FIG. 3.

eEF1A interacts with proteasome subunit Rpt1. (A) Pre1-FLAG was purified from wild-type (WT; lanes 1, 3, 6, and 8), rpn2 (lanes 2 and 7), rpt1 (lanes 4 and 9), and rpt6 (lanes 5 and 10), and the copurification of eEF1A with the proteasome was determined. The strain background for rpn2 differed from that harboring rpt6 and rpt1 mutations. (B) rpt1 was grown at 24, 30, and 37°C, and Pre1-FLAG was precipitated. The levels of eEF1A and Rpt1 were examined. Stability of the proteasome at high temperature (37°C) was unaffected, as determined by the constant levels of Rpt1 that was precipitated from the wild-type and rpt1 strains. (C) Rpt1 was isolated from Escherichia coli and 100 ng of the purified protein was resolved in the right lane. Rpt1 was applied to immobilized GST-eEF1A, GST-Rpn8, GST-S5a, and GST, and >90% of the input Rpt1 bound GST-eEF1A. (D) The positions of purified Rpt1 (panel 3) and GST-eEF1A (panel 2) in Superose 6 are compared to bovine serum albumin (panel 1). Ten-fold less Rpt1 and GST-eEF1A were combined (to minimize aggregation or nonspecific binding), incubated at 4°C for 2 h, and resolved in Superose 6 (panels 4 and 5). Fractions were incubated with glutathione-Sepharose (GST pulldown [GST-PD]) and Rpt1 was coprecipitated with GST-eEF1A (panels 6 and 7). Dashed lines indicate the position of free eEF1A. IP, immunoprecipitation.