Chl4p and iml3p are two new members of the budding yeast outer kinetochore

Abstract

Kinetochore proteins contribute to the fidelity of chromosome transmission by mediating the attachment of a specialized chromosomal region, the centromere, to the mitotic spindle during mitosis. In budding yeast, a subset of kinetochore proteins, referred to as the outer kinetochore, provides a link between centromere DNA-binding proteins of the inner kinetochore and microtubule-binding proteins. Using a combination of chromatin immunoprecipitation, in vivo localization, and protein coimmunoprecipitation, we have established that yeast Chl4p and Iml3p are outer kinetochore proteins that localize to the kinetochore in a Ctf19p-dependent manner. Chl4p interacts with the outer kinetochore proteins Ctf19p and Ctf3p, and Iml3p interacts with Chl4p and Ctf19p. In addition, Chl4p is required for the Ctf19p-Ctf3p and Ctf19p-Iml3p interactions, indicating that Chl4p is an important structural component of the outer kinetochore. These physical interaction dependencies provide insights into the molecular architecture and centromere DNA loading requirements of the outer kinetochore complex.

Figure 1

Phenotypes of chl4 mutants. (A) Chromosome loss phenotype visualized using the SUP11 system. A nonessential chromosome fragment carrying SUP11 can suppress the ade2-101 mutation that leads to accumulation of a red pigment in the cells (Koshland and Hieter, 1987). Thus, cells containing the chromosome fragment are white, whereas those that have lost it are red. Wild-type (YPH1124), chl4Δ (YPH1534), chl4-20 (YPH1535), and chl4-61 (YPH1536) strains containing a nonessential chromosome fragment marked with URA3 were grown in SC-uracil medium and then plated on YPD plates. (B) Benomyl sensitivity. Wild-type (YPH500), chl4Δ (YPH1539), chl4-20 (YPH1540), and chl4-61 (YPH1541) strains were spotted in fivefold dilutions on YPD plates containing the amount of benomyl indicated on the left.

Figure 2

Chl4p interacts with CEN DNA in an Ndc10p- and Ctf19-dependent, but Ctf3p-independent manner. (A) Chl4p interacts with CEN DNA. ChIP assay performed by immunoprecipitation of Myc-tagged Chl4p, Ctf19p, or Ndc10p followed by multiplex PCR analysis. Lanes 9–12, dilutions (2.5-fold) of one of the total templates, showing that PCR is in the linear range. Strains are untagged (YPH499), Chl4p-Myc (YPH1542), Ctf19p-Myc (YPH1550), and Ndc10p-Myc (YPH1555). (B) The interaction of Chl4p with CEN DNA depends on Ndc10p. ChIP assay performed by immunoprecipitation of Myc-tagged Chl4p or Ndc10p from wild-type, ndc10-2, or chl4Δ strains, followed by multiplex PCR analysis. For the temperature-sensitive assay, wild-type and ndc10-2 strains were grown to log phase at 25°C, and half of the culture was then shifted to 37°C for 3 h. Strains are untagged (YPH499), Chl4p-Myc (YPH1542), Chl4p-Myc ndc10-2 (YPH1543), Ndc10p-Myc (YPH1555), and Ndc10p-Myc chl4Δ (YPH1556). (C) The interaction of Chl4p with CEN DNA depends on Ctf19p. ChIP assay performed by immunoprecipitation of Myc-tagged Chl4p or Ctf19p from wild-type, ctf19Δ, or chl4Δ strains, followed by multiplex PCR analysis. Strain are untagged (YPH499), Chl4p-Myc (YPH1542), Chl4p-Myc ctf19Δ (YPH1544), Ctf19p-Myc (YPH1550), and Ctf19p-Myc chl4Δ (YPH1551). (D) The interaction of Chl4p with CEN DNA is independent of Ctf3p. ChIP assay performed by immunoprecipitation of Myc-tagged Chl4p or Ctf3p from wild-type, ctf3Δ, or chl4Δ strains, followed by multiplex PCR analysis. Strains are untagged (YPH499), Chl4p-Myc (YPH1542), Chl4p-Myc ctf3Δ (YPH1545), Ctf3p-Myc (YVM218), and Ctf3p-Myc chl4Δ (YPH1552). In A–D, T, total lysate; IP, immunoprecipitated fraction.

