Negative Regulation of the Novel norpAP24 Suppressor, diehard4, in the Endo-lysosomal Trafficking Underlies Photoreceptor Cell Degeneration

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 norpAP24 suppressor, diehard4, is responsible for the inability of endo-lysosomal rhodopsin trafficking and retinal degeneration in Drosophila models of retinal dystrophies. We found that diehard4 encodes Osiris 21. Loss of its function suppresses retinal degeneration in norpAP24, rdgC306, and trp1, but not in rdgB2, suggesting a common cause of photoreceptor death. In addition, the loss of Osiris 21 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.


Introduction
Retinitis pigmentosa is the most common form of retinal degeneration and the major cause of human blindness [1]. Most degenerative retinal dystrophies are caused by various genetic mutations. Malfunctioning of phototransduction is the predominant cause of retinal dystrophies, in that most genes involved in the functioning and regulation of the phototransduction cascade are directly or indirectly related to retinal degeneration syndromes [2,3]. Therefore, it is not surprising that rhodopsin-1, the major visual pigment, was the first molecule identified as a target for such mutations [4,5].
Drosophila norpA (phospholipase C, PLC) acts as a central effector molecule in phototransduction [6]. It has been used as an invertebrate model for studying molecular mechanisms of retinal degeneration caused by malfunctioning of the phototransduction cascade [7]. Interestingly, cGMP phosphodiesterase, which relays the signal from G-proteins in vertebrate phototransduction, is also known to trigger retinal degeneration in mouse models [8][9][10]. The loss of norpA function essentially shuts down the phototransduction cascade, resulting in a failure to raise intracellular Ca 2+ levels through light-sensitive channels. Thus, Ca 2+ -dependent enzymes required for rhodopsin recycling cannot be activated, resulting in the formation of the stable rhodopsinarrestin complex [11][12][13][14]. It has been reported that excessive endocytosis followed by the formation of stable rhodopsin-arrestin complexes and accumulation of internalized rhodopsin in late endosomes trigger apoptosis in norpA mutant photoreceptor cells [12].
The ''granule group'' genes in Drosophila have been known for their vital role in lysosomal biogenesis and functioning [15,16]. A previous study found that the functional loss of the ''granule group'' genes resulted in rhodopsin accumulation in the Rab7positive late endosomes and triggered retinal degeneration in norpA mutant photoreceptor cells [12,17]. Therefore, the accumulation of internalized rhodopsin in late endosomes and impaired endolysosomal trafficking clearly causes retinal degeneration in both the norpA and the ''granule group'' mutant photoreceptors. However, the molecular basis of this pathologic accumulation remains unknown.
The role of excessive endocytosis of light-activated rhodopsin on saturating the capacity of the trafficking machinery for the endolysosomal progression, resulting in the accumulation of endocytosed rhodopsin in the late endosomes remains controversial. Alternatively, previously unknown regulatory mechanisms prevent endocytosed rhodopsin from further movement toward lysosome. A growing number of evidences support the fact that the eukaryotic cell utilizes active regulatory mechanisms in monitoring and maintaining the intracellular membrane balance of the endolysosomal system [18][19][20][21]. Therefore, it is imperative to identify genetic components underlying rhodopsin accumulation and present epistatic evidences that possibly override the endolysosomal blockage in phototransduction mutants.
Triplo-lethal (Tpl) locus, cytologically defined as the 83D4-E2 region in chromosome 3 in Drosophila, was identified as a sole locus responsible for both triplo-lethality and haplo-lethalith in segmental aneuploids [22]. Point mutations responsible for the Tpl phenotype remain unidentified [23], although the Ell product, a transcription elongation factor, was found to be a suppressor of the Tpl phenotype [24]. Therefore, it is proposed that this phenotype is caused by a gene cluster that shows at least partial redundancy and its dosage is critical to its function [25].
The Osiris gene family was identified in an effort to explain the Tpl phenotype as an effect of a gene cluster. This is a large conserved family, with most genes (20 of 23) located within the Tpl locus [26]. Although the cellular function of the Osiris family of proteins is currently unknown, all members share characteristic features, including endoplasmic reticulum signal sequences, a pair of cysteine residues near the amino terminus, a putative transmembrane domain, an AQXLAY motif, and a number of endocytic signaling motifs such as YXXØ [26,27].
