Characterization of Mif2p by Western analysis and indirect immunofluorescence. (A) Western analysis of Mif2p in whole cell extracts from logarithmically growing yeast strains (30°C). Mif2p was detected using anti-Mif2p antiserum and chemiluminescence. Mif2p migrates as at ∼94 kD; Mif2–HAp migrates at ∼102 kD. (Lane 1) Wild-type strain 5371-10-2 with pRS316, CEN6 URA3 vector (); (lane 2) 5371-10-2 [pRS202], 2μ URA3 vector; (lane 3) 5371-10-2 [pPM4], pRS316–MIF2; (lane 4) 5371-10-2 [pPM44], pRS202–MIF2; (lane 5) PM1202-7B, mif2::HIS3 [pPM4]; (lane 6) PM1203–1C, mif2::HIS3 [pPM102], pRS316–MIF2–HA. Molecular masses of protein standards are indicated (MW). (B–E) Localization of Mif2p in wild-type diploid cells (BP5050; 30°C) by indirect immunofluorescence. (B) Mif2p staining as visualized by affinity-purified anti-Mif2p; (C) Total DNA as detected by DAPI staining; (D) MTs as visualized by anti-α-tubulin; (E) DIC image of field. Brighter foci of Mif2p staining are indicated by arrowheads.

 Coimmunoprecipitation of CEN DNA with Mif2p. (A) Formaldehyde cross-linked chromatin (2 hr fixation) prepared from wild-type strain 5371-10-2 was immunoprecipitated with anti-Mif2p antibodies in the absence or presence of recombinant 6-His–Mif2 fusion protein. Some reactions were prepared in duplicate. Total input material and coimmunoprecipitated DNA were analyzed by slot blot hybridization with the indicated probes. The percent of total input material for a given locus present in an immunoprecipitate is indicated to the right of each panel. The variable low levels of material detected by the TUB2 and total genomic DNA probes probably reflect contaminating RNA, although noncentromeric chromatin may exhibit some insolubility as compared to centromeric chromatin. (B) Cross-linked chromatin (2 hr fixation) prepared from mif2::HIS3 strains expressing either wild-type MIF2 or epitope-tagged MIF2–HA from a centromere-based plasmid was immunoprecipitated with anti-Mif2p antiserum or anti-HA antibody. Coimmunoprecipitated DNA was analyzed by slot blot hybridization using a CEN3-specific probe. (C) Time course of fixation. Cross-linked chromatin prepared from the MIF2–HA strain after various times of fixation was mock-treated (shaded circle) or immunoprecipitated with anti-Mif2p antiserum (black square) or anti-HA (black diamond) and analyzed as in B. The percent of total input CEN3 DNA that coimmunoprecipitated with Mif2–HAp was plotted. Identical results were obtained in B and C using a CEN16 probe (data not shown). That CEN DNA recovery was consistently greater in immunoprecipitates prepared with authentic Mif2p antibodies as compared to anti-HA presumably reflects the polyclonal nature of these sera.

 Interaction of Mif2p with segregation-incompetent derivatives of CEN3. (A) Schematic of the CEN3 genomic locus and three segregation-incompetent CEN3 derivatives that were individually integrated at the URA3 locus of wild-type yeast strain MS10. The predicted sizes of CEN3-containing AluI restriction fragments or PCR products are listed. Shading indicates the pBluescript polylinker present in the integrating vector pRS306 (). Hatching indicates pBR322 sequences present in the cen3–BCT2 construct. Arrowheads designate positions of PCR primers (see Materials and Methods). (B) Crosslinked chromatin (1 hr fixation) prepared from the URA3:: cen3–BCT2, URA3::CEN3–CDEI+II, and URA3:: CEN3–CDEIII strains was mock treated (lanes 2,3) or immunoprecipitated with anti-Mif2p antiserum (C223) in the absence (lanes 4,5) or presence of recombinant Mif2 protein (lanes 6,7). Reactions were prepared in duplicate. Total input material (T) and coimmunoprecipitated DNA were digested with AluI and analyzed by Southern blot hybridization with a CEN3-specific probe. Coimmunoprecipitated DNAs were loaded in sixfold excess relative to the total. The asterisk in each panel indicates the position of the AluI fragment diagnostic for the ectopic locus. Background hybridization reflects prior sonication of the chromatin.

