeast essentially as previously described (Kim et al., 1999), except that the lysis buffer contained 50 mM Hepes-KOH (pH 7.4), 200 mM KCl, 10% glycerol (vol/vol), 1% NP-40 (vol/vol), 10 mM EGTA, 2 mM DTT, 50 mM NaF, 0.1 mM Na3VO4, 5 mM ß-glycerophosphate, and the following protease inhibitors (Sigma-Aldrich): 2 µg/ml each of antipain, leupeptin, pepstatin A, chymostatin, and aprotinin; 10 µg/ml of phenanthroline; 16 µg/ml of benzamidine-HCl; and 1 mM PMSF. Cell lysis was performed in glass tubes (13 x 100 mm), each containing 0.1 ml of cell suspension.
E. coli cells (BL21 or BL21[DE3]) expressing GST or GST fusion proteins were harvested and rinsed once in 10 ml of ice cold washing buffer, which consisted of 50 mM Hepes-KOH (pH 7.4) and 200 mM KCl. Cells were resuspended in 1.5 ml of buffer B, which consisted of 50 mM Hepes-KOH (pH 7.4), 200 mM KCl, 1% NP-40 (vol/vol), 1 mM EDTA, 2 mM DTT, and the protease inhibitors listed above. Cells were resuspended in 1.5 ml of the same buffer and lysed in a French press at 16,000 psi. Cell debris was removed by a 10-min centrifugation at 20,000 g, thus generating the supernatant fraction. The supernatant was added to a 1.5-ml microcentrifuge tube that contained 0.1 ml of a 50% slurry (vol/vol) of glutathione-agarose beads. After a 2-h incubation at 4°C with constant agitation, the glutathione-agarose beads were harvested, followed by two 5-min washes with 1 ml of lysis buffer B. The proteins bound on the glutathione-agarose beads were eluted by the addition of 0.1 ml of elution buffer (50 mM Tris-HCl [pH 8.0], 50 mM KCl, 15 mM reduced glutathione; Sigma-Aldrich). The binding of His6Dam1 to GSTIpl1 and GSTSli15 was performed essentially as described previously (Kim et al., 1999).
Protein samples were electrophoretically separated in SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated with 2,000-fold diluted rabbit anti-GST antibodies (Molecular Probes, Inc.), 1,000-fold diluted mouse anti-HA or anti-Myc ascites fluid (both from BAbCO), 100,000-fold diluted anti-G6PDH antibodies (Sigma-Aldrich), 1,000-fold diluted affinity-purified guinea pig anti-Dam1 antibodies (Cheeseman et al., 2001), or 2,000-fold diluted affinity-purified rabbit anti-Duo1 antibodies (Hofmann et al., 1998). Proteins recognized by primary antibodies were visualized by the ECL chemiluminescent system (Amersham Pharmacia Biotech).
Dam1 immunoprecipitation and phosphatase treatment
Cells from 50 ml of a log phase culture were harvested and washed with sorbitol buffer (1.3 M sorbitol, 0.1 M KPO4, pH 7.5) and incubated for 50 min with lyticase. Cells were pelleted gently and resuspended in lysis buffer E (1% SDS [wt/vol], 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) with protease inhibitors, 1 mM PMSF, and phosphatase inhibitors (10 mM sodium pyrophosphate, 10 mM sodium azide, 10 mM NaF, 0.4 mM Na3VO4). The resuspended cells were sonicated 10 times for 10 s each and pelleted at maximum speed in a microcentrifuge. The supernatant was diluted 10-fold with dilution buffer (1% Triton [vol/vol], 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0). Approximately 15 µl of anti-Dam1 antibody precoupled to 20 µl of protein A Affi-gel beads (Bio-Rad Laboratories) was added. The beads were incubated overnight at 4°C and then washed three times with dilution buffer. Sample buffer was added directly to half of the sample. The remaining sample was treated with 800 u of phosphatase (New England Biolabs, Inc.) for 2 h at 30°C, according to the manufacturer's guidelines, and sample buffer was then added. Both samples were then run on a 12% polyacrylamide gel.
Ipl1 kinase assay
Unless otherwise stated, the reaction mixture consisted of 5 µl kinase buffer (60 mM MgCl2, 60 mM MnCl2, 6 mM DTT, 0.6 mM Na3VO4, 30 mM ß-glycerophosphate), 5 µl [-32P]ATP (160 µM), 10 µl GSTIpl1 or GSTIpl1D245N (
0.3 µg) in elution buffer (see above), and 10 µl substrate (
5 µg of bovine MBP [Sigma-Aldrich] or
1 µg of GST fusion protein) in elution buffer. After 15 min at 30°C, reactions were stopped by adding 10 µl of 4x sample buffer. Protein samples were electrophoretically separated in SDS-PAGE. The stoichiometry of phosphate incorporated into GSTSli15 was determined by scintillation counting of the phosphorylated GSTSli15 band that had been excised from the dried SDS-PAGE.
Microtubule binding assay
Purified bovine brain tubulin (60 µM) in PME buffer (80 mM K-Pipes, pH 6.8, 1 mM EGTA, 1 mM MgCl2) was thawed and centrifuged in a microcentrifuge for 5 min at 4°C to remove insoluble protein. To assemble microtubules, GTP was added to a final concentration of 1 mM, glycerol was added to a final concentration of 25% (vol/vol), and the reaction was incubated at 35°C for 30 min. After assembly, taxol was added to a final concentration of 20 µM to stabilize microtubules. Microtubules were then diluted in PME buffer containing 1 mM GTP and 10 µM taxol.
For cosedimentation assays, GST and GST fusion proteins were purified from E. coli and dialyzed into PME buffer, diluted fivefold with PME buffer, and then centrifuged for 20 min at 60,000 rpm and 25°C in a TLA100 rotor (Beckman Coulter). 20 µl of soluble protein was added to variable concentrations of taxol-stabilized microtubules (05 µM) in 40-µl reactions. The reactions were incubated for 20 min at 25°C to allow binding to occur, and then centrifuged as above to pellet the microtubules. Pellets and supernatants were fractionated on SDS-PAGE gels and probed by immunoblotting with anti-GST antibodies.
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Footnotes |
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Acknowledgments |
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This work was supported in part by National Institutes of Health grants to C.S.M. Chan (GM45185) and G. Barnes (GM47842) and a National Science Foundation Graduate Research fellowship to I.M. Cheeseman.
Submitted: 4 May 2001
Revised: 5 October 2001
Accepted: 8 October 2001
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References |
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