Previously, we screened for norpA P24 suppressors by random mutagenesis. The screening had the advantage of the yeast sitespecific recombination FLP-FRT system and could identify both essential and nonessential genes [28]. Here we report that the novel norpA P24 suppressor, diehard4 (die4), is responsible for the inability of endo-lysosomal rhodopsin trafficking and retinal degeneration in norpA P24 mutants. We found that die4 encodes Osiris 21 (Osi21). A loss of function of Osi21 suppresses retinal degeneration in various phototransduction mutants. In addition, the loss of function shifts the membrane balance between endosomes and lysosomes, resulting in the facilitated degradation of endocytosed rhodopsin. Our results demonstrate that the existence of negative regulation in vesicular traffic between endosomes and lysosomes. This mechanism may trigger retinal degeneration in phototransduction mutants.

Results
The Novel norpA P24 Suppressor, die4, Encodes Osiris 21 Drosophila norpA encodes eye-specific phospholipase C and acts as a central effector in phototransduction [6]. The norpA photoreceptor has been used as a model system for studying progressive retinal dystrophies in humans because the loss of its function leads to rapid light-dependent retinal degeneration as a result of excessive endocytosis of stable rhodopsin-arrestin complexes and accumulation of internalized rhodopsin in late endosomes [11,12,14]. Previous studies [29] have shown that the norpA P24 (a strong hypomorphic allele of norpA [30]) photoreceptor showed progressive retinal degeneration ( Figure 1A-B). Its degenerative phenotype appeared within three days and was obvious within four days upon constant light exposure. We found that wild-type (Canton-S) flies showed no sign of retinal degeneration even after seven days of constant light exposure ( Figure 1F), indicating that norpA P24 degeneration was strongly dependent on light.
The die4 mutant was previously identified as a norpA P24 suppressor from a genetic screen by using eye-specific FLP-FRT mosaic flies [31], delaying degeneration several days ( Figure 1G). The die4 mutant was generated using ethyl methanesulfonate (EMS) mutagenesis, possibly bearing multiple mutations. In addition, mosaic screening enables identification of both lethal and non-lethal mutations. Because of these complexities, we used multiple mapping methods to identify the exact mutation responsible for the suppressive phenotype of the die4 mutant. We previously reported that the mutation in the cytological region of 32D5 to E4 of the die4 chromosome, is responsible for the suppressive phenotype [31]. Although the die4 chromosome is homozygous lethal, this mutation is viable, in that the genomic deficiency, Exel6028, failed to complement the suppressive phenotype of die4, and was still viable (Table S1).
In this context, we performed a complementation test of die4 with 11 genes deleted in Exel6028 to identify EMS-induced mutations responsible for the suppressive phenotype ( Figure S1A, Table S2). We identified that the loss of Osi21 (CG14925) is responsible for the suppressive phenotype of die4, in that the Minos transposon-inserted allele of Osi21, Mi{ET1} Osi21 [32], failed to complement die4 and the introduction of the genomic fragment encompassing Osi21 reversed the suppressive effect of die4/ Mi{ET1} Osi21 at the deep pseudopupil (DPP) level ( Figure 1G, Table S2). Sequence analysis of the die4 chromosome revealed significant amino acid changes (G149S, M181T, and F229L) in Osi21 (Figure 2, Figure S1). These results were confirmed by targeted knock-down of Osi21 using RNAi method ( Figure 1C). Therefore, the suppressive effect of die4 on norpA P24 -triggered retinal degeneration is due to the loss of Osi21 function. We therefore conclude that die4 is a loss-of-function allele of Osi21.
