Mutational analysis of cysteine-string protein function in insulin exocytosis.
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.
Zhang
H
H
Division de Biochimie Clinique, Département de Médecine Interne, and Département de Biochemie Médicale, Centre Médicale Universitaire, CH 1211 Genève 4, Switzerland.
Kelley
W L
WL
Chamberlain
L H
LH
Burgoyne
R D
RD
Lang
J
J
D002478:Cells, Cultured; D005089:Exocytosis / Q000502:physiology*; D050956:HSP40 Heat-Shock Proteins; D006801:Humans; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D008565:Membrane Proteins / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics*; D017354:Point Mutation; D013347:Subcellular Fractions / Q000737:chemistry
D050956:HSP40 Heat-Shock Proteins; D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C088460:cysteine string protein