Surprisingly, we did not see changes in the levels of chaperones at P10. This discrepancy is likely due to the fact that steady-state levels reflect both protein synthesis and degradation, and under conditions of stress, heat shock proteins are induced. For select CSPα interactome members, we determined that their mRNA levels were unchanged in wild-type and CSPα KO brains, indicating that the observed decreases in protein levels occurred posttranscriptionally (Figures S2D and S2E). The 15 proteins whose levels we validated in the CSPα KO can either be direct clients of the CSPα chaperone complex or indirectly decreased due to secondary changes. To determine

which of these proteins are direct clients, we tested if they bind either CSPα or Hsc70 in a nucleotide-dependent check details manner. In the presence of ATP, clients typically bind the Hsc70-DnaJ cochaperone complex with low affinity, while ADP promotes a high-affinity interaction (Kampinga and Craig, 2010). We expressed CSPα and Hsc70 as GST fusions and carried out GST pull-downs with wild-type mouse brain Ulixertinib cost homogenates (Figure 3A), using the Hsc70 binding protein Stip

1/HOP as a positive control. Our results revealed that dynamin 1 binds CSPα, suggesting that it is a client of this chaperone complex. Importantly, dynamin 1 behaves like a prototypical Hsc70-DnaJ chaperone client in that it binds CSPα, and not Hsc70, and its binding is ADP dependent. Consistent very with previously published work, SNAP-25 binds both CSPα and Hsc70 (Figure 3A) (Chandra et al., 2005 and Sharma et al., 2011). Additionally, we could show that BASP1 is an Hsc70 binding protein and rule out that complexin I, NSF, and synucleins are direct CSPα clients. Based on our proteomic and biochemical analysis, we narrowed our analysis

of CSPα clients to dynamin 1 and SNAP-25. We confirmed the interactions of both dynamin 1 and SNAP-25 with the CSPα chaperone complex in vivo by immunoprecipitating dynamin 1 and SNAP-25 with CSPα from brain homogenates in the presence of nucleotides (Figure 3B). Again, the binding of dynamin 1, but not of SNAP-25, to CSPα is promoted by addition of ADP. We also showed these interactions with proteins heterologously expressed in HEK293T cells (Figure 3C). These results indicate that dynamin 1 and SNAP-25 are both direct clients of the Hsc70-CSPα chaperone complex but probably have different sites of interaction. We therefore tested binding of dynamin 1 and SNAP-25 to CSPα and Hsc70 with purified proteins. As seen in Figure 3D, dynamin 1 is recruited to this complex via CSPα binding, while SNAP-25 is recruited via Hsc70 (Sharma et al., 2011). Previous work has shown that binding of purified SNAP-25 to Hsc70 is stabilized in the presence of ADP-β-S (Sharma et al., 2011). Based on these findings, we predicted that the two clients may have different effects on the nucleotide binding domain of Hsc70 and therein its ATPase activity.