The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.
- protein kinase A
- protein phosphorylation
Abbreviations used: CSP, cysteine string protein; Hsc70, heat-shock cognate protein of 70kDa; PKA, cAMP-dependent protein kinase; GST, glutathione S-transferase; S10A, CSP mutant bearing the site-directed replacement of Ser10 with alanine; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor.
- The Biochemical Society, London ©2002