Cysteine-string proteins (Csps) are vesicle proteins involved in neurotransmission. They contain at least four domains: an N-terminal J-domain which can interact with the chaperone Hsc70, an adjacent linker region, the defining cysteine rich domain and a variable C terminus. As the relevance of these domains for the function of Csps in exocytosis is unknown, we have performed a mutational analysis of Csp domains using insulin release by large dense core vesicles (LDCVs) as a model of regulated exocytosis. All mutants were apparently palmitoylated and their subcellular distribution was similar to endogenous Csp. Point mutations within the highly conserved HPD motif of the J-domain abolished activation of Hsc70. However, these mutations altered the effect of Csp on exocytosis only after additional truncation of the extreme C terminus as found in the Csp splice variant Csp2. Furthermore, the strikingly conserved linker region adjacent to the J-domain was important for Csp function in exocytosis, but not for the activation of Hsc70 ATPase. The effects of Csp wild-type or mutants were preserved in permeabilized cells excluding an effect on transmembrane ion fluxes. These observations demonstrate a functional difference between the two isoforms and suggest a role for the J-domain co-chaperone function as well as for the newly defined linker region in LDCV exocytosis.
Mutational analysis of cysteine-string protein function in insulin exocytosis
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H. Zhang, W.L. Kelley, L.H. Chamberlain, R.D. Burgoyne, J. Lang; Mutational analysis of cysteine-string protein function in insulin exocytosis. J Cell Sci 1 May 1999; 112 (9): 1345–1351. doi: https://doi.org/10.1242/jcs.112.9.1345
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