ATP hydrolysis in D1 dissociates Drg1 from Rlp24C. (A–C) SPR analyses with GST-Rlp24C immobilized on a sensor chip. The data shown are from a representative experiment out of two (B) or three (A and C) biological replicates. (A) The exchange in D1 but not D2 results in decreased release from the GST-Rlp24C fusion. 100 nM of Drg1 (WT), Drg1EQ1, or Drg1EQ2 proteins were injected over the sensor chip in the presence (+) or absence (−) of ATP. RU were recorded and plotted over time. (B) For Scatchard analysis, different concentrations of Drg1 or Drg1EQ1 were injected in the presence of AMP-PNP. The response at the end of the association phase was determined for each concentration from the sensorgrams. The RU per nanomole concentration of Drg1 (left) or Drg1EQ1 (right) were plotted as a function of RU. (C) Comparison of the interaction between Rlp24C and Drg1 (left) or Drg1EQ1 (right) in the presence of ATP or AMP-PNP. (D) ATP hydrolysis in D1 is important for the release of Drg1 from Rlp24C in vitro. GST pull-down experiments using GST-Rlp24C as bait. GST-Rlp24C was immobilized on glutathione agarose and incubated with 60 µg Drg1 or Drg1EQ1 in the presence of the indicated nucleotide. PNP, AMP-PNP; −, incubation in the absence of nucleotide. The eluates were analyzed by SDS-PAGE and Coomassie staining.