Regulation of Sli15/INCENP, kinetochore, and Cdc14 phosphatase functions by the ribosome biogenesis protein Utp7

Abstract

The Sli15-Ipl1-Bir1 chromosomal passenger complex is essential for proper kinetochore-microtubule attachment and spindle stability in the budding yeast Saccharomyces cerevisiae. During early anaphase, release of the Cdc14 protein phosphatase from the nucleolus leads to the dephosphorylation of Sli15 and redistribution of this complex from kinetochores to the spindle. We show here that the predominantly nucleolar ribosome biogenesis protein Utp7 is also present at kinetochores and is required for normal organization of kinetochore proteins and proper chromosome segregation. Utp7 associates with and regulates the localization of Sli15 and Cdc14. Before anaphase onset, it prevents the premature nucleolar release of Cdc14 and the premature concentration of Sli15 on the spindle. Furthermore, Utp7 can regulate the localization and phosphorylation status of Sli15 independent of its effect on Cdc14 function. Thus, Utp7 is a multifunctional protein that plays essential roles in the vital cellular processes of ribosome biogenesis, chromosome segregation, and cell cycle control.

Figure 1.

Utp7 associates with Sli15, Bir1, and centromere DNA. Cells were incubated at 26°C or, where indicated, shifted to 37°C for 3 h. (A) Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibodies, using extracts from wild-type and mutant cells. CEN16 and CIT3 sequences were amplified by PCR (28 cycles) from the total input chromatin (IN), antibody-immunoprecipitated samples (+), or mock-treated no-antibody controls (−). (B) Sli15-Myc was immunoprecipitated (IP) from extracts of wild-type cells that expressed Utp7-HA or from extracts of utp7-26-HA cells that also expressed Utp7-26-HA from a 2μ plasmid. Proteins were analyzed by immunoblotting (IB). (C and D) Similar to B, but Ipl1-Myc or Bir1-Myc was immunoprecipitated from wild-type cells that expressed Utp7-HA. (E) Extracts from cells expressing Utp7-HA or mutant Utp7-26-HA (from chromosomal locus) were immunoblotted with anti-HA antibodies.

Figure 2.

utp7-26 mutant cells missegregate chromosomes. (A) Cells growing exponentially at 26°C were shifted to 37°C for 3 h. Budding index (UB, unbudded; SB, small-budded; LB, large-budded) of 100 cells each was scored. (B) Microtubule- and DNA-stained images of large-budded cells are shown. (C) Cells were arrested in G1 by α-factor at 26°C for 2 h, and then shifted to 37°C in the absence of α-factor but the presence of 15 μg/ml nocodazole. The budding index of 100 cells was scored at each time point.

Figure 3.

Robust expression of GST-Utp7 leads to chromosome missegregation but not ribosome biogenesis defects. (A) Expression of GST or GST-Utp7 was induced for 4 h at 30°C in exponentially growing wild-type cells that expressed Sli15-Myc, followed by ribosome profile analysis of extracts. (B) Cells in A were processed for DNA and microtubule staining. The spindle length of 100 large-budded cells and the chromosome segregation defect of 100 anaphase cells were scored. Short = typical metaphase spindle; fully elongated = typical telophase spindle. (C) Expression of GST or GST-Utp7 was induced in wild-type diploid cells heterozygous for SLI15-MYC and IPL1-HA. Extracts from these cells were immunoblotted with anti-Myc, anti-HA, or anti-G6PDH antibodies. (D) Extracts from cells that were incubated at 26°C or shifted to 37°C for 3 h were immunoblotted with anti-Ipl1 or anti-G6PDH antibodies.

Figure 4.

Sli15 and other kinetochore proteins mislocalize in utp7-26 cells. (A) Chromatin immunoprecipitation (ChIP) was performed with the antibodies shown, using extracts from wild-type, utp7-26, or sli15-3 cells that were incubated at 26°C and then shifted to 37°C for 3 h. PCR was performed as described in Fig. 1. (B) Immunoblotting of extracts from some of the cells used in A.

Figure 5.

Sli15 mislocalizes on microtubules in utp7-26 cells. (A) Cells expressing Sli15-Myc were incubated at 26°C and then shifted to 37°C for 3 h. Representative DNA-, microtubule-, and Sli15-Myc-stained images from unbudded (UB), preanaphase (PA), early anaphase (EA), and late-anaphase (LA) cells are shown. Arrows in some images mark Sli15-Myc signal on cytoplasmic microtubules. (B) Images of preanaphase cells from A are shown at higher magnification and with slight changes in contrast. (C) Similar to A, except cells were first incubated at 26°C for 1 h in the presence of HU and then shifted to 37°C for 2 h. (D) Chromatin immunoprecipitation (ChIP) was performed with anti-Myc antibodies, using extracts from cells shown in C. PCR was performed as described in Fig. 1.

Figure 6.

Utp7 associates with and regulates the localization of Cdc14 and Net1. (A) Cells expressing Nop1-HA in combination with Cdc14-Myc or Net1-Myc were first incubated at 26°C for 1 h in the presence of HU and then shifted to 37°C for 2 h. DNA was stained with DAPI; Nop1-HA with anti-HA antibodies; Cdc14-Myc and Net1-Myc with anti-Myc antibodies. (B) Chromatin immunoprecipitation (ChIP) was performed with anti-Myc antibodies, using extracts from cells shown in A. PCR was performed as described in Fig. 1. (C) Cdc14-Myc was immunoprecipitated (IP) from extracts of wild-type cells that expressed Utp7-HA or from extracts of utp7-26-HA cells that also expressed Utp7-26-HA from a 2μ plasmid. Cells were incubated at 26°C or shifted to 37°C for 3 h. Proteins were analyzed by immunoblotting (IB). (D) Similar to C, except that Net1-Myc was immunoprecipitated from cells that expressed Net1-Myc instead of Cdc14-Myc.

Figure 7.

Utp7 regulates Sli15 localization and phosphorylation by Cdc14-dependent and -independent mechanisms. (A) Cells that expressed Sli15-Myc were incubated at 26°C and then shifted to 37°C for 3 h. Representative DNA-, microtubule-, and Sli15-Myc-stained images of cells in telophase are shown. (B) Similar to A, except cells were first incubated at 26°C for 1 h in the presence of HU and then shifted to 37°C for 2 h. (C) CDC14-TAB6 cells that expressed Sli15-Myc were incubated at 26°C. Images of a large-budded preanaphase cell (top row) and a telophase cell (bottom row) are shown, with arrows marking Sli15-Myc signal on cytoplasmic microtubules. (D) Chromatin immunoprecipitation (ChIP) was performed with anti-Myc antibodies, using extracts from cells shown in B before and after the 2-h shift to 37°C. PCR was performed as described in Fig. 1. (E) Immunoblotting was performed with anti-Myc and anti-G6PDH antibodies, using extracts from cells shown in B before and after the 2-h shift to 37°C.

Figure 8.

Model for the regulation of Sli15 phosphorylation and localization by Utp7. APP = another protein phosphatase. In an alternative model, Utp7 positively regulates an unknown kinase that phosphorylates Sli15.