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• W1971378151 abstract "Article1 October 1997free access Fission yeast Cut2 required for anaphase has two destruction boxes Hironori Funabiki Hironori Funabiki Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606 Japan Present address: Department of Physiology, Box 0444, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0444 USA Search for more papers by this author Hiroyuki Yamano Hiroyuki Yamano ICRF Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Koji Nagao Koji Nagao Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606 Japan Search for more papers by this author Hirofumi Tanaka Hirofumi Tanaka Tokyo University of Pharmacy and Life Science, Horinouchi 1432–1, Hachioji, Tokyo, 192-03 Japan Search for more papers by this author Hideyo Yasuda Hideyo Yasuda Tokyo University of Pharmacy and Life Science, Horinouchi 1432–1, Hachioji, Tokyo, 192-03 Japan Search for more papers by this author Tim Hunt Tim Hunt ICRF Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Mitsuhiro Yanagida Corresponding Author Mitsuhiro Yanagida Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606 Japan Search for more papers by this author Hironori Funabiki Hironori Funabiki Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606 Japan Present address: Department of Physiology, Box 0444, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0444 USA Search for more papers by this author Hiroyuki Yamano Hiroyuki Yamano ICRF Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Koji Nagao Koji Nagao Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606 Japan Search for more papers by this author Hirofumi Tanaka Hirofumi Tanaka Tokyo University of Pharmacy and Life Science, Horinouchi 1432–1, Hachioji, Tokyo, 192-03 Japan Search for more papers by this author Hideyo Yasuda Hideyo Yasuda Tokyo University of Pharmacy and Life Science, Horinouchi 1432–1, Hachioji, Tokyo, 192-03 Japan Search for more papers by this author Tim Hunt Tim Hunt ICRF Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Mitsuhiro Yanagida Corresponding Author Mitsuhiro Yanagida Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606 Japan Search for more papers by this author Author Information Hironori Funabiki1,2, Hiroyuki Yamano3, Koji Nagao1, Hirofumi Tanaka4, Hideyo Yasuda4, Tim Hunt3 and Mitsuhiro Yanagida 1 1Department of Biophysics, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto, 606 Japan 2Present address: Department of Physiology, Box 0444, School of Medicine, University of California San Francisco, San Francisco, CA, 94143-0444 USA 3ICRF Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK 4Tokyo University of Pharmacy and Life Science, Horinouchi 1432–1, Hachioji, Tokyo, 192-03 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:5977-5987https://doi.org/10.1093/emboj/16.19.5977 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The fission yeast Schizosaccharomyces pombe cut2+ gene is essential for sister chromatid separation. Cut2 protein, which locates in the interphase nucleus and along the metaphase spindle, disappears in anaphase with the same timing as mitotic cyclin destruction. This proteolysis depends on the APC (Anaphase-Promoting Complex)–cyclosome which contains ubiquitin ligase activity. The N-terminus of Cut2 contains two stretches similar to the mitotic cyclin destruction box. We show that both sequences (33RAPLGSTKQ and 52RTVLGGKST) serve as destruction boxes and are required for in vitro polyubiquitination and proteolysis. Cut2 with doubly mutated destruction boxes inhibits anaphase, whereas Cut2 with singly mutated boxes can suppress cut2 mutations. Strong expression of the N-terminal 73 residues containing the destruction boxes leads to the accumulation of endogenous cyclin and Cut2, and arrests cells in metaphase, whereas the same fragment with the mutated boxes does not. Cut2 proteolysis occurs in vitro using Xenopus mitotic extracts in the presence of functional destruction boxes. Furthermore, Cut2 is polyubiquitinated in an in vitro system using HeLa extracts, and this polyubiquitination requires the destruction boxes. Introduction Ubiquitin-mediated proteolysis is involved