Structural features and sequence of the OSI21 protein.

<p>The 282 amino acid long OSI21 protein has at least four distinct motifs/domains, including an endosome/lysosome sorting signal (blue), a 2-Cys region (red), a DUF1676 domain (green), and a YXXØ motif (yellow). Sequencing analysis identified three significant amino acid changes (checked blue) in DIE4, presumably causing a functional loss of <i>Osi21</i>.</p

The loss of <i>Osi21</i> function suppresses <i>rdgC³⁰⁶</i>, <i>trp¹</i>, but not <i>rdgB²</i>.

<p>The suppressive effect of <i>Osi21</i> loss was examined in deep pseudopupil (DPP) (A, D, G) and electron micrograph level (B–C, E–F). For DPP analysis, newly eclosed flies were exposed to 2900 lux of constant light in the 25°C incubator. Maintenance of DPP was scored each day. The fraction of flies with DPP was plotted using 100 flies of each genotype in triplicate. Data are shown as the mean ±SE. (A) <i>rdgC³⁰⁶</i> and <i>die4; rdgC³⁰⁶</i>, (D) <i>trp¹</i> and <i>die4; trp¹</i>, and (G) <i>rdgB²</i> and <i>rdgB²; die4</i>. For electron micrograph, flies in each genotype were exposed to 2900 lux of constant light in the 25°C incubator. Four-day old <i>rdgC³⁰⁶</i> (B) and <i>die4; rdgC³⁰⁶</i> (C), Eight-day old <i>trp¹</i> (E) and <i>die4; trp¹</i> (F) were sacrificed for analysis. Compared to the completely deformed ommatidial structure of single mutants, double mutants showed well preserved rhabdomeric and ommatidial structures.</p

Molecular identification of <i>die4</i>.

<p>(A–F) Morphology of sectioned ommatidium exposed to constant light was observed by electron microscopy. The <i>norpA^P24</i> flies showed progressive retinal degeneration upon exposure to constant light (A–B). The loss of <i>Osi21</i> function by specific knock-down by RNAi rescued retinal degeneration caused by <i>norpA^P24</i> (C). Compared Exel6028 heterozygote (D), retinal degeneration in <i>norpA^P24</i> flies was also rescued by generating transheterozygote flies with genomics deficiency Exel6028 and <i>die4</i> (E). Wild-type (Canton-S) flies showed no sign of retinal degeneration upon extended light exposure (F). (G) Newly eclosed flies reared under constant light. The fraction of flies with deep pseudopupil was plotted using approximately 20 flies, in triplicate, of <i>norpA^P24</i>, <i>norpA^P24</i>; <i>die4</i>, <i>norpA^P24</i>; <i>die4/Exel6028</i>, <i>norpA^P24</i>; <i>die4/Mi{ET1}^Osi21</i>, <i>norpA^P24</i>; <i>die4/Mi{ET1}^Osi21</i>; <i>[Osi21]⁺</i>. The <i>die4/Mi{ET1}^Osi21</i> transheterozygote flies were protected from <i>norpA^P24</i> degeneration as <i>die4/Exel6028</i> and the protection was reverted by the introduction of the genomic fragment encompassing the <i>Osi21</i> gene. Data are shown as the mean ±SE in triplicate. (A) <i>w, norpA^P24; Rh1::Gal4/+</i>, 3 days, (B) <i>w, norpA^P24; Rh1::Gal4/+</i>, 4 days, (C) <i>w, norpA^P24; Rh1::Gal4/UAS::Osi21-RNAi</i> , 4 days, (D) <i>w, norpA^P24; Exel6028/Cy</i>, 4 days, (E) <i>w, norpA^P24; die4/Exel6028</i>, 4 days, (F) Canton-S, 7 days. Light intensity, 2900 lux, Scale bar, 1 µm.</p

Negative Regulation of the Novel <i>norpA^P24</i> Suppressor, <i>diehard4</i>, in the Endo-lysosomal Trafficking Underlies Photoreceptor Cell Degeneration