Osi21 was identified as an Osiris family protein, without known cellular functions, on the basis of sequence homology [26]. Computational analysis, as described by Shah et al. [27], was performed using its amino acid sequence, which revealed that OSI21 includes (1) an endosome/lysosome sorting signal, (2) a two-Cys region, (3) duf1676 (Pfam family: PF07898), and (4) a YXXØ motif ( Figure 2) as predicted by previous studies [26,27,33]. Interestingly, Osi21 is located on the 2L chromosome. Thus, Osi21 is not linked to the cluster of 20 Osiris family genes that are located in the Triplo-lethal region (Tpl) of the 3R

Author Summary
Malfunctioning of phototransduction is the major cause of human blindness. Without functional phototransduction, rhodopsin-1, the major visual pigment, is rapidly endocytosed and accumulated in late endosomes. Impaired lysosomal delivery of endocytosed rhodopsin and its degradation has been reported to trigger progressive and light-dependent retinal degeneration in Drosophila models. It is intriguing why endocytosed rhodopsin accumulates in late endosomes instead of being delivered to lysosomes for degradation. Is this attributable to a saturation of rhodopsin endocytosis, which impedes the delivery capacity of the cell? To investigate the underlying mechanisms of rhodopsin accumulation in late endosomes, we used a suppressor of phototransduction mutants, which was identified previously from our unbiased genetic screen. This suppressor, called diehard4, shifts the membrane balance between late endosomes and lysosomes, resulting in the facilitated degradation of endocytosed rhodopsin. Our results clearly demonstrate that a previously unknown mechanism of negative regulation is actively engaged in vesicular traffic between endosomes and lysosomes in fly photoreceptors. We showed that eliminating such blockage alone was enough to rescue retinal degeneration in phototransduction mutants. From these results, we anticipate that the identification of additional components and an in-depth description of this molecular machinery will aid in therapeutic interventions of various retinal dystrophies and neurodegenerative disorders. chromosome and is responsible for the Tpl phenotype, suggesting that its cellular function differs from that of the other Osiris family proteins.
The Loss of Osi21 Function Suppresses Retinal Degeneration in norpA P24 , rdgC 306 , and trp 1 , but not in rdgB 2 Mutant Photoreceptors Drosophila rdgC encodes rhodopsin-specific phosphatase and requires rhodopsin recycling [34,35]. The loss of rdgC function leads to light-and age-dependent retinal degeneration as a result of excessive endocytosis of stable rhodopsin-arrestin complexes [14]. Because the rdgC mutant photoreceptor cells share the cause of retinal cell death with norpA P24 , we used rdgC 306 , the loss-offunction rdgC mutant, to test the effect of die4 on retinal cell death. DPP analysis and histological analysis using electron microscopy showed that die4 protects retinal degeneration due to rdgC 306 ( Figure 3A-C, Figure S2), suggesting that Osi21 is not a specific regulator in norpA-triggered retinal degeneration but plays an essential role in retinal degeneration caused by intracellular accumulation of cytotoxic rhodopsin.
We also examined the effect of die4 on retinal degeneration due to the loss of trp function, a light-sensitive Ca 2+ channel [36]. Drosophila trp 1 was recovered as a spontaneously occurring temperature-sensitive loss-of-function mutant at a temperature of 24uC [36,37] and is known to show light-enhanced retinal degeneration [38]. Although a dysfunction in Ca 2+ fluctuation was suggested as a cause of its retinal degeneration phenotype, its mechanism of degeneration remains unknown. Interestingly, we found that the loss of die4 function protects retinal degeneration caused by trp 1 at the DPP and ultrastructural levels ( Figure 3D-F, Figure S2). This result suggests that intracellular accumulation of cytotoxic rhodopsin also causes retinal degeneration in trp 1 mutant photoreceptor cells.
We used rdgB 2 photoreceptors as a negative control to assume the functions of die4 because cytoplasmic rhodopsin aggregation is not involved in retinal degeneration in rdgB 2 photoreceptors [39]. As expected, die4 was unable to suppress rdgB 2 -triggered retinal degeneration ( Figure 3G). Combined together, these double mutant analyses suggest that intracellular rhodopsin aggregation triggers unregulated cell death in norpA P24 , rdgC 306 , and trp 1 photoreceptors, and that Osi21 is a key regulator in the formation of rhodopsin aggregation, wherein the loss of Osi21 function suppresses retinal degeneration in these mutant photoreceptor cells.

Osi21 Negatively Regulates Late Endosomal Membrane Traffic toward Lysosomes
Previous studies found that norpA and rdgC mutant photoreceptor cells die due to excessive endocytosis of rhodopsin-arrestin complexes and accumulation of endocytosed rhodopsin in late endosomes [11,12,14]. These findings indicate that the inability of rhodopsin transport and degradation through the endo-lysosomal system cause unregulated cell death in norpA and rdgC mutant photoreceptors. In this context, we tested the possibility that Osi21 acts as a regulator that maintains membrane homeostasis between endosomes and lysosomes in which the functional loss of Osi21 shifts the membrane balance of the endo-lysosomal system.
