Overexpression of Cysteine-String Proteins inDrosophilaReveals Interactions with Syntaxin

Abstract

Cysteine-string proteins (CSPs) are associated with secretory vesicles and critical for regulated neurotransmitter release and peptide exocytosis. At nerve terminals, CSPs have been implicated in the mediation of neurotransmitter exocytosis by modulating presynaptic calcium channels; however, studies of CSPs in peptidergic secretion suggest a direct role in exocytosis independent of calcium transmembrane fluxes. Here we show that the individual expression of various CSP isoforms in Drosophila similarly rescues the loss of evoked neurotransmitter release at csp null mutant motor nerve terminals, suggesting widely overlapping functions for each isoform. Thus, the structural difference of CSP variants may not explain the opposing putative functions of CSP in neurotransmitter and peptide exocytosis. Consistently, the individual overexpression of each CSP isoform in wild-type Drosophila shows similar effects such as impaired viability and interference with wing and eye development. The dominant effects caused by the overexpression of CSP are suppressed by the simultaneous overexpression of syntaxin-1A but not by the coexpression of SNAP-25. Although overexpression of CSP itself has no apparent effect on the synaptic physiology of larval motor nerve terminals, it fully suppresses the decrease of evoked release induced by the overexpression of syntaxin-1A. A direct protein-protein interaction of CSP with syntaxin is further supported by coimmunoprecipitations of syntaxin with CSP and by protein binding assays using recombinant fusion proteins. Together, the genetic and biochemical interactions of CSP and syntaxin-1A suggest that CSP may chaperone or modulate protein-protein interactions of syntaxin-1A with either calcium channels or other components of the regulatory machinery mediating depolarization-dependent neurotransmitter exocytosis.

Genetic Analysis of SolubleN-Ethylmaleimide-Sensitive Factor Attachment Protein Function inDrosophilaReveals Positive and Negative Secretory Roles

“…7A). This phenotype is indicative of reduced neurohormonal secretion, and similar phenotypes have been observed after expression of neurotoxic proteins using the elav C155 -GAL4 driver (Nie et al, 1999). Overexpression of SNAP in the compound eye using the GMR-GAL4 driver in conjunction with the UAS-SNAP-1 transgene results in mildly rough eyes containing patches of necrotic tissue (Fig.…”

Molecular determinants of cysteine string protein modulation of N-type calcium channels

“…Although CSP has been proposed to play a role in the recruitment of calcium channels (Chen et al, 2002), CSP has also been proposed to play an inhibitory role in depolarization dependent calcium entry (Dawson-Scully et al, 2002), which is consistent with our results . In addition to G proteins and calcium channels (Chen et al, 2002;Leveque et al, 1998;Wu et al, 1999;Magga et al, 2000), several other targets of CSP chaperone activity have been proposed including syntaxin (Nie et al, 1999;Evans et al, 2001;Wu et al, 1999), VAMP (also called synaptobrevin) (Leveque et al, 1998), synaptotagmin I (Evans and Morgan, 2002), αGDI (Sakisaka et al, 2002) and CFTR (Zhang et al, 2002). Further experimentation is required to establish the role of CSP in controlling the aggregation state and conformation of these possible target proteins and the nature of the conflicting results regarding the role of CSP in exocytosis.…”

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…112 In addition, CSP may enhance channel function via a recruitment of channels to the plasma membrane. 113 CSP also indirectly regulates channel activity by virtue of its competition with syntaxin 1A for the channel, 114,115 and perhaps by its direct interactions with syntaxin 1A.…”