in several important biological events, such as cell cycle progression, signal transduction and the stress response (e.g. Hershko, 1996). Ubiquitin is conjugated to lysine residues of a target protein by ubiquitinating machinery which consists of E1, E2 and E3. Polyubiquitinated proteins are then degraded by the 26S proteasome in an ATP-dependent manner. The budding and fission yeast mutants of the 26S proteasome are blocked at metaphase (Ghisiain et al., 1993; Gordon et al., 1993), consistent with the notion that ubiquitin-mediated proteolysis is required for both sister chromatid separation and inactivation of mitotic cyclin-dependent kinases (CDKs) in order for cells to undergo anaphase and to exit from the M phase (Holloway et al., 1993; Surana et al., 1993). Mitotic exit and sister chromatid separation also fail to occur in mutants of the 20S anaphase-promoting complex (APC)–cyclosome, which contains the activity for ubiquitin ligase (Irniger et al., 1995; King et al., 1995; Sudakin et al., 1995; Tugendreich et al., 1995). The best studied substrates of ubiquitin- and APC–cyclosome-mediated proteolysis are mitotic cyclins. The N-terminal portion of mitotic cyclins is dispensable for binding to and activation of p34cdc2, and is solely required for mitotic destruction (Murray et al., 1989). When non-degradable B-type cyclins carrying deletions in the destruction boxes were expressed, the cell cycle was blocked in late anaphase with high H1 kinase activity (Murray et al., 1989; Holloway et al., 1993; Surana et al., 1993; Sigrist et al., 1995; Rimmington et al., 1996; Yamano et al., 1996). The onset of sister chromatid separation (anaphase) can occur in the presence of non-degradable B-type cyclin. Final exit from mitosis requires inactivation of the Cdc2 kinase–cyclin B complex. To degrade mitotic cyclins, the 20S APC–cyclosome is necessary as it has the E3 ubiquitin ligase activity for cyclins (King et al., 1995; Sudakin et al., 1995). The APC–cyclosome in budding yeast contains several (perhaps eight or more) subunits including Cdc16 and Cdc27 (Peters et al., 1996; Zachariae et al., 1996), which are known to be essential for mitotic progression. Similar proteins have been found in fission yeast and mammals, and shown to be required for the exit from mitosis and sister chromatid separation (Hirano et al., 1988; O'Donnell et al., 1991; Samejima and Yanagida, 1994; Tugendreich et al., 1995; Yamashita et al., 1996; Yamada et al., 1997). The 20S APC–cyclosome probably exists in all eukaryotes, and was thought to be required for the destruction of mitotic cyclins and other unidentified proteins (e.g. Holloway et al., 1993). The region in mitotic cyclins required for proteolysis contains an amino acid motif called the destruction box which is necessary for regulated proteolysis (Glotzer et al., 1991). The consensus for this motif is RXALGXIXN. While R and L residues are conserved in all destruction boxes of A- and B-type cyclins, N residues are only conserved in B-type cyclins (Glotzer et al., 1991). The destruction box of fission yeast cyclin B Cdc13 is RHALDDVSN (Yamano et al., 1996). The destruction box sequences thus appear to be quite variable except for the minimal RXXL. The exact role of the destruction box is unknown, although it is needed for ubiquitination of B-type cyclin in vitro (Glotzer et al., 1991; King et al., 1996). Frog B-type cyclins whose destruction boxes were substituted by A-type ones were ubiquitinated in frog egg extract; however, they were not destroyed efficiently (Klotzbücher et al., 1996). This suggests that ubiquitination is a necessary step for programmed cyclin destruction, but is not by itself sufficient. Triggering proteolysis probably involves a mechanism still to be identifed. The budding yeast Pds1 and the fission yeast Cut2 show cell cycle-regulated proteolysis, and are the targets of the 20S APC–cyclosome complex (Cohen-Fix et al., 1996; Funabiki et al., 1996b). Their destruction is required for the progression of anaphase, and is dependent upon the presence of functional APC–cyclosome complex. In the mutants pds1 and cut2, cyclin destruction takes place, while sister chromatid separation