Mapping of direct interactions of NAP57 and SHQ1 with the components of the R2TP complex. All panels, except G (which is a glutathione bead pull-down), are amylose resin pull-downs as in Figure 1G–K. (A) Incubation of pontin and reptin alone and together with MBP-NAP57 and control MBP-MCP. Note recombinant reptin (lane 2) includes a minor band that migrates closely to pontin and is apparently a read-through product because it also binds to NAP57 (lane 8). (B) Incubation of pontin and reptin combined with MBP-NAP57 constructs identifies the NAPΔcat domain as docking site (lane 9). (C) Pontin and reptin bind also individually to NAPΔcat. (D) Pontin and reptin, alone and together, bind to NAP57 without its charged and unstructured terminal extremities, MBP-NAP 31–422. (E) Pontin and reptin alone and together bind to MBP-SHQ1. (F) Pontin and reptin alone and together bind to the CS domain of SHQ1. Note MBP-CS migrates between pontin and reptin (arrow). (G) Glutathione-S-transferase (GST) fusions of pontin and reptin combined incubated with the CS domain of SHQ1 and its SSD individually (lanes 1–4) and together (lanes 5,6). A minor band from the fusion proteins that migrates right below the SSD is marked (black square). (H) Incubation of PIH1D1 with MBP-NAP57 constructs identifies NAPΔcat as the binding domain (lane 7). (I) RPAP3 fails to bind to MBP-NAP57 alone (lane 2) and in the context of the other three R2TP components (R2P), which are retained (lane 4). Note a bacterial heat shock protein that sometimes copurifies with MBP-NAP57 and PIH1D1 migrates below RPAP3 (asterisk), and two lower bands contaminate the RPAP3 preparations (black dots). (J) Neither PIH1D1 (lane 3) nor RPAP3 (lane 4) bind to MBP-SHQ1. Note equal background level binding of PIH1D1 to control MBP-MCP (lane 6). (K) Unlike S100 (lane 8), neither pontin and reptin combined, PIH1D1, RPAP3, nor altogether (R2TP) release SHQ1 from MBP-NAP57, irrespective of 1 mM ATP addition. However, pontin, reptin, and PIH1D1, when present, are retained by the complex (lanes 3,4,6,7).
