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. 1999 Jun 28;145(7):1381-94.
doi: 10.1083/jcb.145.7.1381.

Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae

J H Kim  1 , J S Kang, C S Chan
Affiliations

Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae

J H Kim et al. J Cell Biol. .

Abstract

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.

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Figures

Figure 1
Figure 1
Cytological phenotype of ipl1-2 and sli15-3 mutants. ipl1-2 (CCY108-15C-1) and sli15-3 (CCY1060-1D) cells were incubated at 37°C for 4 h and processed for immunofluorescence microscopy. Phase-contrast, DAPI-stained, and anti-tubulin– stained images are shown. Top panels: uneven chromosome segregation; middle panels: nuclear migration defect; bottom panels: monopolar spindle.
Figure 2
Figure 2
Defective sister chromatid segregation in ipl1-2 and sli15-3 cells. Wild-type (WT) (CCY1076-28B), ipl1-2 (CCY1077-6C), and sli15-3 (CCY1078-37B) cells that had the centromere region of chromosome V marked by the binding of TetR-GFP to Tet operator sites were incubated at 37°C for 2 h and briefly fixed. Pairs of DAPI-stained and TetR-GFP images taken from large-budded cells are shown.
Figure 3
Figure 3
Summary of sister chromatid separation/segregation defects in ipl1-2 and sli15-3 cells. The localization of TetR-GFP (small dots) and chromosomal DNA masses (shaded) were examined in wild-type (WT) (CCY1076-28B), ipl1-2 (CCY1077-6C), and sli15-3 (CCY1078-37B) cells that were incubated at 26°C (0 h) or for 2 h at 37°C. Uneven segregation of chromosomal DNA masses was indicated by shaded areas of different sizes. For each sample, 100 large-budded cells that were in early anaphase and 100 large-budded cells that were in late anaphase or early G1 were scored. No distinction was made between the mother and bud of these large-budded cells. Whether the centromere region of chromosome V (marked by TetR-GFP) present on sister chromatids appeared separated (sep.) or unseparated (unsep.) was indicated.
Figure 4
Figure 4
Abnormal distribution of Nuf2-GFP in ipl1-2 and sli15-3 cells. DNA (DAPI), microtubules (anti-tubulin), and Nuf2-GFP were localized in wild-type (CCY766-9D-1) (a–f), ipl1-2 (CCY915-13C-5) (g–o), and sli15-3 (CCY1083-8B) (p–x) cells that had been incubated at 37°C for 2 h and briefly fixed.
Figure 5
Figure 5
Summary of Nuf2-GFP distribution. The localization of Nuf2-GFP (dots) and chromosomal DNA masses (shaded) were examined in wild-type (WT) (CCY766-9D-1), ipl1-2 (CCY915-13C-5), and sli15-3 (CCY1083-8B) cells that were incubated at 26°C (0 h) or for 3 h at 37°C. Uneven distribution of Nuf2-GFP was indicated by dots of different sizes within the same cell. Uneven segregation of chromosomal DNA masses was indicated by shaded areas of different sizes. For each sample, 100 large-budded cells that were in preanaphase and 100 large-budded cells that were in late anaphase or early G1 were scored. No distinction was made between the mother and bud of these large-budded cells. Whether the distribution of Nuf2-GFP appeared abnormal is indicated.
Figure 6
Figure 6
Subcellular localization of GFP-Ipl1 and GFP-Sli15. The DIC, DAPI-stained, and GFP-fusion protein images were obtained from unfixed wild-type diploid (DBY1830) cells that carried the low copy number plasmid pCC959 (encoding GFP-Ipl1) or pCC1060 (encoding GFP-Sli15).
Figure 8
Figure 8
Suppression of sli15-3 by increased dosage of IPL1 and GLC8. Suspensions of sli15-3 mutant cells (CCY1060-1D) carrying different plasmids were spotted on YEPD and allowed to grow at the indicated temperatures for 1.75 d. The plasmids used were high copy number control plasmid pSM217, low copy number SLI15-plasmid pCC977, low copy number IPL1-plasmid pCC100, high copy number glc7-Δ186–312–plasmid pCC418, and high copy number GLC8-plasmid pCC638.
Figure 7
Figure 7
In vivo association of Sli15 with Ipl1. (A) Copurification of HA-Ipl1 with GST-Sli15. GST and GST-Sli15 were affinity-purified in the presence of phosphatase inhibitors (NaF and NaVO4) with glutathione-agarose beads from a yeast strain (TD4) that carried the plasmids pEG(KT) (encoding GST) and pCC1128 (encoding HA-Ipl1) or the plasmids pCC1061 (encoding GST-Sli15) and pCC1128. Aliquots of proteins from total protein extracts (total) or from the affinity-purified (bound) fractions (after 1, 3, or 5 rounds of washing with a buffer containing 200 mM KCl) were analyzed by immunoblotting, using antibodies against GST or the HA-epitope. The enlarged images shown at the bottom are from films that had been exposed for different times. (B) HA-Ipl1 is phosphorylated in vivo. Crude yeast extracts prepared in the presence or absence of the phosphatase inhibitors NaF and NaVO4 from a yeast strain (TD4) that carried the GST-Sli15 plasmid pCC1061 and the HA-Ipl1 plasmid pCC1128 were analyzed by immunoblotting with antibodies against the HA-epitope. (C) Coimmunoprecipitation of HA-Ipl1 with Sli15-Myc. Sli15-Myc was immunoprecipitated with antibodies against the Myc-epitope from a yeast strain (TD4) that carried the plasmids pCC1128 (encoding HA-Ipl1) and pCC1192 (encoding Sli15) or the plasmids pCC1128 and pCC1193 (encoding Sli15-Myc). Aliquots of proteins from total protein extracts (total) or from the immunoprecipitated fractions (anti-Myc IP) were analyzed by immunoblotting, using antibodies against the Myc-epitope or the HA-epitope. (D) Direct binding of His6-Ipl1 to GST-Sli15. GST and GST-Sli15 were affinity-purified with glutathione-agarose beads from an E. coli strain (BL21) that carried pGEX-2T (encoding GST) or pCC1062 (encoding GST-Sli15). Proteins in a crude extract (total) from an E. coli strain (TOP10) that carried pCC1167 (encoding His6-Ipl1) or pRBD2-nonO (encoding His6-NonO-C) were allowed to bind to the immobilized GST or GST-Sli15. Aliquots of proteins from the crude extract (total) or from the fractions that bound to GST or GST-Sli15 were analyzed by immunoblotting, using antibodies against GST, Ipl1, or NonO.
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