Mrc1-Tof1-Csm3 complex. The 3 subunits of the MTC complex (Mrc1-Tof1-Csm3) were co-expressed in yeast. Genes were integrated into the chromosome of strain OY01 (ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 bar1Δ MATa pep4::KANMX6) with Mrc1Flag integrated at the Ade2 locus, untagged Tof1 at the His3 locus, and HisCsm1 at the Leu2 locus, each under control of the Gal1/10 promoter. Cells were grown under selection at 30°C in SC glucose, then split into 18L YP-glycerol and grown to OD600 of 0.7 at 30°C before induction for 6 hr upon addition of 20 g of galactose/L. After 6 hr, cells were harvested by centrifugation, resuspended in a minimal volume of 20 mM HEPES, pH 7.6, 1.2% polyvinylpyrrolidone, and protease inhibitors and frozen by dripping into liquid nitrogen. Purification of MTC was performed by lysis of 18 L equivalent of frozen cells with a SPEX cryogenic grinding mill. Ground cell powder was thawed in the cold room and resuspended to 25 ml final volume with 5X FLAG binding buffer (1x is 250 mM K glutamate, 50 mM HEPES pH 7.5, 1 mM EDTA pH 8.0) plus protease inhibitors and stirred slowly for 30’. Cell debris was removed by centrifugation (19,000 r.p.m. in a SS-34 rotor for 1 hr at 4°C) and the supernatant was collected and mixed with 1.5 ml anti-Flag M2 affinity resin (Sigma) equilibrated in 1X FLAG binding buffer with 10% glycerol. The mixture was rotated on an orbital platform in the cold room at 30 rpm for 1 hr. To collect the bound proteins, anti-FLAG resin was pelleted at 1000 X g in 50 ml conical tubes and washed 5 times with 5 ml of FLAG binding buffer followed by centrifugation. After the final wash step, the anti-Flag affinity resin was resuspended in 2 ml of FLAG binding buffer with 10% glycerol, loaded onto a gravity column and washed twice with 7.5 ml of FLAG binding buffer containing 10% glycerol. Bound protein was eluted with the same buffer containing 0.2 mg/ml 3X FLAG peptide (EZ Biolab, Carmel, Indiana USA). Eluted protein was concentrated to 0.75 ml of 1.5 mg/ml protein and injected onto a 24 ml Superose 12 gel filtration column equilibrated in 2X PBS with 10% glycerol in two separate runs of 0.5 ml and 0.25 ml. Fractions were analyzed on a 7.5% SDS-PAGE gel and MTC-containing fractions were pooled, aliquoted, flash frozen in liquid nitrogen, and stored at −80°C.