YEPD media.

Extract preparation and quantification of protein levels: Cultures (1.7 ml) of tagged strains were grown in 96-well format to log phase, and total cell
extracts were examined by SDS–polyacrylamide gel electrophoresis (PAGE)/western blot analysis. Before the quantitativ ... e SDS–PAGE/western blot analysis, strains were ordered on the basis of estimates of TAP abundance from a preliminary dot-blot analysis. In order to provide a standard for the conversion of western signals to absolute protein levels, a TAP-tagged protein (Escherichia coli initiation factor A, INFA) was overexpressed in E. coli and purified to homogeneity. Yeast extracts containing serial dilutions of INFA ranging from 500 attomoles (which was the limit of detection, see Supplementary Fig. S1) to 25 picomoles were run on a gel along with extracts from 25 different yeast TAP-tagged strains representing the full range of observed protein signals (a second TAP-tagged protein (initiation factor B) was also analysed to ensure that the observed TAP signal was not influenced by the fusion protein). Comparison of the signals generated by these 25 proteins to the known standards allowed the creation of a conversion factor between the observed western blot signals and absolute protein levels. Based on the number of cells (1x10^7) used for the SDS–PAGE/western blot analysis, the protein levels were then converted to measurements of protein molecules per cell. Cultures were grown to OD of ~0.7 at 30 oC and pelleted cells were lysed by the addition of 50 µL of a boiling SDS solution (50mM Tris-HCl , pH 7.5, 5% SDS, 5% glycerol, 50mM DTT, 5mM EDTA, Bromophenol Blue, 2µg/mL Leupeptin, 2µg/mL Pepstatin A, 1µg/mL Chymostatin, 0.15 mg/mL Benzamidine, 0.1 mg/mL Pefabloc, 8.8 µg/mL Aprotanin, 3µg/mL Anitpain). Lysed cells were centrifuged and the supernatant extract was stored at –80 oC. 13 µL aliquots of the SDS-lysed extracts were loaded on 26 well, 4-15% gradient acrylamide Tris-HCl Criterion precast gels (Bio Rad). The gels were run at 200 Volts for 70 minutes and transferred using Trans-blot SD semi-dry transfer cell (BioRad) onto PVDF membranes at a constant current of 160 mA per gel for 120 minutes. Before transfer, the activated PVDF membranes and the gels were soaked in 39mM Glycine, 48mM Tris-HCl, 0.375% SDS with and without 20% methanol respectively in order to facilitate transfer while preventing bleed through. Analysis of a number of randomly chosen blots indicated that the large majority of the protein samples were transferred onto the PVDF membrane. The blots were probed using an affinity purified rabbit polyclonal antibody raised against the calmodulin binding peptide. This antibody can detect the TAP tag with great sensitivity as it can bind CBP as well as the Protein A segment of the tag through interaction with its Fc region. The blots were subsequently probed with a horse radish peroxidase (HRP) conjugated Goat secondary antibody (Jackson ImmunoResearch) against rabbit IgG and reacted with SuperSignal West Femto Maximum Sensitivity Substrate ECL (BioRad) and the chemiluminescence of the bands corresponding to the tagged proteins were detected and quantified using a CCD camera (Alpha Innotech). Transfer efficiency was monitored by Ponceau S staining all membranes and including “Magic Mark” (invitrogen) molecular weight standards, which contain IgG binding domains allowing visualization by Western blotting.