Features of the MPS2 encoded protein. (A) Deduced amino acid sequence of the MPS2 encoded protein. The putative transmembrane segment (residues 311–327) is boxed. Potential Cdc28p phosphorylation sites are indicated in bold letters, and potential destruction box sequences are underlined. The point mutation found in the mps2–1 allele at amino acid 39 is highlighted (E to K). (B) Coiled-coil probability plot. The x-axis is the position of Mps2p amino acids and the y-axis is the probability (p) of being in a coiled-coil using the COILS program (http://www.ch.embnet.org/software/COILS form.html), based on the Lupas algorithm (Lupas et al., 1991).

Deletion of MPS2 results in abnormal ploidy. (A) A representative tetrad from the mps2Δ(116–387)::HIS3/MPS2 strain (AM610) grown for 14 d at 23°C. (B) Histogram of relative DNA content from each of the spore clones shown in A.

Mps2p levels show little fluctuation during the cell cycle. (A) An overnight culture of MCL 94 cells containing pTet-9xmycN-MPS2 grown in the presence of 5 μg/ml doxycycline (repressing conditions) was diluted in the same selective medium containing 50 or 100 ng/ml doxycycline to allow a low constitutive expression from the tet promoter. Cells were grown overnight to early log phase at 30°C (asynchr.) and then divided into two separate cultures: one culture was left untreated (−) and the other was arrested in the G1 phase with α-factor (0.2 μM final concentration) for 2 h (+). Equal quantities of total cell protein were then subjected to SDS-PAGE, and proteins were transferred to membranes to determine the levels of 9xmycN-Mps2p by immunoblot analysis. 9xmycN-Mps2p is absent when cells are grown in the presence of fully repressing concentrations of doxycycline (5 μg/ml) or from cells that do not contain the 9xmycN-MPS2 plasmid. The G1 arrest was verified by flow cytometric analysis. (B) Wild-type cells expressing both 9xmycN-Mps2p and Pds1-HAp (MCL123) were grown to early log phase at 24°C (asynchr.) and then divided into two separate cultures: one culture was left untreated (exp) and the other was arrested in the G1 phase with α-factor (0.2 μM) for 2 or 5 h. After 2 h, >90% of the cells were already arrested in G1 phase as determined by flow cytometry analysis, as were cells treated with α-factor for 5 h. After this time, cells were released from the arrest by washing out the α-factor. Samples were taken at the indicated time points and analyzed for 9xmycN-Mps2p, Cdc28p, and Pds1-HAp by immunoblot analysis. Cell and nuclear morphology and DNA content were analyzed by propidium iodide staining.

9xmycN-Mps2p fractionates as an integral membrane protein. A whole-cell extract was prepared from cells expressing 9xmycN-Mps2p from its normal promoter (MCL120) by gentle spheroplast lysis (see MATERIALS AND METHODS) and then fractionated as described in MATERIALS AND METHODS. (A) Cell fractionation: the same amount of protein from the whole-cell extract and S13, P13, P100, and S100 fractions were subjected to SDS-PAGE and immunoblotted with anti-myc, anti-Sec61p, anti-Dpm1p, and anti-hexokinase antibodies successively. (B) Solubilization of Mps2p from the P13 fraction prepared from MCL120. The pellet was extracted with lysis buffer (control) or lysis buffer containing 1 M NaCl, 50 mM Tris (pH 7.5)-10 mM EDTA, 0.2 M Na₂CO₃ (pH 11), 1% Triton X-100, or 6 M urea as described in MATERIALS AND METHODS. The mixtures were incubated on ice for 10 min and then centrifuged to produce a pellet fraction (P) and a supernatant fraction (S). Equivalent samples of the pellet and supernatant were analyzed by immunoblotting with anti-myc or anti-Sec61 antibodies. (C) Total solubilization of Mps2p protein from the P13. The P13 pellet was extracted with lysis buffer (control) or lysis buffer containing 1% Triton X-100, 1% Triton X-100 plus 6 M urea, or 1% Triton X-100 plus 1 M NaCl. Pellet (P) and supernatant (S) were separated and analyzed by immunoblotting.

Localization of GFP-Mps2p in living cells. Several cells are shown in a, in which GFP-Mps2p (strain MCL175) localizes in one or two discrete foci coincident with the nuclear DNA. The cell with two distinct spots of fluorescence is a small budded cell. The cells shown in b reveal that GFP-Mps2p also localizes to the nuclear periphery. Several cells are shown with one bright spot and a fainter signal at the nuclear periphery (arrows). A higher magnification of a budded cell is shown in c with two bright spots and a perinuclear localization. The GFP-Mps2p was visualized via autofluorescence of GFP, and the DNA was visualized with Hoechst. A single plane of focus is shown. Bar, 1 μm.

9xmycC-Mps2p colocalizes with Spc42p-GFP, but not Nup49p-GFP. (A) A strain expressing both 9xmycC-Mps2p and Spc42p-GFP (SMY28–2b) was prepared for indirect immunofluorescence microscopy (see MATERIALS AND METHODS). The Spc42p-GFP was visualized via autofluorescence of GFP, the 9xmycC-Mps2p was detected using a Texas Red-conjugated 2° antibody, and the DNA was visualized with DAPI. Four different cells are shown at a high magnification to show the detail of colocalization. The bright spots of 9xmycC-Mps2p (red) and the Spc42p-GFP (green) signals overlap, as seen in the combined image (yellow). The Spc42-GFP and 9xmycC-Mps2p signals at the lower SPBs in images b and d do not completely overlap in these focal planes; however, Spc42p and Mps2p are not expected to be in the same exact position in the organelle. In addition, the amount of overlap depends on the ability to indirectly label Mps2p at any given SPB. (B) Two different cells from a strain expressing both 9xmycC-Mps2p and Nup49p-GFP (SMY1892) are shown at a high magnification. There are bright spots of 9xmycC-Mps2p fluorescence observed along the nuclear periphery that are devoid of Nup49p-GFP signal. All panels in this Figure represent a single optical plane that was deconvolved using the Slidebook software package (see MATERIALS AND METHODS). Bar, 1 μm in both panels.

GFP-Mps2p localizes to the SPB. Cells expressing GFP-Mps2p were prepared for immunoelectron microscopy using anti-GFP antibodies and colloidal gold-conjugated secondary antibodies. Thin sections of four different cells are shown. C, cytoplasm; N, nucleoplasm; NE, nuclear envelope; MTs, microtubules; SPB, spindle pole body. Arrowheads show the localization of individual gold particles in b and d. Gold particles were observed in all 12 SPBs examined. Bar, 0.1 μm.