mps3Δ75–150 mutants have defects in telomere tethering. (A) Yeast cells expressing GFP-LacI and Nup49-GFP fusions were tagged with ∼256 copies of the LacO_R at telomere VI-R, VIII-L, or XIV-L. The subnuclear position of the telomere was scored with respect to the distance from the nuclear envelope in a single plane image and assigned a position into one of three zones of equal volume (see text for details). Bar, 5 μm. (B) The distribution of telomere ends in zone 1 (black), 2 (white), and 3 (gray) was determined in asynchronously growing wild-type and mps3Δ75–150 cells at 30°C for each telomere. The dotted line at 33% corresponds to a random distribution. Confidence values (P) for the χ² test were calculated for each dataset between random and test distributions. The number of cells examined in each dataset is indicated (n).

mps3Δ75–150 mutants are defective in S phase telomere tethering. The number of telomere ends in each zone of the nucleus was specifically examined in unbudded G1 cells and small- to medium-budded S-phase cells in an asynchronously growing culture of wild-type and mps3Δ75–150 mutant cells expressing GFP-LacI and Nup49-GFP fusions along with copies of the LacO_R at telomere VI-R or XIV-L. The dotted line at 33% corresponds to a random distribution. Confidence values (P) for the χ² test were calculated for each dataset between random and test distributions. The number of cells examined in each dataset is indicated (n).

LexA-Sir4_839–934 can recruit chromosomes to the nuclear periphery in the presence of the Mps3 acidic domain. (A) Schematic of ∼256 LacO_R and four lexA^op binding sites integrated at ARS607, which is located on the arm of chromosome VI ∼50 kb from the centromere and 70 kb from the telomere. (B) Wild-type, mps3Δ75–150, and esc1Δ cells that contained this version of chromosome VI as well as a galactose-inducible LexA-sir4_839–934-NLS (SLJ2651, SLJ2652, and SLJ2653, respectively) were grown overnight in YEP + 2% raffinose at 30°C to mid-log phase. Cultures were divided and 2% dextrose was added to repress expression (−) and 2% galactose was added to induce expression of LexA-sir4_839–934-NLS for 2 h at 30°C. Localization of arm VI to zone 1 (black), 2 (white), and 3 (gray) was then analyzed. The dotted line at 33% corresponds to a random distribution. Confidence values (P) for the χ² test were calculated for each dataset between random and test distributions. The number of cells examined in each dataset is indicated (n). (C) Schematic of ∼256 LacO_R and four lexA^op binding sites integrated at ARS609, near telomere VI-R. The subtelomeric sequence of telomere VI-R has also been truncated by the insertion of an ADE2 reporter linked to copies of the TG_1–3 telomeric repeats (Hediger et al., 2002). (D) Localization of truncated telomere VI-R in wild-type, mps3Δ75–150, and esc1Δ cells that contained a galactose-inducible LexA-sir4_839–934-NLS (SLJ2647, SLJ2648, and SLJ2649, respectively), determined as described in B. (E) Expression of the telomeric ADE2 gene in strains from D was monitored by streaking cells to SD plates containing 10 μg/ml adenine and 2% dextrose or 2% galactose/2% raffinose to repress (−) or induce the expression of LexA-sir4_839–934-NLS, respectively. After growth for 3 d at 30°C, plates were incubated for 1 wk at 4°C to allow the red pigment to develop. Expression of ADE2 results in white colored cells and blocks the accumulation of the red pigment in this strain background; this occurs in cells that have lost telomeric silencing. Bar, 1 cm.

The Mps3 N terminus is able to rescue the silencing defect of mps3Δ75–150 mutants. A 2-μm plasmid containing no insert, MPS3, mps3 1–75, mps3 1–150, or mps3 1–181 was transformed into the mps3Δ75–150 mutant (SLJ2516), and silencing at telomere V-R, HMR, and the rDNA was assayed as described in Fig. 7.

