Elucidating the role of the Hsc70 chaperone in SNARE complex assembly.

Abstract

The complex mechanism of synaptic vesicle fusion with the plasma membrane for neurotransmitter release is initiated by the formation of the SNARE complex at the presynaptic terminal. SNARE is a proteinaceous complex composed of four helices contributed by three proteins: one helix from syntaxin, two from SNAP-25 (both at the plasma membrane) and, one helix from synaptobrevin (at the vesicle). SNAP-25 is an intrinsically disordered protein that is prone to aggregation. The fusion process is tightly regulated and requires the constitutively expressed Hsp70 chaperone (Hsc70) and its co-chaperones CSPα (J protein) and Hsp110 (nucleotide exchange factor). However, the precise molecular details by which the Hsc70 system participates in the SNARE complex formation remain unknown. SNAP-25 must neither aggregate nor fold prematurely before SNARE formation. We hypothesize that Hsc70 and CSPα cooperate to chaperone SNAP-25 to keep it in folding competent state for SNARE complex formation and facilitates the bringing of the membranes to proximity for exocytosis. To test this hypothesis, we employed a bottom-up approach and studied the interaction between Hsc70 and CSPα with SNAP-25 in vitro. Using a peptide array that spans the sequence of SNAP-25, we identified three potential Hsc70-interacting sequences and designed peptides containing these sequences to test binding in solution. We characterized the interaction of SNAP-25 peptides with Hsc70 and CSPα using a combination of biochemical and biophysical techniques, such as native-PAGE, binding affinity by fluorescence anisotropy, ATP-hydrolysis activity of Hsc70, and NMR. Our peptide binding data show different affinities of SNAP-25 peptides with Hsc70 in solution. We have tentatively identified an Hsc70 binding site within SNAP-25 that is likely to represent the site used in the cell. Importantly, we showed that the aggregation of SNAP-25 is delayed in the presence of Hsc70 and CSPα.