Figure 1

Isolation of CCT substrate complexes. (A) Affinity purification of CCT–3CBP and CCT–6CBP on calmodulin resin. CaM-resin-purified CCT was analysed by 10% SDS–PAGE. (Lane I) CCT–3CBP complex: the eight CCT subunits migrating around the 60 kDa marker are indicated by the square bracket. (Lane II) CCT–6CBP complex: subunit Cct6p migrating at 70 kDa as a consequence of the inserted tag is indicated by an arrow. (Lane III) CCT–6CBP complex as used for in-house MALDI-MS and PMF-MS analysis. Molecular weight markers in kilodaltons are indicated on the left, Vid27p and actin are indicated as a reference (see also Supplementary Table S1). (B) Purification of CCT–3CBP in the presence and absence of ATP. CCT was incubated 5 mM ATP at room temperature during the CaM affinity purification procedure. EDTA eluates of CCT were applied to 10–40% sucrose gradients. The panels +ATP wash and -ATP wash show four adjacent sucrose gradient fractions from each 20S CCT peak electrophoresed on 10% SDS gels followed by Coomassie (fractions 1–4) or silver staining (fraction 2 from each peak). (C) Histograms of the CCT–3CBP-bound polypeptides plotted against their MudPIT score (Tables I). Comparisons between the ATP-treated (red bars) and untreated CCT samples (gray bars) show the proteins that are removed by 5 mM ATP treatment during the CaM chromatography step.