fails to occur. It is of considerable interest how simultaneous proteolysis of cyclins and Pds1 or Cut2 can occur, as their proteolysis seems to be a key regulatory aspect for coordinated progression of anaphase (sister chromatid separation) and mitotic exit (cyclin destruction). The fission yeast cut2+ gene is required for sister chromatid separation in anaphase (Hirano et al., 1986; Uzawa et al., 1990). In temperature-sensitive (ts) cut2-364, sister chromatids are only partly separated (Funabiki et al., 1996a). Most cell cycle events, except for sister chromatid separation, can occur, as evidenced in ts cut2-cdc11 double mutant cells where cytokinesis is blocked by cdc11, leading to a huge polyploid nucleus. The cut2+ gene is essential for viability and encodes a protein of 301 amino acids which interacts with Cut1 to form the large complex (Uzawa et al., 1990; Funabiki et al., 1996a). Cut2 is degraded in anaphase and becomes highly unstable in G1-arrested cells (Funabiki et al., 1996b). Its level recovers rapidly in S phase. The degradation of Cut2 is dependent on the presence of the cut9+ gene required for the formation of the 20S APC–cyclosome complex (Yamada et al., 1997). Furthermore, Cut2 appears to contain the sequences essential for proteolysis in the first 80 amino acids, since truncation of this region leads to stabilization of Cut2 in G1-arrested cells (Funabiki et al., 1996b). Expression of the non-degradable truncated mutant (Cut2Δ80) blocks sister chromatid separation but not other cell cycle events. Furthermore, grafting the N-terminal region of the fission yeast cyclin Cdc13 gene to that of N-truncated Cut2 allows suppression of the ts phenotype of cut2-364 (Funabiki et al., 1996b). Cut2 proteolysis is hence essential for the onset of anaphase. Cut2 may not be a glue-like protein postulated to link sister chromatids until anaphase (Holloway et al., 1993; Irniger et al., 1995). Deletion of the glue protein would result in premature sister chromatid separation. However, cut2 null mutant cells failed to separate sister chromatids (Funabiki et al., 1996a,b). Cut2 is thought to have a positive role in bringing cells to normal metaphase, but it must be degraded to enter anaphase. In the present study, we provide evidence that Cut2 contains two destruction boxes in its N-terminal region. Construction of Cut2 with mutagenized boxes clearly indicated that both boxes were implicated in proteolysis of Cut2. They also served as the essential destruction boxes in vitro for the Xenopus mitotic extracts. Therefore, the two destruction boxes, though their consensus sequences are only loosely conserved, can be recognized by an in vitro proteolytic system of an evolutionarily distant organism. We further show that Cut2 is polyubiquitinated in a manner depending on the presence of destruction boxes in an in vitro system using HeLa cell extracts prepared 1 h after the release from colcemid block. In this in vitro system, the N-terminal 73 amino acid-containing fragment can also be polyubiquitinated. These results indicate that the destruction boxes are required for polyubiquitination of Cut2. Results Failure of sister chromatid separation by a Cut2 destruction box mutant The amino acid sequence of Cut2 (Uzawa et al., 1990; Funabiki et al., 1996b) reveals two stretches in the N-terminal region (33RAPLGSTKQ and 52RTVLGGKST) that are similar to the consensus for the destruction box of mitotic cyclins (Glotzer et al., 1991; Yamano et al., 1996; Figure 1A). To examine whether these sequences are required for regulated instability of Cut2, two arginine (33R and 52R) and two leucine (36L and 55L) residues conserved in all the destruction boxes of mitotic cyclins were mutated to alanine (A). The resulting mutant genes having substitutions in the first destruction box (33AAPAGSTKQ), in the second box (52ATVAGGKST) or in both boxes are designated cut2dm1, cut2dm2 and cut2ddm, respectively. The cut2 mutant gene, cut2Δ80, with deletions of the first 80 residues, was described previously by Funabiki et al. (1996b). Figure 1.Induced overexpression of wild-type and mutant Cut2. (A) This diagram indicates genes encoding the full-length Cut2 (cut2+), N-terminal 80 amino acid deletion (cut2Δ80), substitution mutations in the first destruction box (cut2dm1), the second destruction box (cut2dm2) or both destruction boxes (cut2ddm). In the substituted destruction box mutants, 33R, 52R, 36L and 55L are replaced by A as indicated in the figure. These genes were placed downstream of the weak inducible promoter REP81. (B) The wild-type strain carrying one of the above plasmids was plated in the absence or presence (2 μM) of thiamine at 33°C. Overexpression took place in the absence of thiamine. Colonies were made in cells carrying a plasmid with the wild-type cut2+ gene or a vector plasmid, but colony formation was inhibited in cells carrying the mutant cut2 genes. The growth inhibition effects of cut2Δ80 and cut2ddm were the strongest, whereas cut2dm1 showed the least effect. (C) Wild-type cells carrying plasmid with the cut2ddm mutant gene were cultured in liquid EMM2 medium at 33°C for 12 h in the absence of thiamine and stained by DAPI. Cells displayed a failure of sister chromatid separation. The frequent cut phenotype was observed. This phenotype was identical to that obtained by overproducing the deletion cut2Δ80 (Funabiki et al., 1996b). Bar, 10 μm. Download figure Download PowerPoint The wild-type and mutant genes were placed downstream of the weak inducible nmt1 promoter (REP81) and overexpressed in wild-type Schizosaccharomyces pombe cells in the absence of thiamine (Basi et al., 1993). While the wild-type Cut2 protein overproduced under the control of the promoter REP81 in wild-type cells did not inhibit colony formation at all, overproduced mutant proteins did so strongly (Figure 1B). The degree of inhibition by cut2dm1 was the lowest among the mutants examined, whereas both cut2Δ80 and cut2ddm showed a very strong inhibitory effect. Fluorescence microscopic observation of liquid cultures of wild-type cells carrying plasmids with the REP81-cut2Δ80, -cut2dm1, -cut2dm2 or -cut2ddm gene in the absence of thiamine indicated that sister chromatid separation failed to occur, followed by the cut phenotype where cytokinesis took place in the absence of nuclear division (Figure 1C, micrograph taken from cut2ddm). This cytological phenotype greatly resembled that of cut2Δ80 (Funabiki et al., 1996b). Inability of the double destruction box mutant to suppress cut2 null and ts mutant cells To test whether Cut2 with the destruction box mutations was functional, a ts cut2 mutant was transformed by plasmids carrying the wild-type gene (cut2+), cut2dm1, cut2dm2, cut2ddm or cut2Δ80 under the control of the REP81 promoter. Colonies were produced at 26°C by all transformants in the repressed condition (+ thiamine, data not shown). Transformants were then plated at 36°C in the presence or absence of thiamine (Figure 2). Temperature-sensitive mutant cells carrying a plasmid with the wild-type cut2+ or the single destruction box mutant genes (cut2dm1 and cut2dm2) placed downstream of REP81 were found to be viable in the repressed condition, indicating that reduced production of single mutants cut2dm1 and cut2dm2 could suppress the ts phenotype of cut2-364. However, the double destruction box mutant cut2ddm and the N-terminal deletion cut2Δ80 did not suppress this phenotype. Although single destruction box mutant proteins (Cut2dm1 and Cut2dm2) might be destroyed less efficiently than the wild-type Cut2, they could support colony formation if expressed at a very low level in the cut2 mutant. However, Cut2ddm was found to be no longer functional, and inhibited colony formation as did Cut2Δ80. An unexpected finding was that, in the derepressed condition (−thiamine), Cut2dm1 did not block colony formation and could suppress the ts phenotype (Figure 2, −thiamine). A possible explanation for the phenotypic difference between wild-type and ts cut2 mutant is that, while Cut2dm1 would become toxic in the wild-type, because cells containing the wild-type Cut2 should move through anaphase rapidly, it would not become toxic in the absence of wild-type Cut2, because anaphase might be slowed down by or ‘adapted’ to the mutant Cut2dm1. Figure 2.The substitution mutant cut2ddm fails to rescue cut2-364. Mutant