 Interaction of centromere proteins Cbf1p and Ndc10p with CEN3 DNA. Cross-linked chromatin (2 hr fixation) was prepared from a set of epitope-tagged CBF1–HA strains in which either pRS306 [vector (polylinker)] or segregation-incompetent CEN3 derivatives (Fig. A) were integrated at the URA3 locus. Chromatin was mock-treated (lanes 2,3) or immunoprecipitated with anti-HA [specific for Cbf1-HAp; (lanes 4,5)], an affinity-purified anti-Ndc10p antibody (lane 6), or anti-Mif2p antiserum (lane 7). Aliquots of total input material (≈3 μl chromatin solution) and coimmunoprecipitated DNA (≈30 μl chromatin solution) were analyzed by PCR with primers specific for the authentic CEN3 locus (A), the integrated constructs (B–E), or three noncentromeric loci on yeast chromosome III (F) (see Materials and Methods for details). Shown are negative images of the ethidium bromide-stained products resolved by PAGE. Size standards are indicated to the left of each panel. Expected sizes of the PCR products are in parentheses. A and F are from the URA3::cen3–BCT2 strain; identical results were obtained for the other three strains (data not shown).

 Interactions of Mif2p, Ndc10p, and Cbf1p are limited to the CEN DNA. The CEN3 (A) and CEN16 (B) chromosomal regions are represented. Stippling corresponds to noncoding DNA; orientation and position of open reading frames near CEN16 are indicated. Cross-linked chromatin (2 hr fixation) prepared from a CBF1–HA strain was immunoprecipitated with anti-Mif2p, anti-Ndc10p, anti–HA (specific for Cbf1–HAp), or mock treated. Total input material, a 1:24 dilution, thereof, and coimmunoprecipitated DNAs were analyzed by PCR as in Fig. , using primers specific for the indicated series of short, overlapping, CEN-flanking fragments (rectangles). Shading indicates the relative enrichment in the immunoprecipitates of DNA corresponding to those intervals and is based on the negative images of ethidium bromide-stained PCR products shown below each schematic. Data shown are for chromatin prepared from a URA3::cen3–BCT2 strain (A) and a URA3::CEN3–CDEI+II strain (B).

 Model for assembly of the budding yeast CKC in vivo. Conserved centromeric DNA elements, CDEI (8 bp) and CDEIII (25 bp), indicated as hatched boxes, flank AT-rich CDEII (78–87 bp). CDEIII is both necessary and sufficient for the interaction of Mif2p and Ndc10p with CEN DNA in vivo. Ndc10p is a component of the multiprotein CDEIII-binding complex, CBF3, as are Cbf3Bp, Ctf13p, and Skp1p. CDEIII is also necessary, though not sufficient, for interaction of the CDEI-binding protein, Cbf1p, with CEN DNA in vivo. The observed in vivo dependence of the Cbf1p–CEN DNA interaction on CDEIII suggests that centromere assembly initiates with binding of factors at CDEIII (e.g., CBF3 components, indicated in gray, and Mif2p), which in turn nucleate further centromere assembly. Nucleosomes are represented by shaded spheres. Potential repressive chromatin that would preclude independent association of some factors (e.g., Cbf1p) is indicated by the horizontal arrow. Other factors comprising the mature CKC might include one or more MT-binding proteins (; ), CENP-A-related Cse4p (), and mitotic checkpoint proteins (). Ordered nucleosomal arrays surround the mature CKC in budding yeast (). Genetic evidence for possible looping in the mature centromere is discussed in . For further explanation, see text.