Fission Yeast Slp1: An Effector of the Mad2-Dependent Spindle Checkpoint

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Science  13 Feb 1998:
Vol. 279, Issue 5353, pp. 1045-1047
DOI: 10.1126/science.279.5353.1045


Mad2 is a component of the spindle checkpoint, which delays the onset of anaphase until all chromosomes are attached to the spindle. Mad2 formed a complex with Slp1, a WD (tryptophan–aspartic acid)–repeat protein essential for the onset of anaphase. When the physical interaction between the two proteins was disrupted, the spindle checkpoint was no longer functional. Post-anaphase events such as chromosome decondensation and the next round of DNA replication were not delayed even when the spindle assembly was incomplete. This relief of dependence appears to be a result of deregulation of ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex.

Duplicated chromosomes segregate equally in anaphase. The spindle checkpoint delays the onset of anaphase until all chromosomes are attached to the spindle (1) and prevents production of cells with missing or extra chromosomes. Mad2 is a component of the spindle checkpoint conserved in eukaryotes (2). It associates with unattached kinetochores and disappears as mitosis proceeds normally. Fission yeast Mad2 causes a metaphase arrest when it is overexpressed (3). Thus, Mad2 has a key function in a signaling pathway that delays the onset of anaphase in response to a spindle defect. However, the nature of this function has not yet been described.

The fission yeast slp1 + gene encodes a protein containing repeating units that often end with the sequence Trp-Asp (WD repeat). The slp1 + gene is essential for cell growth, and a temperature-sensitive mutant (slp1-362) was arrested before anaphase (4). Slp1 is a member of a family of proteins that includes Fzy, Cdc20, p55CDC (5), Hct1/ Cdh1, and Fzr (6). Members of this family may function as regulators of the ubiquitin-dependent proteolysis during mitosis (6). We isolated the fission yeast Mad2 in the yeast two-hybrid system (7-9) withslp1 + as bait. Slp1 and Mad2 interacted strongly and gave rise to histidine-prototrophic growth (Fig.1A) and increased β-galactosidase (LacZ) enzymatic activity (Fig. 1B) in a specific manner. The physical interaction between the two proteins was confirmed with a fusion protein of glutathione-S-transferase with Slp1 (GST-Slp1), and Mad2 protein translated in vitro in the presence of35S-methionine (10). 35S-labeled Mad2 bound to GST-Slp1, but not to GST (Fig. 1C). We generated a series of truncated Slp1 mutants and tested them for binding activity in the yeast two-hybrid system. F6, a 66–amino acid polypeptide, interacted with Mad2 (Fig. 1D). Deletion of 29 amino acids (Δ118–146) resulted in a loss of the binding activity (Fig. 1D), suggesting that these amino acids are important for binding to Mad2.

Figure 1

Binding of Mad2 to Slp1. (A and B) Yeast two-hybrid system. A tester strain was transformed by the combination of plasmids as indicated. In the transformants with pGBT-slp1 and pGAD-mad2, two reporter genes,HIS3 and LacZ, were highly expressed. The specific activity of β-galactosidase (indicated as Miller units) was determined as described (22). Similar results were obtained in three independent experiments. (C) Binding of in vitro–translated mad2 gene product labeled by35S (lane 1) to GST protein or a fusion protein, GST-Slp1, which were bound to glutathione–Sepharose 4B beads. The proteins bound to GST (lane 2) or to GST-slp1 (lane 3) were separated by polyacrylamide gel electrophoresis. (D) Each box represents a WD (Trp-Asp) repeat motif in Slp1. Truncated Slp1 fragments are shown below. The tester strain was transformed with pGAD-mad2 and pGBT8 carrying each of the truncated slp1 +fragments. F6R bears F6 fragment in the reverse orientation. The transformants were streaked on the media containing (+) or lacking (−) histidine.

Deletion of mad2 + (11) (Fig.2A) did not cause noticeable defects under normal growth conditions. The cut7 + gene encodes a kinesin, and the cut7 mutant fails in interdigitating the mitotic spindles (12). Thecut7 mutants arrest as septated cells with overcondensed chromosomes, implying that loss of cut7 + is normally recognized by the spindle checkpoint and results in a delay of chromosome decondensation. Therefore, Δmad2 was expected to abolish the metaphase arrest seen in the cut7 mutant at the restrictive temperature. Indeed, there was no indication of chromosome overcondensation in the double mutants, cut7Δmad2, over a period of 4.5 hours after the shift to the restrictive temperature (Fig. 2, C and D). Their nuclei appeared noncondensed and hemispheric. In the cut7 Δmad2mutants, the dependence of chromosome decondensation on formation of a mitotic spindle was relieved. In contrast, 3 hours after the shift, 60% of the cut7 single-mutant cells exhibited overcondensed chromosomes that appeared dense and fibrous. Most of the double-mutant cells contained nearly 4N DNA content 2 hours after the shift (Fig.3), indicating that the double mutant can initiate the next round of DNA replication even when the spindle assembly is incomplete. Thus, the fission yeastmad2 + functions as a component of the spindle checkpoint. These results are consistent with the previous report (3).

