Sgt1 dimerization is required for yeast kinetochore assembly

Abstract

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1 and Hsp90 help assemble the core kinetochore complex CBF3 by activating the CBF3 components Skp1 and Ctf13. In this study, we show that Sgt1 forms homodimers by performing in vitro and in vivo immunoprecipitation and analytical ultracentrifugation analyses. Analyses of the dimerization of Sgt1 deletion proteins showed that the Skp1-binding domain (amino acids 1-211) contains the Sgt1 homodimerization domain. Also, the Sgt1 mutant proteins that were unable to dimerize also did not bind Skp1, suggesting that Sgt1 dimerization is important for Sgt1-Skp1 binding. Restoring dimerization activity of a dimerization-deficient sgt1 mutant (sgt1-L31P) by using the CENP-B (centromere protein-B) dimerization domain suppressed the temperature sensitivity, the benomyl sensitivity, and the chromosome missegregation phenotype of sgt1-L31P. These results strongly suggest that Sgt1 dimerization is required for kinetochore assembly.

FIGURE 1.

Sgt1 forms dimers. A, Sgt1 associates with Sgt1 in vivo. Lysates of yeast cells that expressed HA-Sgt1 (plasmid pB1354) and either Sgt1-Myc (Y1649, left) or untagged Sgt1 (YPH499, right) were subjected to immunoprecipitation (IP) with anti-Myc antibodies. Immunoblot analyses were performed with anti-HA or anti-Myc antibodies. T, total lysates; S, supernatant; P, precipitate. B, an analytical ultracentrifugation of Sgt1 is shown. Absorbance scans at 280 nm at equilibrium are plotted versus the distance from the axis of rotation Sgt1. Shown is protein at a concentration of 5μm centrifuged at 4 °C for at least 24 h at 16,000 rpm (black), 20,000 rpm (red), 24,000 rpm (blue), and 30,000 rpm (cyan). The solid lines represent the best fit of all the data sets to a monomer-dimer self-association model with a dissociation equilibrium constant of 20 nm. The root mean square deviation of the fit is 0.0040 absorbance units. C, the sedimentation velocity profiles (fringe displacement) of Sgt1 were fitted to a continuous sedimentation coefficient distribution model (c(s)) as well as a hybrid continuous/discrete distribution model (not shown). The root mean square deviation of the fit is 0.018 fringes.

FIGURE 2.

Sgt1-binding domain of Sgt1. A, the indicated mutant proteins were radiolabeled, mixed, and incubated for 1 h at 30 °C with an extract that contained unlabeled HA-Sgt1 (21, 24). HA-Sgt1 was immunoprecipitated (IP) by using anti-HA antibodies that had been conjugated to Sepharose. The immunoprecipitates were eluted and subjected to SDS-PAGE, and the radioactive bands were identified by autoradiography. HA-Sgt1 was detected by immunoblotting with anti-HA antibodies. T, total lysates (6% of the starting material); DS, deletion proteins. The numbers above the lanes indicate the positions of the N- and C-terminal amino acids (aa) in each Sgt1 deletion protein. B, an illustration of the Sgt1 deletion proteins used in the Sgt1 binding assays and a summary of the results of these assays are shown. The results of our previous study of Sgt1 binding to Skp1 and Hsc82 are shown for comparison only (24). Analyses of the Sgt1 binding ability of the Skp1-binding domain (Skp1-BD), Hsc82-binding domain (Hsc82-BD), and Sgt1-binding domain (Sgt1-BD) are shown. +, binding activity; –, no significant binding activity; +/–, weak binding activity; VR1, variable region 1; CS, CHORD protein- and Sgt1-specific; VR2, variable region 2; SGS, SGT1-specific. ND indicates analyses were not performed. C, the N-terminal domain of Sgt1 is sufficient for dimerization. Essentially the same experiments as shown in A were conducted, but HA-tagged mutant proteins were used in place of HA-tagged full-length Sgt1. HA-tagged Sgt1 mutant protein was expressed in the in vitro translation system but was not radiolabeled, whereas untagged Sgt1 mutant proteins were radiolabeled. HA-tagged Sgt1 was detected by immunoblotting with anti-HA antibodies. T, total lysates (6% of the starting material).

FIGURE 3.

Effect of Hsp90 on Sgt1 Dimerization. A, recombinant MBP-Hsp90 induces Sgt1-Skp1 binding. The indicated amounts of MBP-Hsp82 were added to ∼300 ng of His-Sgt1 and GST-Skp1 and incubated at 30 °C for 1 h before immunoprecipitation (IP) with anti-His antibodies. Input refers to total lysates (6% of starting material); Pellet refers to the immunoprecipitate. B, increased levels of Hsp82 stimulate Sgt1-Sgt1 binding moderately. The experiment is essentially the same as the one described in A, except that GST-Sgt1 was used in place of GST-Skp1. His-Sgt1 or GST-Sgt1 was detected by anti-His or anti-GST antibodies. C, Hsp90 is required for Sgt1-Skp1 binding but not Sgt1-Sgt1 binding in vitro. Upper, HA-Skp1-FLAG-Sgt1 binding was monitored in the presence of the Hsp90 inhibitor geldanamycin. In each reaction, equal amounts of HA-Skp1 and FLAG-Sgt1 proteins pretreated with DMSO or GA were immunoprecipitated with anti-HA antibody. Proteins in the immunoprecipitates were detected by tag-specific antibodies. Lower, similar to the experiment in the upper panel, except that HA-Sgt1 was used instead of HA-Skp1 in the Sgt1-Sgt1 binding assay. D, Sgt1-Sgt1 association is unaltered in hsp82 mutants, but Sgt1-Skp1 binding is reduced. Lysates were collected for the mutant strain hsp82-T101I (strain Y1653) that expressed HA-Sgt1 and Myc-Sgt1. Before lysis, the cells were grown either at 25 °C only or at 25 °C and shifted to 37 °C for 3 h. Lysates were subjected to immunoprecipitation with anti-Myc antibodies. Bound fractions were analyzed by using anti-HA, anti-Myc, and anti-Skp1 antibodies.

