Figure 5.

The C terminus of Mps2 interacts with the Mps3 SUN domain. (A) Shown is the C-terminal sequence of Mps2 after the transmembrane domain (amino acids 328–387). Aromatic amino acids are shown in magenta. (B) A nonsense codon was inserted after the sequence encoding the transmembrane domain in MPS2 (codon 328) to generate an allele that lacked the C-terminal domain. This allele, mps2ΔC, a wild-type copy of MPS2 or an empty vector were integrated in single copy into the LEU2 locus of a MPS2 deletion covered by a URA3-based plasmid containing wild-type MPS2 (SLJ1901). The ability of each to rescue the mps2Δ was tested by plating fivefold serial dilutions of cells on 5-FOA plates (right). As a control, cells were stamped onto SD-URA plates (left). Plates were incubated at 30°C for 2 d. Identical results were obtained at 16, 23, 34, and 37°C (not depicted). (C) Similarly, a nonsense codon was inserted after the indicated amino acid in MPS2 to generate the C-terminal (Ct) truncation alleles depicted. Mutant genes were transformed into strains containing a deletion of MPS2 covered by a URA3-based plasmid containing wild-type MPS2 (SLJ1901) plus a TRP1-based 2μ plasmid containing nothing (−), MPS2, or MPS3. The ability of each mps2 mutant to rescue the deletion in the presence of vector, MPS2, and MPS3 was tested by plating a series of six fivefold dilutions on 5-FOA. After 4 d at 30°C, plates were scored as follows: ++, growth at the fourth dilution; +, growth at the third dilution; +/−, growth at the first and second dilution; −, no growth above background. mps2-381 cells grew at 23 and 30 but not at 37°C (ts) in the absence of a rescuing plasmid. (D) Mps2 C-terminal deletion mutants were also expressed as MBP fusion proteins in bacteria and mixed with 1 μg purified 6xHis-Mps3SUN. Mps2 fusion proteins were immunoprecipitated using anti-Mps2 antibodies, and the level of each was determined by Western blotting with anti-MBP antibodies (top). Binding to the Mps3 SUN domain was tested by Western blotting with anti-Mps3 antibodies (bottom). (E) mps2-381 cells (SLJ2006) were transformed with the indicated gene on a 2μ plasmid carrying a TRP1 marker (pRS424), and the ability of the 2μ plasmid to restore growth to the mutant at 37°C was tested by plating a series of six fivefold dilutions of cells on SD-TRP plates. Plates were incubated for 2 d at 37°C or 3 d at 23°C. (F) The indicated gene on a 2μ URA3 plasmid (pRS202 or pRS426) was transformed into mps2-381 cells (SLJ2006) and analyzed for its ability to restore growth at 37°C in a serial dilution assay as described in C. mps2-381 growth at 37°C was not suppressed by 2μ SPC42, 2μ SPC29, 2μ CNM67, 2μ NUD1, 2μ KAR1, or 2μ SFI1.