A charged prominence in the linker domain of the cysteine-string protein Cspα mediates its regulated interaction with the calcium sensor synaptotagmin 9 during exocytosis.

The cochaperone cysteine-string protein (Csp) is located on vesicles and participates in the control of neurotransmission and hormone exocytosis. Csp contains several domains, and our previous work demonstrated the requirement of the Csp linker domain in regulated exocytosis of insulin in rodent pancreatic β cells. We now address the molecular details to gain insight into the sequence of events during exocytosis. According to pulldown experiments and in vitro binding assays, Cspα interacts indirectly with SNAP-25 and directly with the calcium sensor synaptotagmin 9 (Syt9), which could be an intermediate between the chaperone and the t-SNARE. The C(2)A calcium binding domain of Syt9 and the linker domain of Cspα constituted the minimal interacting module. FRET-FLIM experiments confirmed the interaction between Syt9 and Cspα. Moreover, the point mutation E93V in the linker domain of Cspα significantly reduced the interaction between the two proteins. Molecular modeling revealed that this point mutation abolished a charged prominence on the surface of Cspα required for interaction. Strikingly, free calcium in the physiological low micromolar range enhanced the interaction between Syt9 and the linker domain of Cspα in vitro. These data indicate that Cspα interacts with Syt9, and such a complex may be relevant in the calcium-mediated control of a late stage of exocytosis by triggering the specific recruitment of a folding catalyst at the fusion point.