To test our hypothesis, we examined whole mounts of Drosophila retinas by confocal microscopy. We found that the loss of Osi21 function minimally affected the Rab5-positive vesicles (early endosomes) ( Figure 4A   For DPP analysis, newly eclosed flies were exposed to 2900 lux of constant light in the 25uC 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 6SE. (A) rdgC 306 and die4; rdgC 306 , (D) trp 1 and die4; trp 1 , and (G) rdgB 2 and rdgB 2 ; die4. For electron micrograph, flies in each genotype were exposed to 2900 lux of constant light in the 25uC incubator. Four-day old rdgC 306 (B) and die4; rdgC 306 (C), Eight-day old trp 1 (E) and die4; trp 1 (F) were sacrificed for analysis. Compared to the completely deformed ommatidial structure of single mutants, double mutants showed well preserved rhabdomeric and ommatidial structures. doi:10.1371/journal.pgen.1003559.g003 The reduced Rab7-positive vesicles in Osi21 knock-down photoreceptor cells may be attributed to Gal4 titration due to the existence of a second UAS promoter of in the Osi21 knockdown construct. Thus, we used the w; Rhi1::Gal4, UAS::YFP-Rab7/ +; UAS::RFP-arf72A/+ as a titration control and showed that the second UAS promoter did not affect YFP-Rab7 expression ( Figure 4F). Quantification of each vesicle clearly showed that among the Rab5-, the Rab6-and the Rab7-positive vesicles, the loss of Osi21 function only affected the Rab7-positive vesicles ( Figure 4K). Minimal increase of the Rab5-positive area in Osi21 knock-down photoreceptors may be the secondary effect caused by a reduction in Rab7-positive vesicles. Accordingly, biochemical analysis of isolated endo-lysosomal vesicles using Iodixanol density gradients (See Text S1) showed that immunoreactivities of Rab7, which was colocalized with endocytosed rhodopsin, were shifted toward the lower density fractions by the loss of Osi21 function ( Figure S3), suggesting a reduced fraction of late endosomes in Rab7-positive vesicles [40,41]. These results suggest that the loss of Osi21 function specifically affects the membrane balance between late endosomes and lysosomes.
Because the specific shift of membrane balance between late endosomes and lysosomes raised a strong possibility of direct regulation of Osi21 on membrane homeostasis of the endolysosomal system, we examined the subcellular localization of the OSI21 protein. Newly eclosed flies were reared in a light/dark cycled incubator and were exposed to bright light (2900 lux) for 90 min to induce massive rhodopsin endocytosis and its accumulation in late endosomes. The subcellular localization of OSI21-GFP from whole mount ommatidia was examined using confocal microscopy. We assumed that the OSI21-GFP is functional because its expression counterbalanced the suppressive effect of Osi21-RNAi in the DPP level (Data not shown). We found the OSI21-GFP localization partially overlapped with Lysotracker staining ( Figure 5A and E, Pearson's correlation coefficient: 0.617). In addition, majority of Osi21-GFP colocalized with endocytosed Rh1-RFP ( Figure 5B and E, Pearson's correlation coefficient: 0.635), suggesting that Osi21 functions directly on the endolysosomal membrane system in a way that Osi21 negatively regulates late endosomal membrane traffic toward lysosomes, resulting in rhodopsin accumulation in late endosomal compartments.

The Loss of Osi21 Function Results in the Facilitated Degradation of Endocytosed Rhodopsin
Changes in membrane balance between late endosomes and lysosomes may also affect the dynamics of vesicular traffic and the rate of rhodopsin degradation, in which the loss of Osi21 function facilitates rhodopsin traffic toward lysosomes and its lysosomal degradation, resulting in a delay of retinal degeneration in norpA P24 photoreceptor cells. In fact, reduced rhodopsin content due to vitamin A deprivation or mutation in the rhodopsin gene rescued norpA-triggered retinal degeneration [42]. Our analysis by confocal microscopy showed that, compared to the norpA P24 photoreceptor cells ( Figure 5C), norpA P24 mutant photoreceptor cells with the Osi21-RNAi transgene showed greatly proliferated lysosomes ( Figure 5D-E). These lysosomes were colocalized with endocytosed rhodopsin (Pearson's correlation coefficient: 0.604). Although we often found that small amounts of endocytosed rhodopsin escaped Osi21 blockage ( Figure 5C, arrowhead) and was colocalized with the lysosome (Pearson's correlation coefficient: 0.342), there were less lysosomes in the control norpA P24 photoreceptor cells. Moreover, majority of lysosomes did not colocalized with endocytosed rhodopsin, indicating that such colocalization reflected regular lysosomal turnover. These results raise the possibility that the loss of Osi21 function facilitates the rhodopsin degradation in lysosomes.