cut2-364 was transformed with plasmid REP81 carrying the wild-type cut2+ or cut2 mutant genes. Transformants obtained at 26°C in the presence of thiamine were plated at 36°C in the presence or absence of thiamine. Plasmids carrying the wild-type cut2+, mutant cut2dm1 or mutant cut2dm2 gene suppressed the ts phenotype of cut2–364 in the presence of thiamine. However, plasmids carrying cut2Δ80 or cut2ddm failed to suppress the ts phenotype of cut2-364 in the presence or absence of thiamine. Download figure Download PowerPoint A similar test was carried out for the gene-disrupted strain (cut2Δ), which carried these plasmids. To this end, heterozygous diploid cells in which one of the chromosomal cut2+ genes was disrupted by S.pombe ura4+ (Grimm et al., 1988) were transformed by plasmids. When haploid segregants obtained by sporulation of these transformed diploid cells were tested for their viability in the medium lacking uracil at 33°C, only suppressed cut2Δ spores could grow. Results of colony formation were basically identical to those for ts cut2 mutants (data not shown), indicating that Cut2 protein with one destruction box was capable of supporting colony formation. Metaphase block by overproduction of the N-terminal fragment In Xenopus extracts, the N-terminal fragment of cyclin B acts as a competitive substrate for degradation of the endogenous full-length cyclin B (Holloway et al., 1993). Yamano et al. (1996) recently showed that overproducing the N-terminal fragment of Cdc13 (S.pombe cyclin B) led to inhibition of the metaphase–anaphase transition and also to the degradation of endogenous Cdc13. If Cut2 is a substrate for the same proteolytic machinery employed for destroying mitotic cyclins, massive overproduction of the N-terminal fragment of Cut2 might also prevent the destruction of endogenous Cut2 and cyclin B, thus preventing both sister chromatid separation and the inactivation of p34cdc2. We examined the phenotype of wild-type cells carrying a plasmid with the N-terminal 73 residues of Cut2 (the gene designated cut2N73; Figure 3A) placed under the strong REP1 promoter in the absence of thiamine (Maundrell, 1990). Overproduction of Cut2N73 and Cut2N73dm1 fragments blocked colony formation in the absence of thiamine (Figure 3B). In contrast, overproduction of Cut2N73dm2 and Cut2N73ddm allowed the formation of colonies in the absence of thiamine (Figure 3B). Thus the growth-inhibitory effect of the N-terminal fragment requires the presence of the second destruction box sequence. We interpret these results as showing that the two destruction boxes are not equal, and that the second box is more important than the first one. Figure 3.Overproduction of the N-terminal fragments. (A) Construction of clones coding for the N-terminal 73 amino acid fragment of Cut2 (cut2N73), with mutations in the first destruction box (cut2N73dm1), in the second destruction box (cut2N73dm2) and in both destruction boxes (cut2N73ddm). These fragments were placed under the control of the strong nmt promoter REP1 (Maundrell, 1990). The names of the resulting plasmids are indicated. (B) Wild-type strains carrying the plasmids described above were plated in the presence or absence of thiamine (2 μM) at 33°C. No colony was produced by a plasmid carrying the N73 fragment without a mutation in the absence of thiamine, but colonies were made by cells overproducing double destruction box mutant fragments. Download figure Download PowerPoint Cell cycle phenotypes of cells overproducing the wild-type (Cut2N73) and mutant (Cut2N73ddm) N-terminal fragments were then examined (Figure 4). At 14 h after the removal of thiamine, the level of the N73 fragments (p11) increased sharply (Figure 4A). The level of Cdc13 (p63) also increased when Cut2N73 was overproduced. The upper band of endogenous Cut2, which appeared only in mitosis (Funabiki et al., 1996b), was seen. When the mutant Cut2N73ddm fragment was overproduced, however, the level of Cdc13 (p63) did not increase. For the endogenous Cut2 (p42), the mitotic upper band did not appear, but the level of endogenous p42 slightly increased. Figure 4.Metaphase arrest by overproducing the Cut2N73 fragment. Wild-type