FIGURE 4.

The TPR domain of Sgt1 is important for its kinetochore function. A, the N-terminal region of Sgt1 that contains the TPR domain is important for Sgt1-Sgt1 and Sgt1-Skp1 interactions. HA-Sgt1, HA-sgt1-3, or HA-sgt1-5 was expressed in Sgt1-Myc, sgt1-3-Myc, or sgt1-5-Myc cells (Y1650, Y1651, and Y1652, respectively). Cells were either grown at 25 °C only or at 25 °C and shifted to 37 °C for 3 h before harvesting. The lysates were subjected to immunoprecipitation with anti-Myc antibodies conjugated to Sepharose. Anti-HA or anti-Myc antibodies were used to detect HA-tagged or Myc-tagged proteins, respectively, in the immunoprecipitates (IP). Skp1 was detected by anti-Skp1 antibodies. B, shown is an alignment of TPR motifs in Sgt1 with those in Sti1, a typical TPR protein (27). Identical residues are in black boxes; similar residues are in gray boxes; and the indicated residues, except for Ala-67, are those whose significance for the kinetochore function was described in our previous reports (21, 24). The starting amino acid position of each residue is shown in parentheses. C, all three TPR motifs of the Sgt1 TPR domain are important for kinetochore function. Strains (Y1662–1666 and Y1668) in which SGT1 was deleted were designed to express pRS414-HA-Sgt1 (plasmid pB180) and pRS415-Myc-Sgt1 (plasmid pB1367) or the indicated point mutations in pRS414-HA (plasmids pB1361–1365) and pRS415-Myc (plasmids pB1368–1372). Lysates were subjected to immunoprecipitation with anti-Myc antibodies conjugated to Sepharose (lower panels). Error bars indicate the S.D. of two independent results. The signals in the upper panels were quantified, and binding activity was defined as the ratio of the amount of coprecipitated protein to the amount of input protein. Specifically, the binding activity of wild-type Sgt1 was defined as 1, and the binding activity of each mutant was normalized to that value.

FIGURE 5.

Sgt1 dimerization is required for the kinetochore function. A, fusion of the CENP-B-DD with an Sgt1 mutant protein results in partial recovery of a lost phenotype. The 59 C-terminal amino acids of human CENP-B, in which the dimerization domain resides, were fused with Sgt1 (CENP-B-DD-Sgt1) and with Sgt1-L31P (CENP-B-DD-Sgt1-L31P). Both HA- and Myc-tagged constructs for each (wild-type and mutant) were expressed in sgt1Δ cells (Y1677 and Y1678, respectively). Untagged (without CENP-B-DD), HA-tagged, and Myc-tagged constructs were used for each (wild-type and mutant) and were expressed in sgt1Δ cells (Y1662 and Y1663, respectively). Cell lysate from all four strains were subjected to immunoprecipitation (IP) with anti-Myc antibodies conjugated to Sepharose. Anti-HA and anti-Myc antibodies were used to detect HA- and Myc-tagged CENP-B-DD-Sgt1, CENP-B-DD-Sgt1-L31P, Sgt1, and Sgt1-L31P. Skp1 was detected with anti-Skp1 antibodies. The asterisk indicates a nonspecific band. HA-Sgt1 and HA-Sgt1-L31P protein in Input lanes were very close to the nonspecific band. B, cells that expressed CENP-B-DD fused with Sgt1 (Y1675) or Sgt1-L31P (Y1676) were streaked on yeast extract-peptone-dextrose plates. The plates were also inoculated with a strain that expressed wild-type Sgt1 (Y1655) and a strain that expressed sgt1-L31P without CENP-B-DD (Y1656). Plates were incubated at 25 °C and 37 °C. Pictures were taken after 3 days of growth. C, the indicated strains (the same as in B) were grown on yeast extract-peptone-dextrose plates containing 15 mg/ml benomyl or DMSO only. The numbers of cells that were spotted onto each plate (left to right) were ∼5 × 10⁴, 1 × 10⁴, 2 × 10³, and 4 × 10². The plates were incubated at 30 °C for 3 days. D, shown are the results of a colony color-sectoring assay to analyze the chromosome missegregation phenotype of cells expressing Sgt1-L31P (Y1670) and CENP-B-DD-Sgt1-L31P (Y1680).