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. 2001 Nov 15;20(22):6371-82.
doi: 10.1093/emboj/20.22.6371.

Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint

L Sironi  1 , M Melixetian, M Faretta, E Prosperini, K Helin, A Musacchio
Affiliations

Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint

L Sironi et al. EMBO J. .

Abstract

Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms incompatible complexes with Mad1 and Cdc20, neither of which requires Mad2 oligomerization. A monomeric point mutant of Mad2 can sustain a cell cycle arrest of comparable strength to that of the wild-type protein. We show that the interaction of Mad2 with Mad1 is crucial for the localization of Mad2 to kinetochores, where Mad2 interacts with Cdc20. We propose a model that features the kinetochore as a 'folding factory' for the formation of a Mad2-Cdc20 complex endowed with inhibitory activity on the anaphase promoting complex.

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Figures

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Fig. 1. (A) Purification of Mad2 and Mad1485–718–Mad2 (lanes 1 and 2, respectively) by IMAC. (B) SEC profile of the material eluted after IMAC. Mad1–Mad2 is contaminated with free Mad2, resulting from higher expression levels of this protein relative to Mad1. By SDS–PAGE, peak 1 (P1) contains only Mad1–Mad2. The excess of Mad2 is contained in peak 2 (P2). (C) The P1 peak shown in (B) was concentrated and analysed by SEC, and a single peak appeared (thick line). The elution volume can be compared with that of known protein standards (thin dashed lines). (D) Mad1–Mad2 was incubated with urea concentrations of 1, 2 or 4 M (lanes 2–4), and NaCl concentrations of 1, 2, 3 or 4 M (lanes 6–9), and metal-affinity beads were added. Mad1 still co-purified with Mad2 after this treatment, indicating that the interaction between these proteins is very strong.
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Fig. 2. (A) His-Mad2wt (thick line) was purified by IMAC and analysed by SEC. The elution profile was compared with that of known standards (MWM, thin dashed line). (B and C) SEC analysis of Mad2ΔC and Mad2ΔN. (D) The ability of Mad2wt (wt), 5- and 10-residue N-terminal deletions of Mad2 (ΔN5 and ΔN10, respectively) and Mad2ΔC (ΔC) to interact with Mad1 was evaluated using the co-expression system described in the text, followed by IMAC purification. Note that Mad2ΔC is unable to interact with Mad1.
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Fig. 3. (A) Mad2R133A (thick line) is monomeric by gel filtration. (B) The ability of Mad2R133A to interact with Mad1 was evaluated using the co-expression system described in the text, followed by IMAC purification, and separation of the bound proteins by SDS–PAGE. Similarly to Mad2wt, the Mad2R133A mutant co-purifies with Mad2. (C) Elution profile of Mad1–Mad2R133A (thick line) compared with the elution profile of Mad1–Mad2wt (thin dashed line). (D) The P1 peak shown in (C) was concentrated and analysed by SEC. A single peak appeared (thick line). There is no disruption of the Mad1–Mad2R133A complex upon SEC, as indicated by the complete absence of free Mad1 or Mad2R133A upon SDS–PAGE separation (bottom panel). Thirteen 100 µl fractions (1–13) were collected and analysed, starting at an elution volume of 0.8 ml. (E) The forms of Mad2 indicated were incubated either with GST (lanes 1–4) or GST–Cdc20111–160 on GSH–agarose beads. After washing, bound proteins were resolved by SDS–PAGE. Mad2ΔC (lane 6) was unable to bind to Cdc20, whereas the N-terminal deletion mutant (lane 7) and Mad2R133A (lane 8) bound like the wild-type protein (lane 5). The results were confirmed by western blotting using an anti-His tag antibody.
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Fig. 4. (A) Mad2wt was incubated with a synthetic peptide containing residues 111–154 of Cdc20, and the mix separated by SEC (left panel, thick line). The content of the peaks was visualized by SDS–PAGE (central panel). The right-hand site of the figure is a cartoon representing our interpretation of the results. (B) When the same experiment was carried out with purified Mad1–Mad2wt complex (running as a single peak, Figure 1C), two new peaks appeared. P2 contains Mad2 and the Cdc20 peptide, while P3 contains an excess of peptide. (C) The same experiment was carried out with the purified Mad1–Mad2R133A complex (running as a single peak, Figure 3D). Relative to the wild-type complex shown in (B), we observe the absence of P2. Thus, the appearance of P2 in (B) is probably due to Mad2 oligomerization.
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Fig. 5. (A) The expression levels of Mad2R133A and Mad2ΔC are similar to that of Mad2wt. HeLa cells were transfected with Myc-tagged Mad2 expression plasmids; 24 h after transfection the cell lysates were prepared and analysed on a 12% polyacrylamide gel, followed by western blotting using Mad2 antibody (Santa Cruz). (B) Mad2R133A localizes to kinetochores in nocodazole-arrested cells, while the C-terminal deletion mutant does not. PTK1 cells were co-transfected with H2B–GFP and Myc-tagged Mad2 expression plasmids; 24 h after transfection the cells were treated with nocodazole for another 24 h, then fixed and stained with anti-Myc antibody (9E10; in red). Chromosomes were visualized by H2B–GFP fluorescence (in green).
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Fig. 6. (A) Mad2R133A associates with Cdc20 and CDC27 in nocodazole-arrested cells. HeLa cells were transfected with Mad2 expression vectors and 24 h later treated with nocodazole for 16 h.The cells were lysed and Mad2 was immunoprecipitated with the antibodies indicated. The immunoprecipitates were analysed by western blotting using αMyc antibody (9E10). (B) Mad2R133A co-localizes with Cdc20 at kinetochores in nocodazole-arrested cells. Mad2 expression plasmids were transfected into PTK1 cells and the cells were treated with nocodazole for 24 h. Myc-tagged Mad2 was visualized with Cy3-conjugated antibody (in red). Cdc20 was visualized using anti-Cdc20 mouse monoclonal antibody.
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Fig. 7. Mad2R133A cooperates with Mad1 to arrest the cells in mitosis. HeLa cells were synchronized by double thymidine block, co-transfected with Mad1, Mad2 and H2B–GFP expression plasmids during release from first thymidine block. The nuclear morphology (A) and mitotic indexes (B) were analysed by H2B–GFP fluorescence 12, 14 and 16 h after release from double thymidine block.
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Fig. 8. Models of spindle checkpoint function. (A) The Mad1–Mad2 complex contains ‘core’ Mad2 subunits (orange) forming a strong interaction with Mad1, and loosely associated subunits (dark grey) that are associated via Mad2 oligomerization. Cdc20 interacts with the loosely associated Mad2 subunits, whose stock is replenished by the addition of new subunits. (B) The Mad1–Mad2 complex is tight. Association of Cdc20 with Mad2 occurs upon destabilization of the Mad1–Mad2 complex. A new cycle starts when a new Mad2 molecule binds Mad1. The cycle is interrupted as a consequence of spindle attachment, and a stable Mad1–Mad2 complex is formed that does not permit further recruitment of Mad2. The precise regulatory mechanism impinging on the recruitment and release of Mad2 to/from Mad1 is not known at present.
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