Figure 3

Interdependence of kinetochore protein localization. (A–E) Indicated proteins were tagged with YFP and imaged as described under MATERIALS AND METHODS. Spc29p-CFP was included in the genetic background as an SPB marker, which allowed determination of the relative location of the kinetochore. In all panels, upper images are cells with short spindles and lower images are cells with long spindles. The left frame has the YFP signal, the middle frame has merged YFP and CFP signals, and the right frame is the corresponding differential interference contrast image. In the color images, YFP is pseudocolored green and CFP is pseudocolored red. A threefold enlargement of the YFP and CFP signals in cells with short spindles, indicated by 3X, is shown at the bottom of each panel. The relevant genetic background (wild-type or kinetochore mutant) is indicated on top of each panel. (F) For colocalization, Chl4p was tagged with YFP and Mif2p was tagged with CFP in a wild-type strain and imaged as in A–E. All strains are homozygous diploids of (A) Chl4p-YFP in wt (YPH1569) and ctf19Δ (YPH1570); (B) Ctf19p-YFP in wt (DHY201), chl4Δ (YPH1571), and iml3Δ (YPH1583); (C) Ndc10p-YFP in wt (YVM1176), ctf19Δ (YPH1572), and chl4Δ (YPH1573); (D) Ctf3p-YFP in wt (DHY202), ctf19Δ (YPH1574), and chl4Δ (YPH1575); (E) Iml3p-YFP in wt (YPH1576), chl4Δ (YPH1577), and ctf19Δ (YPH1578); and (F) Mif2p-YFP (YPH1579) and Chl4p-YFP Mif2p-CFP (YPH1580). All strains (except YPH1580) contain Spc29p-CFP. wt, wild-type. Bar, 5 μm.

Figure 3

Interdependence of kinetochore protein localization. (A–E) Indicated proteins were tagged with YFP and imaged as described under MATERIALS AND METHODS. Spc29p-CFP was included in the genetic background as an SPB marker, which allowed determination of the relative location of the kinetochore. In all panels, upper images are cells with short spindles and lower images are cells with long spindles. The left frame has the YFP signal, the middle frame has merged YFP and CFP signals, and the right frame is the corresponding differential interference contrast image. In the color images, YFP is pseudocolored green and CFP is pseudocolored red. A threefold enlargement of the YFP and CFP signals in cells with short spindles, indicated by 3X, is shown at the bottom of each panel. The relevant genetic background (wild-type or kinetochore mutant) is indicated on top of each panel. (F) For colocalization, Chl4p was tagged with YFP and Mif2p was tagged with CFP in a wild-type strain and imaged as in A–E. All strains are homozygous diploids of (A) Chl4p-YFP in wt (YPH1569) and ctf19Δ (YPH1570); (B) Ctf19p-YFP in wt (DHY201), chl4Δ (YPH1571), and iml3Δ (YPH1583); (C) Ndc10p-YFP in wt (YVM1176), ctf19Δ (YPH1572), and chl4Δ (YPH1573); (D) Ctf3p-YFP in wt (DHY202), ctf19Δ (YPH1574), and chl4Δ (YPH1575); (E) Iml3p-YFP in wt (YPH1576), chl4Δ (YPH1577), and ctf19Δ (YPH1578); and (F) Mif2p-YFP (YPH1579) and Chl4p-YFP Mif2p-CFP (YPH1580). All strains (except YPH1580) contain Spc29p-CFP. wt, wild-type. Bar, 5 μm.

Figure 4

Quantification of kinetochore localization signals. (A–E) Kinetochore protein signal strengths of the strains imaged in Figure 3, A–E, were determined as described under MATERIALS AND METHODS. For each graph, the values were normalized to the mean value of the wild-type strain. In B–D, the mean value of the kinetochore signal from cells with a short spindle was used for normalization. Bars represent the standard deviation. The mean value of the signal intensity in wild-type strains and the number of kinetochores or nuclei examined for each panel are as follows, where n1 is the sample size for kinetochore signals in cells with either short or long spindles, n2 is the sample size for kinetochore signals in cells with short spindles, and n3 is the sample size for kinetochore signals in cells with long spindles (sample sizes for determining the nuclear signal strength are equal to either n1 or n2): (A) Mean value (wt) = 2297; for wt, n1 = 78; for ctf19Δ, n1 = 45. (B) Mean value (wt) = 12368; for wt, n2 = 33 and n3 = 26; for chl4Δ, n2 = 36 and n3 = 30; for iml3Δ, n2 = 22 and n3 = 14. (C) Mean value (wt) = 12587; for wt, n2 = 86 and n3 = 62; for ctf19Δ, n2 = 64 and n3 = 38; for chl4Δ, n2 = 82 and n3 = 33. (D) Mean value (wt) = 4035; for wt, n2 = 24 and n3 = 16; for ctf19Δ, n2 = 45 and n3 = 24; for chl4Δ, n2 = 38 and n3 = 19. (E) Mean value (wt) = 5740; for wt, n1 = 74; for ctf19Δ, n1 = 33; for chl4Δ, n1 = 63. wt, wild-type.