In this context, first, we examined the rate of rhodopsin endocytosis and degradation by time course measurements of pulse-chased photoreceptors by confocal microscopy. For the measurements, we expressed RFP-tagged rhodopsin under the control of the hs::Gal4 driver. Newly eclosed norpA P24 and norpA P24 ; Osi21-RNAi flies were kept in complete darkness for 24 h and then subjected to heat-shock. These flies were then kept in the darkness for another day to allow synthesis and transport of Rh1-RFP to the rhabdomere, following which they were exposed to bright light to chase Rh1-RFP. Whole mount photoreceptors were examined by confocal microscopy at 24 h interval for 96 h by typing the photoreceptor based on the progression of rhodopsin endocytosis and degradation ( Figure 6A). We found that the initial movement of endocytosed rhodopsin toward the endosomal system was not different between the norpA P24 and norpA P24 ; Osi21-RNAi photoreceptor ( Figure 6B, see the percentage of Type I and Type II photoreceptors). However, norpA P24 mutant photoreceptor cells with the Osi21-RNAi transgene showed facilitated clearance of endocytosed rhodopsin ( Figure 6B, see the percentage of Type III and Type IV photoreceptors), indicating facilitated degradation of endocytosed rhodopsin through the loss of Osi21 function.
The effect of the Osi21 loss-of-function on the rhodopsin contents in norpA P24 photoreceptors was also examined by western blot analysis by using flies eclosed within 12 h. Flies were reared either in the dark to prevent rhodopsin endocytosis or in 18 h light/8 h dark cycles to stimulate rhodopsin endocytosis. No significant differences in rhodopsin content were observed in the dark-reared norpA P24 mutant photoreceptor cell with the Osi21-RNAi transgene, compared to dark-reared norpA P24 photoreceptors ( Figure 6C, lanes 2-3). However, the rhodopsin content was greatly reduced in the norpA P24 mutant photoreceptor cells with the Osi21-RNAi transgene by the bright light stimulation ( Figure 6C, lanes 4-5). These results suggest that more endocytosed rhodopsin was transported into and degraded by lysosomes because of Osi21 loss-of-function.
Massive influx of Rh1 into the endosomal system may saturate endosomal trafficking machinery, resulting in the late endosomal accumulation of endocytosed rhodopsin. Therefore, we tested the effect of the Osi21 loss-of-function on rhodopsin content when the endosomal trafficking machinery was activated by overexpressing Rab5 or Rab7 ( Figure S4). To induce the maximal rhodopsin endocytosis, newly eclosed flies were exposed to bright light (2900 lux) for 48 h. No significant influence of Rab5 overexpression on the degradation of endocytosed rhodopsin was observed ( Figure 6D, left). However, overexpression of Rab7 synergistically accelerated rhodopsin degradation with the loss of function of Osi21 ( Figure 6D, right). Considering no significant decrease in rhodopsin content was observed with Rab7 overexpression alone, our results suggest that Osi21 negatively regulated rhodopsin transport between late endosomes and lysosomes by interacting with the Rab7-positive trafficking machinery. Therefore, we conclude that Osi21 is a critical negative regulator of vesicular traffic between endosomes and lysosomes. Its functional loss suppresses retinal degeneration in phototransduction mutants by changing the membrane dynamics between late endosomes and lysosomes and by facilitating the degradation of endocytosed rhodopsin.

Discussion
In both vertebrates and invertebrates, malfunctioning of phototransduction may stimulate the cell death machinery, resulting in retinal degeneration [43]. Without functional phototransduction, rhodopsin-1, the major visual pigment, is rapidly endocytosed and accumulated in the late endosomes [12]. Impaired lysosomal delivery of endocytosed rhodopsin and its degradation trigger progressive and light-dependent retinal degeneration in phototransduction mutants [12,17]. However, the mechanism underlying the accumulation of endocytosed rhodopsin in late endosomes, instead of delivering to lysosomes for degradation, remains to be elucidated.