cells carrying a plasmid with the wild-type N-terminal fragment Cut2N73 or the mutant fragment Cut2N73ddm under the control of the strong promoter REP1 were cultured at 33°C for 16 h in the absence of thiamine. (A) Overexpression of the N-terminal fragment (p11) was detected by anti-cut2 (α-Cut2) antibodies. The levels of full-length Cut2, Cdc13 and Cdc2 were determined by anti–cut2 (α-Cut2), anti-cdc13 (α-Cdc13) and anti-PSTAIRE (α-PSTAIRE) antibodies, respectively. (B) Cells overproducing Cut2N73 or Cut2N73ddm at 14 h after the removal of thiamine were stained by DAPI, anti-tubulin (TUB) and anti-sad1 (which stains spindle pole bodies) antibodies. Cells overproducing Cut2N73 were blocked at metaphase, whereas cells overexpressing Cut2N73ddm grew normally, showing interphase and mitotic cells. The bar indicates 10 μm. (C) The frequency of cells containing condensed chromosomes in the cultures expressing Cut2N73 (N73) or Cut2N73ddm (N73ddm) was determined by DAPI staining. (D) The H1 kinase activities dependent upon Cdc13 in the cultures overproducing Cut2N73 or Cut2N73ddm were measured using immunoprecipitation by anti-cdc13 antibodies. Download figure Download PowerPoint Cytological phenotypes were then examined (Figure 4B–D). About 45% of the cells overproducing the Cut2N73 fragment (left panel in B) displayed condensed chromosomes and a short spindle (TUB, anti-tubulin antibody stain; SPB, anti-sad1 stain). Cdc13-dependent histone H1 kinase activity was elevated 5-fold in cells overproducing Cut2N73 (Figure 4D), suggesting that overproduction of the N-terminal fragment arrested cells at metaphase. In sharp contrast, neither the frequencies of cells revealing condensed chromosome (and a short metaphase spindle) nor the level of histone H1 kinase activity increased in cells overproducing the same fragment with mutated destruction boxes (N73ddm). These results clearly demonstrated that overproduction of the Cut2N73 fragment led to the metaphase arrest and that this arrest required the presence of functional destruction boxes. Stability of Cut2ddm in G1-arrested cells Cut2 was shown previously to be highly unstable in G1-arrested cdc10 cells, whereas the N-terminal deletion Cut2Δ80 was stable (Funabiki et al., 1996b). To examine the stability of the mutant Cut2 proteins under these conditions, wild-type Cut2, mutant Cut2ddm and mutant Cut2Δ80 were expressed in G1-arrested cdc10 cells under the control of the weak promoter REP81 and their abundance measured at the indicated times (see Figure 5A). Overproduction started after 11 h in the absence of thiamine, and then cdc10 cells were shifted to 36°C. Cut2ddm did not accumulate as Cut2Δ80. The level of Cut2ddm (the total of native p42 and cleaved p38) was twice that of wild-type Cut2 and one-quarter that of p37 Cut2Δ80 (Figure 5B). Thus Cut2ddm mutant protein was not as stable as Cut2Δ80, and was only slightly more stable than the wild-type in G1-arrested cells (Figure 5A and B). The single destruction box mutants proteins (Cut2dm1 and Cut2dm2) did not accumulate at all in G1-arrested cdc10 cells (data not shown). The level of Cdc13 shown as control was greatly reduced in G1-arrested cells (4 h at 36°C). These results suggested that sequences other than the two destruction boxes might cause the instability in G1-arrested cells. Figure 5.Accumulation of Cut2ddm protein in cdc10 mutant cells. (A) Wild-type cut2+, mutant cut2ddm and mutant cut2Δ80 placed under the control of the weak promoter REP81 were overexpressed in G1-arrested cdc10 mutant cells in which mitotic cyclin Cdc13 and Cut2 were unstable (Funabiki et al., 1996b; Yamano et al., 1996). The cultures of the cdc10 mutant carrying a plasmid with the wild-type cut2+ or mutant cut2 genes were shifted to 36°C at the time (11 h) that overproduction began. Cells were collected at 0 (11) and 4 (15) h after the shift to 36°C (hours after the removal of thiamine), and assayed for the levels of Cut2, Cdc13 and Cdc2 proteins using antibodies α-Cut2, α-Cdc13 and αPSTAIRE. The levels of wild-type Cdc13 and Cut2 were negligible in G1-arrested cell extracts (after 4 h), but the level of mutant Cut2Δ80 was high. The level of cut2ddm