A forward genetic screen in Drosophila melanogaster to identify mutations affecting INAD localization in photoreceptor cells

Abstract

In Drosophila photoreceptors, the multivalent PDZ protein INAD interacts with multiple signaling components and localizes complexes to the rhabdomere, a subcellular compartment specialized for phototransduction. Since this localization is critical for signaling, we conducted a genetic screen of the third chromosome for mutations that result in mislocalization of an INAD-GFP fusion protein. We identified seven mutant lines that fall into two complementation groups, idl (INAD localization) -A and idl-B. We show that idl-A mutants fail to complement with chaoptic (chp) mutants. Since chaoptin is a structural component of the rhabdomere, mislocalization of INAD may be a secondary effect of the retinal degeneration in chp and idl-A mutants. Genetic complementation and DNA sequencing reveal that the two idl-B mutants represent new alleles of trp, a gene encoding the major light-activated channel. The molecular change in each allele affects a highly conserved residue in either an ankyrin domain on the N-terminus or in the S6 transmembrane domain of TRP. These changes lead to the loss of TRP protein. TRP has previously been shown to anchor INAD in the rhabdomeres, therefore the independent identification of two trp alleles validates our screen for INAD-GFP localization. One possibility is that a limited number of proteins are required for localizing INAD-signaling complexes. A similar screen of the X and second chromosomes may be required to find the remaining players involved.