FIG. 3.

Mad3p functions at the effector end of the spindle checkpoint pathway. (A) Mad3p is required for the effects of mad2⁺ overexpression. Wild-type cells (366 strain) and spindle checkpoint mutants (mad1Δ [DMSP074], mad3Δ [DMSP001], bub1Δ [DMSP037], and bub3Δ [DMSP035]) were transformed with pREP3X-mad2⁺. Transformants were then spotted onto EMM plates with and without the addition of thiamine. Plates were photographed after 3 days of growth at 30°C. (B) Mad2p protein levels are not affected by the mad3 deletion. Wild-type and checkpoint mutant strains were grown as indicated (± pREP3x-mad2⁺, ± thiamine), and whole-cell extracts were prepared and separated by SDS-PAGE. Immunoblots were carried out with polyclonal anti-Mad2p antibodies and anti-BiP antibodies as a loading control. Mad2p levels were induced in all strains containing pREP3x-mad2+ in the absence of thiamine. (C) Mad2p-induced metaphase arrest is Mad3p dependent. Cultures of the strains indicated were fixed and stained for the presence of mitotic spindles with antitubulin antibodies. Only the mad3Δ culture failed to accumulate short spindles when Mad2p was overexpressed.