FIG. 2.

Cyclin E newly translated in a reticulocyte lysate associates with the CCT complex. (A) Newly translated cyclin E fractionates at 900 kDa and in the presence of ATP chases to a 150-kDa fraction. Cyclin E mRNA was translated in a reticulocyte lysate in the presence of [³⁵S]methionine. One aliquot was chased with HK/glc (closed circle) and the other was chased without HK/glc (open circle). Samples were then applied to a Superose 12 gel filtration column run, and fractions were analyzed by SDS-PAGE. Radioactivity incorporated into cyclin E was quantitated with a PhosphorImager. The amount of cyclin E recovered in each fraction is shown as a percentage of the total recovered from the column. (B) Newly translated cyclin E coimmunoprecipitates with CCT. Cyclin E was translated as described for panel A and was chased at 30°C in the presence (+) or absence (−) of 5 mM EDTA. The lysates were then immunoprecipitated by using anti-cyclin E monoclonal antibody, polyclonal anti-CCTβ serum, or polyclonal anti-Cdk2 antibody. A similar experiment performed with β-actin is shown for comparison.