IUScholarWorks at Indiana University South Bend Retinophilin Is a Light-Regulated Phosphoprotein Required to Suppress Photoreceptor Dark Noise in Drosophila

a Light-Regulated Required to Suppress Photoreceptor Dark Noise in Photoreceptor cells achieve high sensitivity, reliably detecting single photons, while limiting the spontaneous activation events responsible for dark noise. We used proteomic, genetic, and electrophysiological approaches to characterize Retinophilin (RTP) (CG10233) in Drosophila photoreceptors and establish its involvement in dark-noise suppression. RTP possesses membrane occupation and recognition nexus (MORN) motifs, a structure shared with mammalian junctophilins and other membrane-associated proteins found within excitable cells. We show the MORN repeats, and both the N- and C-terminal domains, are required for RTP localization in the microvillar light-gathering organelle, the rhabdomere. RTP exists in multiple phosphorylated isoforms under dark conditions and is dephosphorylated by light exposure. An RTP deletion mutant exhibits a high rate of spontaneous membrane depolarization events in dark conditions butretainsthenormalkineticsofthelightresponse.PhotoreceptorslackingneitherinactivationnorafterpotentialC(NINAC)myosinIII,amotorprotein/kinase,alsodisplayasimilardark-noisephenotypeastheRTPdeletion.WeshowthatNINACmutantsaredepletedforRTP.TheseresultssuggesttheincreaseindarknoiseinNINACmutantsisattributabletolackofRTPand,furthermore,definesanovelroleforNINACintherhabdomere.WeproposethatRTPisalight-regulatedphosphoproteinthatorganizesrhabdomericcomponentstosuppressrandomactivationofthephototransductioncascadeandthusincreasesthesignalingfidelityofdark-adaptedphotoreceptors. staining was seen in the light-adapted sample, and the phosphorylated (cid:3) spot is greatly reduced. E , The RTP region of dark-adapted and light-adaptedfliesinCBB-stainedtwo-dimensionalgel(leftpanels)and 32 Pincorporation(rightpanels)fromtheanalysis performedbyMatsumotoandPak(1984).Themarked (cid:1) spotinthedark-adaptedsampleshowedthehighestlevelof 32 P labeling. Although the 25-year-old experiment showed more limited resolution of the spots than possible with current technology, both results show that RTP phosphorylation is reduced in the light-treated samples. F , PMF analysis of the dark-adapted (cid:1) spotfromthearchivedtwo-dimensionalgelshownin E .Thelabeledtrypticfragmentswerederivedfrom RTP (as identified in C ), demonstrating that the major protein present in the 32 P-labeled (cid:1) spot was RTP.

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