The Mps3 acidic domain is required for peripheral nuclear envelope localization. (A) Schematic of Mps3 showing the acidic domain from amino acids 75–150 as well as the transmembrane domain (tm), regions of coiled-coil (cc), the polyglutamine repeat (pQ), and the SUN domain. (B) Versions of MPS3 in which the indicated N-terminal amino acids were deleted were constructed and transformed into SLJ2039 along with an empty vector and wild-type MPS3. The ability of each to rescue mps3Δ was tested by plating fivefold serial dilutions to 5-FOA. Cells were also stamped to SD-URA. Plates were incubated for 2 d at 30°C. (C) MPS3 (SLJ2059) and mps3Δ75–150 (SLJ2064) cells were grown to mid-log phase at 30°C. Flow cytometric analysis of DNA content and quantitation of budding index indicated that mps3Δ75–150 mutants do not arrest in mitosis. The biphasic peaks represent cells with G1 (1 N) and G2/M (2 N) DNA content. The percentage of large-budded cells in each sample is indicated. (D) Indirect immunofluroescence was performed to visualize microtubules (green), SPBs (anti-Tub4; red), and DNA (DAPI; blue). The percentage of large-budded cells with bipolar mitotic spindles was determined by counting the number of Tub4 signals per cell (n = 200). (E) Protein levels in extracts of wild-type (SLJ001), MPS3-GFP (SLJ2551), mps3Δ75–150–GFP (SLJ2552), and mps3Δ75–150–GFP MPS3 (SLJ2659) cells were determined by Western blotting with anti-GFP antibodies. Glucose–6-phosphate dehydrogenase served as a loading control. (F) Localization of Mps3-GFP and mps3Δ75–150–GFP (green) was determined by confocal imaging of yeast nuclei using avalanche photodiode detectors. Projection images were generated as described in Materials and methods. Arrows indicate the position of SPBs and the percentages indicate the proportion of cells observed with the localization pattern presented. (G) We also examined mps3Δ75–150–GFP localization in cells containing a wild-type copy of mps3 (SLJ2659). Bars, 5 μm.

The Mps3 N terminus is sufficient to restore telomere tethering to mps3Δ75–150 mutants. (A) Schematic of Mps3 containing the domains described in Fig. 1 A and constructs containing the indicated regions of the Mps3 N terminus. These were introduced into mps3Δ75–150 mutants expressing GFP-LacI and Nup49-GFP fusions along with copies of the LacO_R at telomere VI-R or XIV-L. (B) The distribution of telomere ends in zone 1 (black), 2 (white), and 3 (gray) was determined in asynchronously growing cells at 30°C for each telomere. The dotted line at 33% corresponds to a random distribution. Confidence values (P) for the χ² test were calculated for each dataset between random and test distributions. The number of cells examined in each dataset is indicated (n).

Mps3 interacts with Sir4 through its acidic domain. (A) The protein composition of anti-HA immunoprecipitates from wild-type (SLJ001), MPS3-HA3 (SLJ922), and mps3Δ75–150–HA3 (SLJ2529) cells was analyzed by Western blotting with anti-HA and anti-Sir4 antibodies. Lysates were also analyzed directly by Western blotting. (B) Similarly, the protein composition of the anti-HA immunoprecipitates from the indicated deletion strains was analyzed by Western blotting with anti-HA and anti-Sir4 antibodies. (C) Two-hybrid analysis was done with SLJ1644 cells containing the indicated GAL4- activation domain fusions (columns) and SLJ1645 cells containing the indicated GAL4-DNA binding domain fusions (rows). Diploids were selected on SD-LEU-TRP plates at 30°C, and the ability of fusion proteins to interact was assayed by plating cells, which contain a version of HIS3 driven by the GAL1 promoter, on SD-LEU-TRP-HIS plates at 30°C. (D) Localization of Sir4 (anti-Sir4; yellow) and DNA (DAPI; blue) was determined in wild-type (SLJ2059) and mps3Δ75–150 mutants (SLJ2064) enriched in S phase with hydroxyurea. Bar, 5 μm.

mps3Δ75–150 mutants are defective in telomeric silencing. (A) Three different reporter genes are present in the triple silencer strain to allow simultaneous monitoring of silencing at the mating-type locus, rDNA, and telomeres (Ray et al., 2003). Expression of TRP1 in the HMR locus, which contains a deletion in the E element of the E silencer, is monitored by growth on medium lacking tryptophan, where more growth equals less silencing. The expression of CAN1 in the rDNA locus was monitored using negative selection against CAN1 expression so that more growth equals more silencing. Expression of URA3 placed adjacent to the right telomere of chromosome V (indicated by the black dot) is monitored by negative selection on 5-FOA so that more growth equals more silencing. Arrows indicate the direction of transcription of each reporter gene. (B) 10-fold serial dilutions of single yeast colonies of the indicated genotype were spotted onto different media at 30°C. Representative colonies from four assays are shown. SD complete medium shows the total number of cells spotted and the other media (SD-TRP, SD-ARG + CAN, and 5-FOA) show the extent of silencing at HMR, rDNA, and telomere V-R, respectively. Because the CAN1 reporter in the rDNA locus represents a single gene in an array of 100−150 genes, small changes in growth are considered to represent a larger change in silencing compared with the HMR and telomere reporters, which comprise the entire locus (Ray et al., 2003).