The role of Pds1p in mitotic regulation. Pds1p is an inhibitor of the protease Esp1p that activates anaphase by inducing sister chromatid separation. At the onset of anaphase, Pds1p is degraded in a process that involves the APC/C. At least three checkpoint pathways inhibit mitotic progression by affecting the degradation of Pds1p. Pds1p not only inhibits Esp1p but also serves as its activator by promoting the nuclear localization of Esp1p (indicated by the curved arrow). See text for more details.

A screen for synthetic lethal interactions with pds1Δ using a modified SGA analysis. A pds1Δ strain (MATα MFα::MFα_promoterLEU2 can1Δ::MFA_promoterHIS3 his3Δ ura3Δ met15Δ lys2Δ pds1::NAT, S288C background) carrying a centromeric plasmid encoding PDS1 and URA3 (pPDS1) was crossed individually to ∼4700 MATa strains of the yeast deletion collection, each containing a single deletion of a nonessential gene (designated gene_nΔ). Following diploid selection, the strains were sporulated and selected for double mutants (pds1Δ gene_nΔ) containing pPDS1 (for double-mutant selection procedure see ). Of the 4700 crosses, ∼90% gave rise to double-mutant strains containing the plasmid. Of those, 260 strains showed a putative synthetic interaction as scored by the inability to lose pPDS1 (see below). A total of 220 of these strains were further examined by tetrad analysis. For each of these strains, four double-mutant isolates (pds1Δ gene_nΔ) and two single-mutant isolates (gene_nΔ) were assayed by three 10-fold serial dilutions for their ability to survive in the absence of Pds1p function by counterselecting for pPDS1 on 5-FOA media. Because 5-FOA is toxic to cells expressing URA3, only cells that have spontaneously lost pPDS1 would be able to grow on the 5-FOA plates. The inability to grow on 5-FOA, indicative of a requirement for Pds1p for growth, was manifested by either synthetic lethality (for example, see cin8Δ or slk19Δ) or severe synthetic sickness (see chl1Δ or sti1Δ). Double-mutant combinations that were unable to grow on 5-FOA were transformed with pOC78, a centromeric plasmid coding for PDS1 LEU2 HIS3. If the inability of the gene_nΔ pds1Δ pPDS1 strain to grow on 5-FOA was due to a requirement for PDS1 (and not due to the integration of the URA3 gene into the chromosome, for example), then the presence of pOC78 should allow these cells to grow on 5-FOA. Of the 220 strains examined by tetrad analysis, 21 showed a true synthetic interaction. The identity of the gene disruptions in these strains was verified by sequencing. The high false-positive rate, which is in line with recent reports (), may be due to technical challenges associated with the automated procedure () that can be affected by various factors such as growth rate differences and morphology effects on transfer efficiency.

Cell cycle distribution of mutants that show a genetic interaction with pds1Δ. Wild-type and mutant strains in A and B were grown to early log phase, fixed in 70% ethanol, and analyzed by flow cytometry (A) and fluorescent microscopy (B) for cell cycle distribution. (A) Only strains whose flow cytometry profiles are different from wild type are shown. Note the relatively high fraction of cells with 2N DNA content in the mutants shown relative to wild type. (B) Histograms represent the percentage of cells with a large bud and an undivided nucleus, indicative of a G₂/M phase. (C) Images of wild-type, dia2Δ, vps64Δ, and apq12Δ cells. Bright white areas indicate DNA (DAPI staining).

The genetic interactions between pds1Δ and mutants in genes coding for Hsp90-type chaperones reflect a role for the latter in Esp1p activity. (A) Double-mutant strains (pds1Δ gene_nΔ) carrying pPDS1 were transformed with 2μESP1 (described in ) or with vector alone and analyzed for their ability to survive in the absence of Pds1p by growth on media containing 5-FOA. The synthetic lethality of two double mutants, hsc82Δ pds1Δ and sti1Δ pds1Δ, could be rescued by this approach. An example of a double mutant that was not rescued (cin1Δ pds1Δ) is also shown. (B) Percentage of anucleate cells (i.e., cells without a distinct DNA mass as determined by DAPI staining) was determined as described in . (C) An hsc82Δ/HSC82 esp1-1/ESP1 diploid strain was induced to undergo meiosis and form tetrads. The haploid meiotic products (spores) were dissected onto a rich media plate (YPD). The growth of spores from seven such tetrads is shown. The symbols denote the genotypes of these spores as determined subsequently. Open symbols indicate the predicted genotypes of spores that failed to germinate. Similar results were obtained when analyzing a sti1Δ/STI1 esp1-1/ESP1 diploid strain (data not shown).