was higher than that of wild-type but significantly lower than that of cut2Δ80. (B) Cut2ddm seemed to be cleaved to produce the 38 kDa band. The intensities of these bands were summed and are shown. Download figure Download PowerPoint Overproduction of the N-terminal fragment in G1-arrested cells blocks proteolysis of mitotic cyclin The wild-type and mutant N-terminal fragments were overproduced under the control of the strong promoter REP1 in cdc10-arrested cells (Figure 6A). When the mutant fragment Cut2N73ddm (p11) was overproduced, the levels of both p42 (Cut2) and p63 (Cdc13) were lowered in G1-arrested cdc10 mutant cells. In contrast, the degradation of p42 (Cut2) and p63 (Cdc13) was suppressed significantly in cdc10-arrested cells which overproduced the wild-type N-terminal fragment Cut2N73 (p11). cdc10 cells were verified to be in the G1 state by fluorescence-activated cell sorting (FACS) analysis (4 h at 36°C after overproduction; Figure 6B). These results demonstrated that excess N-terminal fragment with the destruction boxes was capable of inhibiting proteolysis of endogenous cyclin and Cut2 in G1-arrested cells. Figure 6.Overproduction of Cut2N73 and Cut2N73ddm in G1-arrested cdc10 mutant cells. cdc10-129 strains carrying the vector plasmid pREP1 or plasmid with the cut2N73 or the cut2N73ddm sequence under the control of the REP1 promoter were cultured in the absence of thiamine for 12 h at 26°C, and then transferred to 36°C. Cultures were taken at 0, 3 and 4 h, and extracts were subjected to SDS–PAGE for immunoblotting. (A) Immunoblot using anti-cut2, anti-cdc13 and anti-PSTAIRE. The p11 band represents the polypeptide produced by a plasmid carrying the cut2N73 or cut2N73ddm gene. After 4 h, p42 (full-length Cut2) and p63 (Cdc13) were degraded in cdc10 carrying the vector plasmid, and considerably degraded in cdc10 producing the mutant p11 fragment Cut2N73ddm, but a significant fraction of p42 and p63 remained in cdc10 producing the wild-type p11 fragment Cut2N73. (B) The DNA contents of the above cells were examined by FACScan. Most cdc10 cells contained 1C DNA after 4 h at 36°C. Download figure Download PowerPoint Proteolysis of Cut2 in vitro using Xenopus egg extracts The S.pombe B-type cyclin, Cdc13, was degraded in Xenopus egg extract with kinetics similar to endogenous frog B-type cyclins, and the proteolysis was destruction box-dependent (Yamano et al., 1996). Using the same Xenopus cell-free system, we tested whether Cut2 destruction box mutant proteins were degraded (Figure 7). 35S-labelled substrates for destruction assays were prepared by translation in a reticulocyte lysate (indicated by ‘TL’ in Figure 7A–F). As shown in a control experiment (Figure 7F), Cdc13 was degraded in the CSF extracts in the presence of Ca2+, but not in the absence of Ca2+ (cyclin destruction is triggered in meiosis II metaphase-arrested CSF extract by the addition of Ca2+). No proteolysis occurred in the interphase extracts. Figure 7.Proteolysis of Cut2 in vitro using Xenopus extracts. Wild-type and mutant Cut2 proteins were 35S-translabelled in reticulocyte lysates and assayed for destruction in Xenopus CSF extracts (in the presence or the absence of Ca2+) or interphase extracts. TL; in vitro translated products as substrate. (A) Cut2; (B) Cut2dm1; (C) Cut2dm2; (D) Cut2ddm; (E) Cut2Δ80; (F) Cdc13. Cdc13 was employed as the control substrate for proteolysis in the Xenopus extracts, and was completely degraded in CSF extracts in the presence of Ca2+. Proteolysis did not take place if Ca2+ was not added or if the interphase extract was used. The destructability of wild-type Cut2 and mutant Cut2 proteins was examined in the same extracts (A–E). The initial protein bands were greatly shifted to the upper positions" @default.
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• W1971378151 date "1997-10-01" @default.
• W1971378151 modified "2023-09-30" @default.
• W1971378151 title "Fission yeast Cut2 required for anaphase has two destruction boxes" @default.
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• W1971378151 doi "https://doi.org/10.1093/emboj/16.19.5977" @default.
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