Figure 5

Chl4p coimmunoprecipitates with outer kinetochore proteins. (A) Chl4p coimmunoprecipitates with Ctf19p and Ctf3p. Anti-Myc immunoprecipitations were performed in a strain containing Myc-tagged Chl4p and HA-tagged Ctf3p and control strains containing one or no tagged proteins. Total lysate (40 μg) and 15% of the immunoprecipitated fraction were loaded on SDS-PAGE gels, and Western blots were used to detect Myc- and HA-tagged proteins, and Ctf19p, with the antibodies indicated on the left. Strains are untagged (YPH499), Chl4p-Myc (YPH1542), Ctf3p-HA (YPH1553), and Chl4p-Myc Ctf3p-HA (YPH1546). (B) Ctf19p is required for the coimmunoprecipitation between Chl4p and Ctf3p. Anti-Myc immunoprecipitations and Western blots were performed as in A in strains lacking Ctf19p. Lanes 7–12 are controls. Strains are untagged (YPH499), Chl4p-Myc Ctf3p-HA (YPH1547), Chl4p-Myc Ctf3p-HA ctf19Δ (YPH1548), Chl4p-Myc ctf19Δ (YPH1544), Ctf3p-HA ctf19Δ (YPH1554), and untagged ctf19Δ (YPH1315). (C) Ctf3p is dispensable for the coimmunoprecipitation between Chl4p and Ctf19p. Anti-Myc immunoprecipitations and Western blots were performed as in A in strains lacking Ctf3p. Lanes 7 and 8 are controls. Strains are untagged (YPH499), Chl4p-Myc (YPH1542), Chl4p-Myc ctf3Δ (YPH1545), and untagged ctf3Δ (YVM112). (D) Chl4p is required for the interaction of Ctf3p with Ctf19p. Anti-Myc immunoprecipitations and Western blots were performed as in A in strains containing Myc-tagged Ctf3p in the presence or absence of Chl4p. Lanes 7 and 8 are controls. Strains are untagged (YPH499), Ctf3p-Myc (YVM218), Ctf3p-Myc chl4Δ (YPH1552), and untagged chl4Δ (YPH1537). In A–D, T, total lysate; IP, immunoprecipitated fraction.

Figure 6

Iml3p is a new outer kinetochore protein. (A) Iml3p interacts with Chl4p and with Ctf19p in a Chl4p-dependent manner. Anti-HA immunoprecipitations and Western blots were performed as in Figure 5 in wild-type, ctf19Δ, or chl4Δ strains containing Myc-tagged Chl4p and HA-tagged Iml3p or control strains containing one or no tagged proteins. Strains are untagged (YPH499), Iml3p-HA (YPH1557), Chl4p-Myc (YPH1542), Iml3p-HA Chl4p-Myc (YPH1560), Iml3p-HA Chl4p-Myc ctf19Δ (YPH1561), Iml3p-HA ctf19Δ (YPH1559), and Iml3p-HA chl4Δ (YPH1558). (B) The interaction of Ctf19p with Chl4p is reduced in the absence of Iml3p. Anti-Myc immunoprecipitations and Western blots were performed as in Figure 5 in wild-type or iml3Δ strains containing either Myc-tagged Chl4p or no tagged protein. Strains are untagged (YPH499), Chl4p-Myc (YPH1542), and Chl4p-Myc iml3Δ (YPH1549). (C) Iml3p interacts with CEN DNA in a Chl4p- and Ctf19p-dependent manner. ChIP assay performed by immunoprecipitation of Myc-tagged Iml3p from wild-type, chl4Δ or ctf19Δ strains, or by immunoprecipitation of Myc-tagged Chl4p or Ctf19p from wild-type or iml3Δ strains, followed by multiplex PCR analysis. Strains are untagged (YPH499), Iml3p-Myc (YPH1562), Iml3p-Myc chl4Δ (YPH1563), Iml3p-Myc ctf19Δ (YPH1564), Chl4p-Myc (YPH1542), Chl4p-Myc iml3Δ (YPH1603), Ctf19p-Myc (YPH1550), and Ctf19p-Myc iml3Δ (YPH1582). In A–C, T, total lysate; IP, immunoprecipitated fraction.

Figure 7

Chl4p interacts with the kinetochore protein Mif2p. Growth of PJ694a/α strains containing the Chl4p-DNA–binding domain fusion in plasmid pOBD2 (or vector alone as control) and the Mif2p-activation domain fusion in plasmid pOAD (or vector alone as control) on selective media. SC medium lacking tryptophan and leucine only selects for the vectors, whereas medium lacking tryptophan, leucine, and histidine selects for the vectors and the two-hybrid interaction. 3-Aminotriazole (3AT) is added to prevent growth due to leaky expression from the HIS3 promoter. Strains are pOBD2-CHL4 pOAD-MIF2 (YPH1565), pOBD2-CHL4 pOAD (YPH1566), pOBD2 pOAD-MIF2 (YPH1567), and pOBD2 pOAD (YPH1568).

Figure 8

Model of the budding yeast kinetochore. (A) Revision of the kinetochore model presented in Measday et al. (2002), which now includes Chl4p and Iml3p as part of the outer kinetochore. Mif2p has also been added to the diagram because of its two-hybrid interaction with Chl4p. (B) Proposed molecular architecture of the proteins that have been examined in this article, deduced from requirements for CEN DNA interaction, localization, and protein–protein interactions. Arrows represent requirements for interaction with CEN DNA, beginning with the complex/protein interacting with CEN DNA, and pointed toward the complex/protein required for the interaction. (a) From Ortiz et al. (1999). (b) From Measday et al. (2002).