Elucidating the Role of the Hsc70 Chaperone System in SNARE Complex Formation

Abstract

Neurons communicate through neurotransmitters that are released into the synaptic cleft. For this release to occur a synaptic vesicle must fuse with the plasma membrane in the presynaptic terminal of neurons. Fusion requires the formation of the SNARE complex, which is composed of four helices from three proteins: one helix from syntaxin, one helix from synaptobrevin, and two from SNAP‐25 (one from the C‐terminal and one from the N‐terminal). This fusion process is tightly regulated and requires an Hsp70 chaperone system including the constitutive cytoplasmic Hsp70, Hsc70, and its co‐chaperones CSPɑ (a J protein) and Hsp110 (a nucleotide‐exchange factor). However, the molecular details of this system are still poorly understood. SNAP‐25 is a disordered protein that is prone to aggregation and premature folding. Therefore, we hypothesize that SNAP‐25 is chaperoned by Hsc70 to ensure that it remains in a state that is competent for SNARE complex formation. Using a peptide array of SNAP‐25, we identified three most probable binding sites of Hsc70 on SNAP‐25. We took the advantage of the fact that the fully formed SNARE complex is SDS‐resistant and used SDS‐PAGE to monitor successful reconstitution of the SNARE complex in vitro. Our preliminary data support our hypothesis in that the addition to Hsc70 facilitates the formation of the SNARE complex. Our goal is to elucidate in detail the molecular interactions of Hsc70 and CSPɑ with the SNARE complex components, notably SNAP‐25, in the formation of the SNARE complex and the initial stages of synaptic vesicle fusion.