The loss of <i>Osi21</i> function facilitates rhodopsin degradation by modulating the endo-lysosomal membrane dynamics in light-induced <i>norpA^p24</i> photoreceptors.

<p>(A–B) Rate of rhodopsin endocytosis and degradation. Movement of light-pulsed RFP-tagged rhodopsin from the rhabdomere was examined by confocal microscopy. Observed photoreceptors were categorized according to the Rh1 localization. (A) Photoreceptor types. Type I (Rh1-RFP is localized in the rhabdomere), Type II (Rh1-RFP is localized in the rhabdomere as well as cytoplasm), Type III (Rh1-RFP is localized in the cytoplasm) and Type IV (Rh1-RFP disappears due to degradation). (B) Percentage of each typed photoreceptor cells was calculated at 24 h intervals for 96 h. In each time point, approximately 30 photoreceptor cells of each genotype from five individuals were examined. The initial movement of endocytosed rhodopsin toward the endosomal system (Type I and the Type II) was not different between the <i>norpA^P24</i> and <i>norpA^P24</i>; <i>Osi21-RNAi</i> photoreceptor. However, facilitated clearance of endocytosed rhodopsin (Type IV) in <i>norpA^P24</i> mutant photoreceptor cells with the <i>Osi21-RNAi</i> transgene was observed. Error bars indicate the SEM. p<0.01 (the Kolmogorov-Smirnov test). (C) The loss of <i>Osi21</i> function in <i>norpA^p24</i> flies leads to the reduced rhodopsin. Flies were reared either in the dark to prevent rhodopsin endocytosis or in the 18 h light/8 h dark cycles to stimulate rhodopsin endocytosis, and were collected within 12 h after eclosion. Western blot analyses were performed to measure the effect of the <i>Osi21</i> functional loss on the degradation of endocytosed rhodopsin. Relative Rh1 level was calculated from triplicated immunoblots. Data are shown as the mean ±SE. ***p<0.01. <i>w¹¹¹⁸</i> (lane1), <i>norpA^p24; Rh1::Gal4/+</i> (dark-reared, lane2) , <i>norpA^p24; Rh1::Gal4/+; UAS:: Osi21-RNAi/+</i> (dark-reared, lane3) , <i>norpA^p24; Rh1::Gal4/+</i> (18 h light/8 h dark, lane4) , <i>norpA^p24; Rh1::Gal4/+; UAS:: Osi21-RNAi/+</i> (18 h light/8 h dark, lane5). (D) Overexpression of Rab7 synergistically affects rhodopsin degradation with the loss of <i>Osi21</i> function. Newly eclosed flies were exposed to bright light (2900 lux) for 48 h before being sacrificed to induce the maximal rhodopsin endocytosis. Western blot analyses were performed to measure the effect of the <i>Osi21</i> functional loss on the degradation of endocytosed rhodopsin in response to the activation of the endosomal trafficking machinery. Relative Rh1 level was calculated from triplicated immunoblots. Data are shown as the mean ±SE. ***p<0.01. <i>norpA^p24; Rh1::Gal4, UAS:: YFP-Rab5/+</i> (lane1) , <i>norpA^p24; Rh1::Gal4, UAS:: YFP-Rab5/+; UAS:: Osi21-RNAi/+</i> (lane2) , <i>norpA^p24; Rh1::Gal4, UAS:: YFP-Rab7/+</i> (lane3) , <i>norpA^p24; Rh1::Gal4, UAS:: YFP-Rab7/+; UAS:: Osi21-RNAi/+</i> (lane4).