<div><p>Rhodopsin has been used as a prototype system to investigate G protein-coupled receptor (GPCR) internalization and endocytic sorting mechanisms. Failure of rhodopsin recycling upon light activation results in various degenerative retinal diseases. Accumulation of internalized rhodopsin in late endosomes and the impairment of its lysosomal degradation are associated with unregulated cell death that occurs in dystrophies. However, the molecular basis of rhodopsin accumulation remains elusive. We found that the novel <i>norpA^P24</i> suppressor, <i>diehard4</i>, is responsible for the inability of endo-lysosomal rhodopsin trafficking and retinal degeneration in <i>Drosophila</i> models of retinal dystrophies. We found that <i>diehard4</i> encodes <i>Osiris 21</i>. Loss of its function suppresses retinal degeneration in <i>norpA^P24</i>, <i>rdgC³⁰⁶</i>, and <i>trp¹</i>, but not in <i>rdgB²</i>, suggesting a common cause of photoreceptor death. In addition, the loss of <i>Osiris 21</i> function shifts the membrane balance between late endosomes and lysosomes as evidenced by smaller late endosomes and the proliferation of lysosomal compartments, thus facilitating the degradation of endocytosed rhodopsin. Our results demonstrate the existence of negative regulation in vesicular traffic between endosomes and lysosomes. We anticipate that the identification of additional components and an in-depth description of this specific molecular machinery will aid in therapeutic interventions of various retinal dystrophies and GPCR-related human diseases.</p></div

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).</p

OSI21 partially localizes on the endo-lysosomal system.

<p>Newly eclosed flies were exposed to bright light (2900 lux) for 90 min. A single ommatidium prepared from fly retina was visualized by confocal microscopy. (A–B) The OSI21 protein was marked with GFP. Each subcellular structure was marked with Lysotracker -Red (lysosomes, A) and Rhodopsin-RFP (endosomes, B). OSI21 partially localizes on lysosomes (Pearson's correlation coefficient: 0.617, A). OSI21 partially localizes on the rhodopsin positive vesicles (Pearson's correlation coefficient: 0.635, B). (A) <i>w; Rhi1::Gal4, UAS::Osi21-GFP/+</i> (B) <i>w; Rhi1::Gal4, UAS::Osi21-GFP/+; UAS::Rh1-RFP/+</i>. (arrowheads) lysosomes or endosomes colocalized with OSI21-GFP. Flies were kept in the 25°C fly culture room, 18 h light/8 h dark cycles. (C–D) Localization of endocytosed rhodopsin with lysosomes. Newly eclosed flies were exposed to bright light (2900 lux) for 90 min. A single ommatidium was prepared from fly retina and examined using confocal microscopy. GFP-labeled rhodopsin and Lysotracker -red were used for visualizing rhodopsin endocytosis and lysosomes. Lysosomes (red) in <i>norpA^p24</i> photoreceptor cells are smaller in number and are not overlapped with endocytosed rhodopsin (green) (Pearson's correlation coefficient: 0.342, C). (Arrowhead in C) Endocytosed rhodopsin escaped <i>Osi21</i> blockage, reflecting regular lysosomal rhodopsin-turnover. On the other hand, lysosomes are greatly proliferated and are colocalized with endocytosed rhodopsin in the <i>norpA^p24</i> mutant photoreceptor with a <i>Osi21-RNAi</i> transgene (Pearson's correlation coefficient: 0.604, D). (Arrowheads) Lysosomes collocalized with endocytosed rhodopsin. (C) <i>norpA^p24; Rh1::Gal4, UAS::Rh1-GFP/+</i> (D) <i>norpA^p24; Rh1::Gal4, UAS::Rh1-GFP/UAS::Osi21-RNAi</i>. (E) Relative number of green-labeled vesicles among red-labeled population was calculated from three representative photoreceptor cells of each genotype in triplicate. Error bars indicate SEM. ***p<0.01, **p<0.05.</p

The loss of <i>Osi21</i> function modulates the membrane balance of the endo-lysosomal system.