Figure 2

Disruption of mad2 + and loss of spindle checkpoint function. (A) Themad2 + gene was disrupted as shown. Total RNAs prepared from Ura segregants (wild-type strains, lanes 2 and 4) and Ura+ segregants (mad2 disruptants, lanes 1 and 3) were analyzed by Northern blotting with themad2 + cDNA as a probe. Each lane contained a similar amount of RNA. (B) Sensitivity to thiabendazole. Cell suspension of a wild-type strain Δmad2 and two independent isolates of the slp1-mr63 mutant were serially diluted and spotted on the complete media containing thiabendazole at the concentrations indicated. (C) Percentages of cells exhibiting overcondensed chromosomes after the shift to the restrictive temperature. (D) Microscopy of cut7,cut7 Δmad2, and cut7 slp1-mr63 at 3 hours after the shift. They were stained with 4,6-diamine-2-phenylindole (DAPI). Bar, 10 μm.

Figure 3

(left). Loss of the spindle checkpoint in cut7 mutant. Fluorescence-activated cell sorter (FACS) analysis of samples taken every hour after the shift to the restrictive temperature. The vertical axes indicate cell counts, and the horizontal axes, DNA content.

Overexpression of mad2 + causes a metaphase arrest (3), perhaps because Mad2 binds to Slp1 and inhibits its activity to promote the transition from metaphase to anaphase. We therefore generated and characterized an slp1 mutant that is resistant to the overproduction of Mad2. The domain of Slp1 important for binding to Mad2 (amino acid position 118 to 146) was mutagenized at random (13). Mutated slp1 genes on an autonomously replicating plasmid were individually transformed into a wild-type strain with ADH-M2, a plasmid that allows the overexpression of mad2 + from the alcohol dehydrogenase (ADH) gene promoter. When yeast cells were transformed with the wild-typeslp1 + gene on the autonomously replicating plasmid and with ADH-M2, the transforming efficiency was lower than that obtained by cotransformation with ADH-2M, a plasmid bearing themad2 + gene in the reverse orientation. In cells expressing mad2 + from the ADH promoter andslp1 + on the autonomously replicating plasmid, Mad2 probably exists in excess over Slp1 and inhibits the growth of the transformants. Three slp1 mutants (slp1-mr27,52, and 63) overcame the overexpression ofmad2 +. When they were expressed along with ADH-M2, the transformation efficiency was comparable to that obtained when they were expressed with ADH-2M. Nucleotide sequence analysis revealed that the slp1 + gene was mutated similarly in all the mutants at Ala-131 and Phe-132 (Fig.4A). One of the mutant slp1genes, slp1-mr63, was used to replace a null allele ofslp1 in a diploid. The resulting heterozygous diploid (+/slp1-mr63) was resistant to the overexpression ofmad2 + (Fig. 4C), indicating thatslp1-mr63 can overcome the overexpression ofmad2 + in a dominant manner (14). The presence of slp1-mr63 was determined by polymerase chain reaction (PCR) followed by digestion with Dra I (15) (Fig.4, A and B). Among the four haploid segregants produced meiotically from the heterozygous diploid, the Mad2-resistant phenotype gene segregated with slp1-mr63 (Fig. 4D). These haploid cells grew normally, suggesting that slp1-mr63 is otherwise biologically active.

