ISOLATION OF GENES ENCODING PHOTORECEPTOR-SPECIFIC PROTEINS BY IMMUNOSCREENING WITH ANTIBODIES DIRECTED AGAINST PURIFIED BLOWFLY RHABDOMS

The proteins which perform and regulate key steps in phototransduction are assumed to be localized in the rhabdomeric membrane of invertebra te photoreceptor cells. We have employed antibodies raised against rha bdoms purified from blowfly eyes in order to isolate copy deoxyribonuc leic acid (cDNA) clones encoding proteins that are required in the pho totransduction machinery. By immunoscreening a Calliphora retinal cDNA library, we obtained clones of genes coding for five different protei ns. As revealed by partial cDNA sequence analysis, three of these gene s represent the Calliphora homologs of Drosophila trp, inaC and InaD, while the other two displayed no homology to known genes. Northern blo t analysis confirmed that trp, inaC and InaD transcripts were present in RNA isolated from the retina, but not in RNA isolated from brain or thorax, Specific antibodies directed against trp, inaC and InaD prote in were raised using recombinantly expressed proteins or synthetic pep tides. Western blot analyses revealed that tip, inaC and InaD protein are specifically associated with the rhabdomeral photoreceptor membran e, Extraction of membranes with buffers of different ionic strengths s uggested that the trp gene product is an integral membrane protein, wh ilst the inaC and InaD gene products are peripherally bound membrane p roteins. This demonstrates that the immunoscreening approach used here can be successfully applied to isolate genes that code for either int egral or peripheral photoreceptor membrane proteins.