Abstract
Ca2+ influxes regulate multiple events in photoreceptor cells including phototransduction and synaptic transmission. An important Ca2+ sensor in Drosophila vision appears to be calmodulin since a reduction in levels of retinal calmodulin causes defects in adaptation and termination of the photoresponse. These functions of calmodulin appear to be mediated, at least in part, by four previously identified calmodulin-binding proteins: the TRP and TRPL ion channels, NINAC and INAD. To identify additional calmodulin-binding proteins that may function in phototransduction and/or synaptic transmission, we conducted a screen for retinal calmodulin-binding proteins. We found eight additional calmodulin-binding proteins that were expressed in the Drosophila retina. These included six targets that were related to proteins implicated in synaptic transmission. Among these six were a homolog of the diacylglycerol-binding protein, UNC13, and a protein, CRAG, related to Rab3 GTPase exchange proteins. Two other calmodulin-binding proteins included Pollux, a protein with similarity to a portion of a yeast Rab GTPase activating protein, and Calossin, an enormous protein of unknown function conserved throughout animal phylogeny. Thus, it appears that calmodulin functions as a Ca2+ sensor for a broad diversity of retinal proteins, some of which are implicated in synaptic transmission.
Highlights
Prior to the current screen, five calmodulin-binding proteins were known to be expressed in the Drosophila retina, four of which, NINAC, TRP, TRPL and INAD, function in phototransduction
Concluding Remarks—With the identification of the calmodulin-binding proteins in the current screen, there exists a minimum of 13 targets for calmodulin in the Drosophila retina
Each of the calmodulin-binding proteins was enriched in the retina
Summary
Isolation of Clones Encoding Calmodulin-binding Proteins—The retinal expression library used to screen for calmodulin-binding clones was prepared using poly(A)ϩ RNA isolated from adult fly retinas (ZAP library; gift from Charles Zuker, UCSD; cDNAs were non-directionally inserted into the EcoRI site). This was not a subtracted retinal-specific library. To obtain addition cDNA sequences encoding the remaining five (CaM kinase I, CRAG, PLX, dUNC13, and CALO), the retinal ZAP library and an adult head ZAP library were rescreened with DNA probes. PPollux.[3] was generated by cloning the original plx isolate, dm[265], into the XhoI/EcoRI sites of pGEX-5X-3 and digesting with BamHI and religating. The induced bacterial cultures were French-pressed, pelleted by centrifugation, and 100-l supernatants (corresponding to 1 ml of the original culture) were incubated with 50 l
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