An eGFP-based genetic screen for defects in light-triggered subcelluar  translocation of the Drosophila photoreceptor channel TRPL.
Signaling at the plasma membrane is modulated by up- and downregulation of signaling proteins. A prominent example for this type of regulation is the Drosophila TRPL ion channel that changes its spatial distribution within the photoreceptor cell. In dark-raised flies TRPL is localized in the rhabdomeral photoreceptor membrane and it translocates to the cell body upon illumination. It has been shown that TRPL translocation depends on the activation of the phototransduction cascade and requires the presence of functional rhodopsin as well as Ca2+-influx through a second lightactivated ion channel, TRP. However, little is known about the cell biological mechanism underlying TRPL translocation. Here we describe a FRT/FLP screen designed to isolate mutants defective in TRPL internalization based on the localization of eGFP-tagged TRPL in the eyes of living flies. We mutated chromosome arms 2L, 2R and 3R and isolated 12 mutants that failed to internalize TRPL. We found that four mutants did not complement genes known to affect TRPL translocation, which are trp, ninaE and inaD. Two of the isolated mutants represent new alleles of trp and ninaE. The trp allele contains a premature stop codon after amino acid 884, whereas the ninaE allele has a mutation resulting in the substitution P193S. As determined biochemically no TRP or rhodopsin protein, respectively, was expressed in the eyes of these mutants. The absence of TRP or rhodopsin in the isolated mutants readily explains the defect in TRPL internalization and proves the feasibility of our genetic screen.
Meyer
Nina E
NE
University of Hohenheim, Institute of Physiology, Department of Biosensorics, Stuttgart, Germany.
Oberegelsbacher
Claudia
C
Dürr
Thomas D
TD
Schäfer
Andrea
A
Huber
Armin
A
D000818:Animals; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism*; D005816:Genetic Complementation Test; D005820:Genetic Testing / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D008027:Light; D010641:Phenotype; D021381:Protein Transport / Q000235:genetics; D050051:Transient Receptor Potential Channels / Q000235:genetics* / Q000378:metabolism*
D029721:Drosophila Proteins; D050051:Transient Receptor Potential Channels; C074192:trpl protein, Drosophila; D049452:Green Fluorescent Proteins