Figure 4

(right).Isolation of slp1-mr63. (A) Nucleotide and amino acid sequences of the region containing the mutation sites (Ala-131 and Phe-132). In the mutants, one of the Dra I sites (shown in parentheses) was disrupted. Dra I digestion of the PCR product yields 84-, 22-, and 112-bp fragments from a wild-type strain and 84- and 134-bp fragments from the mutant. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; F, Phe; K, Lys; L, Leu; N, Asn; P, Pro; R, Arg; T, Thr; V, Val; and Y, Tyr. (B) The region shown in (A) was amplified by PCR and analyzed on a 2.5% agarose gel after Dra I digestion. Templates for PCR were genomic DNAs isolated from the heterozygous diploid (+/slp1-mr63, lane 1) and four segregants (lanes 2 to 5). The diploid yielded two wild-type strains (lanes 2 and 5) and twoslp1-mr63 mutants (lanes 3 and 4). (C) Growth of a wild-type diploid (+/+, top) and the heterozygous diploid (+/slp1-mr63, bottom). They were transformed with 0.5 μg of ADH-2M (left) or ADH-M2 (right). (D) Growth of a wild-type haploid (top) and a haploid carrying theslp1-mr63 mutation (bottom). They were transformed with 0.5 μg of ADH-2M (left) or ADH-M2 (right).

The slp1-mr63 mutation abolished binding to Mad2 in the yeast two-hybrid system (Fig. 5A) and in vivo. Mutant Slp1 protein did not precipitate with GST-Mad2 in fission yeast cell extracts, whereas wild-type Slp1 did (16,17) (Fig. 5B). We also made cells with both theslp1-mr63 and cut7 mutations. At the restrictive temperature, the double mutant (slp1-mr63 cut7) showed no delay of chromosome decondensation in response to thecut7-induced defect (Fig. 2D). Like Δmad2 cut7, the slp1-mr63 cut7 double mutant initiated the next round of DNA replication before the completion of the spindle assembly (Fig. 3). Furthermore, both Δmad2 and slp1-mr63 were hypersensitive to thiabendazole, an inhibitor of the spindle assembly (Fig. 2B). The slp1-mr63 mutant behaved indistinguishably from Δmad2. These results indicate that Slp1 is the primary target of Mad2 and that slp1-mr63, defective in recognizing the signal from Mad2, abrogates the function of the spindle checkpoint.

Figure 5

Failure of Slp1-mr63 to bind to Mad2. (A) F1 fragment (Fig. 1D) of a wild-type (WT) or the slp1-mr63 mutant (mr63) were tested for the ability to interact with Mad2 in the yeast two-hybrid system. (B) Binding of Slp1-mr63 to GST-Mad2. Cell extracts were prepared from the wild-type strain expressing GST (lanes 1 and 4) or GST-Mad2 (lanes 2 and 5). For lanes 3 and 6, cell extracts were prepared from an slp1-mr63 mutant expressing GST-Mad2 and analyzed as described (16). Antibody to GST (Pharmacia) (lanes 1 to 3) or antibody to Slp1 (lanes 4 to 7) was used for protein immunoblotting. Bacterially expressed Slp1 was run in lane 7. Multiple bands (lane 5) probably represent the degradation products of Slp1. These bands, as well as the band of the intact Slp1, became more intense when slp1 + was overexpressed. (C) Growth of the indicated strains transformed with pREP41-Mad2. The strains were streaked on minimum media (PMA) in the absence of thiamine to induce expression. (D) Growth of the indicated strains on complete media at 36°C.

Proteins with the destruction box, such as cyclins, Pds1, Cut2, and Ase1 (18), are degraded by ubiquitin-dependent proteolysis at anaphase. These proteins participate in regulating the timing of exit from mitosis or of initiation of sister chromatid separation. They are ubiquitinated by the anaphase-promoting complex (APC), which exerts the driving force to initiate anaphase (19). Fission yeast cut9 +(20) and cut4 + (21) encode components of the APC. The cut9 and cut4 mutants are hypersensitive to overexpression of mad2 +. When mad2 + is overexpressed in amounts not toxic to wild-type strains, these mutants are unable to grow at the permissive temperature (3) (Fig. 5C). Mad2 probably inhibits the activity of APC indirectly. The cut9 and cut4mutants are no longer hypersensitive to the overexpression ofmad2 + in the background of slp1-mr63(Fig. 5C). In addition, slp1-mr63, as well as Δmad2, partially suppressed the cut4 mutation. At the restrictive temperature, 36°C, the cut4 slp1-mr63mutant and cut4 Δmad2 mutant formed colonies (Fig. 5D) at a relatively slow rate. Thus, loss of function of the spindle checkpoint appears to remove a negative constraint on APC. Thecut9 mutant is synthetically lethal in the background of a recessive mutation, slp1-362 (4). Taken together, the genetic link between Mad2, Slp1, and APC suggests that the three components may act in a single sequential pathway. We propose that fission yeast Slp1 is the effector molecule that receives an anaphase-inhibitory signal from the Mad2-dependent spindle checkpoint.


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