Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae

Abstract

The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.

Figure 1.

Synthetic lethality and synthetic fitness identified by random spore analysis. (A) Colonies from haploid spores selected after meiosis were plated on nonselective medium (Control) and then replica plated to plates containing the indicated drugs. On the left is a cross demonstrating synthetic lethality and on the right are cells from a cross that does not show any synthetic interaction. (B) Colonies from haploid spores selected after meiosis were plated on nonselective medium (Control) and then replica plated to plates containing the indicated drugs. On the left are colonies from a cross showing synthetic fitness and on the right are colonies from a cross demonstrating synthetic lethality.

Figure 2.

Synthetic genetic interactions of spindle checkpoint mutants mad2, mad1, bub3, and mad3. Nodes represent the indicated mutants and edges. All interactions shown were directly tested for synthetic lethality with each query gene, using random spore analysis. The GO process annotation of each interacting gene is denoted by the node color and is described in the key.

Figure 3.

Assays for chromosome transmission and spindle integrity. (A) Chromosome transmission. Five independent colonies of the indicated diploid genotype were patched onto a YPD plate and mated overnight to a lawn of MATa arg4 cells and then printed onto an SD plate to select triploids. The papillae represent cells in the patch that successfully mated. (B) Spindle integrity. Serial 10-fold dilutions of cells of the indicated genotype were spotted onto a YPD plate containing 10 μg/ml benomyl. All strains grew equally well on YPD plates.

Figure 4.

Chromosome transmission and spindle integrity effects within the network. Mutants with enhanced benomyl sensitivity are indicated by red nodes, mutants with elevated frequency of producing maters are indicated by blue nodes, and mutants that both are benomyl sensitive and have enhanced benomyl sensitivity are indicated by green nodes. Gray nodes indicate mutants that neither were benomyl sensitive nor produced an elevated frequency of maters. Yellow nodes are the spindle checkpoint query mutants.