<p>A single ommatidium was prepared from newly eclosed fly retina and was visualized by confocal microscopy. Each subcellular structure was marked with YFP-Rab5 (early endosomes, A–B), GFP-Rab6 (Golgi complexes, C–D), YFP-Rab7 (late endosomes, E–G), and Lysotracker (lysosomes, H–J). (A–B) Compared to the wild-type photoreceptor cell (A), the <i>Osi21</i> knock-down photoreceptor (B) showed slightly reduced levels of Rab5-positive vesicles (early endosomes) both in size and number. (C–D) No significant differences in Rab6-positive vesicles (Golgi complexes) were observed between the wild-type photoreceptor (C) and the <i>Osi21</i> knock-down photoreceptor (D) both in size and number. (E–G) Compared to the wild-type photoreceptor cell (E, F), the <i>Osi21</i> knock-down photoreceptor (G) showed greatly reduced levels of Rab7-positive vesicles (late endosomes) both in size and number. (H–J) Compared to the wild-type photoreceptor cell (H), the <i>Osi21</i> loss-of-function photoreceptor showed proliferated Lysotracker staining (lysosomes) (I–J). Significant difference only in lysosomal number, but not size, between the wild-type (H) and the <i>Osi21</i> loss-of-function photoreceptor (I–J) was observed. (K) Total area marked with the vesicles was quantified from three representative photoreceptor cells of each genotype in triplicate. Error bars indicate SEM. ***p<0.01. (A) <i>w; Rhi1::Gal4, UAS::YFP-Rab5/+</i>, (B) <i>w; Rhi1::Gal4, UAS::YFP-Rab5/+; UAS::Osi21-RNAi/+</i>, (C) <i>w; Rhi1::Gal4, UAS::YFP-Rab6/+</i>, (D) <i>w; Rhi1::Gal4, UAS::YFP-Rab6/+; UAS::Osi21-RNAi/+</i>, (E) <i>w; Rhi1::Gal4, UAS::YFP-Rab7/+</i>, (F) <i>w; Rhi1::Gal4, UAS::YFP-Rab7/+; UAS::RFP-arf72A/+</i>, (G) <i>w; Rhi1::Gal4, UAS::YFP-Rab7/+; UAS::Osi21-RNAi/+</i>, (C) <i>w</i>, (D) <i>die4/Exel6028</i>, (E) <i>Rh1:Gal4/UAS::Osi21-RNAi/+</i>. (arrows) Rab7-positive late endosomes (arrowheads) lysosomes. Flies were kept in the fly culture room, maintaining 25°C, 18 h light/8 h dark cycles.</p

A model for Osi21 function on photoreceptor degeneration in phototransduction mutants.

<p>(Left) In the wild-type photoreceptor, light-activated rhodopsin initiates phototransduction, relays the signal through PLC_β (<i>norpA</i>), and induces Ca²⁺ influx through the cation-specific calcium channel (<i>trp</i>). On the other hand, rhodopsin kinase (RK) rapidly phosphorylates the activated rhodopsin, enabling arrestin (Arr2) binding for inactivation. The Rh1-Arr2 complex requires a specific calcium-dependent phosphatase (rdgC) activity for dissociation. Stable rhodopsin-arrestin complex due to the lack of Ca²⁺ influx results in massive endocytosis, presumably underlying retinal degeneration in the <i>norpA^p24</i>, <i>rdgC³⁰⁶</i>, and <i>trp¹</i> mutant photoreceptor. (Right) In <i>norpA^p24</i> photoreceptors, the formation of stable rhodopsin-arrestin complexes results in its massive endocytosis and accumulation of endocytosed rhodopsin in late endosomes. <i>Osi21</i>may result in the inability of rhodopsin transport to the lysosome for degradation by negatively regulating endo-lysosomal flow.</p

Underlying numerical data of growth rate test of <i>E</i>. <i>coli</i>.

(XLSX)</p

Protein–protein docking to predict the 3D protein structure of AbHeR and AbGS.

AbHeR and AbGS are indicated as transparent cyan and red helices, respectively. The distances between the hydrogen bonds of interacting amino acids of AbHeR and AbGS were calculated by polar interaction tool in PyMOL and indicated using a yellow dotted line. The key active sites of AbGS are present in the 2 AbGS monomers of dodecamer, and the positions of the key active sites of the 2 different monomers in AbGS are indicated in yellow and white text. The positions of amino acids in AbHeR are indicated in blue text. (A, D, and E) Docking parts of key active sites in AbGS and AbHeR. (B and C) GS 3D structure (PDB: 6su3.1.A) in a position with bound Glu. (D and E) The positions of the docking prediction are indicated with white dotted circles. (B and D) Above and (C and E) top views of the positions are shown. (TIF)</p