Figure 3. CHL1 recruits Hsc70 and αSGT to synaptic vesicles but inhibits their association with CSP by forming dimeric CHL1/CSP and trimeric CHL1/Hsc70/αSGT complexes.

A - GST and GST-tagged CSP immobilized on beads were assayed for their ability to pull down Hsc70 and αSGT from a mixture of these proteins in the presence of ADP or ATP with or without CHL1ID. Graphs show mean optical density of the blots ± SEM (n = 6). For quantitation of the binding of Hsc70 and αSGT to CSP (graphs on the left and in the middle, respectively), levels of these proteins bound to CSP in the presence of ADP and absence of CHL1ID were set to 100%. For quantitation of the binding of CHL1ID to CSP (graph on the right), levels of CHL1ID bound to CSP in the presence of ADP were set to 100%. Note that in the presence of ATP, CHL1ID binds to CSP with higher efficiency than in the presence of ADP. CHL1ID inhibits αSGT/CSP complex formation in a nucleotide independent manner and inhibits ATP-dependent formation of the Hsc70/CSP complex. *p<0.05, paired t-test (compared as indicated). B - Scheme (top) shows CHL1ID fragments marked alphabetically that were immobilized on beads via the GST tag and assessed for their ability to pull down Hsc70, CSP or αSGT. Coomassie blue stained gel shows that similar levels of CHL1 fragments were immobilized on beads. Lower molecular weight degradation products of the recombinant proteins are also observed. Binding of Hsc70, CSP and αSGT to CHL1ID fragments was analyzed by Western blot with the corresponding antibodies. Note that amino acids 1105–1130 within CHL1ID are necessary to bind Hsc70 and CSP, while amino acids 1190–1209 are necessary to bind αSGT. Double bands recognized by CSP antibodies represent oligomeric forms of recombinant CSP as described previously [51]. C,D - Molecular complexes in synaptic vesicles (C) and synaptic plasma membranes (D) were separated by PAGE under non-denaturing conditions and probed by Western blot with the indicated antibodies. Two or more proteins were considered in a presumed complex, if their bands in the gel overlapped at the apparent molecular weight calculated to be equal or above the total molecular weight of the sum of the weights of the proteins together. In accordance with this criterion, arrows show overlapping bands of CHL1, Hsc70, and αSGT that presumably represent the CHL1/Hsc70/αSGT complex. Note that CHL1/Hc70/αSGT complex migrates at the two levels in the native gel probably due to the multimerization of its components or presence of additional binding partners in the slower migrating level (black arrows) compared to faster migrating level (grey arrows). The higher molecular weight level is more evident in synaptic vesicles, while lower molecular weight level is more evident in synaptic plasma membranes. Arrowheads show overlapping bands of CHL1 and CSP that presumably represent the CHL1/CSP complex. Only complexes of CHL1 together with Hsc70, CSP or αSGT are marked. Note that trimeric Hsc70/CSP/αSGT complexes are not detectable in the gels since CSP bands do not overlap with αSGT bands. In D, a CHL1 band overlapping with Hsc70 and CSP immumoreactive bands at ∼230 kDa may contain only the CHL1/CSP complex of ∼230 kDa but not CHL1/Hsc70/CSP or CHL1/Hsc70 complexes with molecular weights above 250 kDa. Note SNAP25 and VAMP2 positive bands, which overlap with CHL1/Hsc70/αSGT and CHL1/CSP complexes, respectively. In synaptic plasma membranes, VAMP2 accumulates in a high molecular weight complex with a slightly different molecular weight than that of the CHL1/Hsc70/αSGT complex. E - Graphs show mean levels of activity ± SEM (n = 6) of luciferase reactivated by indicated combinations of chaperones. Values were normalized to luciferase reactivation levels in the absence of the added CHL1ID set to 100%. *p<0.05, paired t-test. F - Schematic model diagram of CHL1-containing chaperone complexes. The Hsc70/CSP/αSGT complex dissociates into the CHL1/CSP complex present in synaptic vesicles and the CHL1/Hsc70/αSGT complex present in synaptic vesicles and plasma membranes. HPD tripeptides within the intracellular domain of CHL1 and the J-domain of CSP are marked in yellow.