In the current study, we used die4, the norpA P24 suppressor, to investigate the molecular basis of the accumulation of rhodopsin in late endosomes in phototransduction mutants. We found that the loss of die4 function delays retinal degeneration in norpA P24 , rdgC 306 and trp 1 , but not in rdgB 2 . These results suggest that, at least, norpA P24 , rdgC 306 , and trp 1 photoreceptor cells die through a shared route. Previous research suggested that the generation of stable rhodopsin-arrestin complexes is the major cause of cell death in norpA EE5 [11] and rdgC 306 [14]. The formation of stable rhodopsinarrestin complexes in the norpA mutant photoreceptor is attributable to its inability to activate the calcium-dependent phosphatase, RDGC, which dephosphorylates rhodopsin (Figure 7). The calcium-dependent phosphatase also remains inactive in the trp 1 photoreceptor upon light exposure since the cation specific calcium channel is lost in trp 1 [36]. Therefore, all three phototransduction mutants share a common feature; the formation of stable rhodopsin-arrestin complexes.
We found that the loss of die4 function delays retinal degeneration in norpA P24 longer than those in rdgC 306 and trp 1 . These results suggest that the blockage of endo-lysosomal trafficking by Osi21 is not the sole cause of retinal degeneration in rdgC 306 and trp 1 mutants. Recently, Sengupta et al. [45] proposed that PI(4,5)P 2 depletion by NORPA underlies retinal degeneration in trp CM and trp 343 mutants. Interestingly, both mutants exhibit faster lightdependent retinal degeneration than trp 1 mutants. Preventing the formation of stable Rh1-Arr2 complexes by red light slows down the retinal degeneration in trp CM and trp 343 mutants comparable to the trp 1 degeneration in white light, suggesting that the endocytosis of Rh1-Arr2 complexes contributes retinal degeneration in trp mutants and different results are attributable in part to the allelic differences. In addition, PI(4,5)P 2 depletion affects arrestin-mediated endocytosis [45], so that Rh1 internalization might be reduced in trp mutants. However, their results raise a strong possibility that prolonged activation of NORPA possibly contributes to degenerative syndromes in both trp and rdgC mutants.
Double mutant photoreceptor cells are eventually degenerated; they lost their DPP with extended exposure to bright light. Although DPP analysis does not provide a measure of the retinal degeneration process, it faithfully measures a complete loss of the ommatidial structure as it reaches the end of the degenerative process. DPP analysis in the current study suggests that the loss of Osi21 function delays the onset of retinal degeneration in norpA P24 , rdgC 306 and trp 1 mutants. However, the loss of Osi21 function slows down the retinal degeneration in norpA P24 , but not in rdgC 306 and trp 1 : the slope of DPP loss was similar to that of the control soon after DPP loss occurred in double mutants. These results also suggest that the activity of Osi21 is not the sole cause of the rdgC and the trp degeneration.
We conclude that Osi21 acts as a negative regulator of endolysosomal membrane traffic between late endosomes and lysosomes. This conclusion is based on the following observations: (1) Both the size and number of the late endosomes are significantly reduced in Osi21 knock-down photoreceptor cells, (2) the lysosomal compartments are greatly proliferated in Osi21 knock-down photoreceptor cells, (3) the OSI21 protein is localized in the endo-lysosomal compartments, (4) the loss of Osi21 function in the norpA P24 mutant photoreceptor facilitates the degradation of endocytosed rhodopsin, and (5) Rab7 overexpression alone fails to affect the rhodopsin content of the norpA P24 photoreceptor. However, overexpression of Rab7 synergistically accelerates rhodopsin degradation with the loss of Osi21 function, suggesting that Osi21 directly interacts with the Rab7-positive trafficking machinery. These results clearly demonstrate that the existence of negative blockage regulating the membrane balance of the endo-lysosomal system, and not the capacity of endo-lysosomal trafficking machinery, causes retinal degeneration in phototransduction mutants.
Heptahelical G protein-coupled receptors (GPCRs) are considered the most diverse and therapeutically important family of receptors [46,47]. Like many vertebrate GPCRs, light-activated rhodopsin-1 in Drosophila is rapidly phosphorylated by a specific kinase, called rhodopsin kinase. Phosphorylated rhodopsin-1 is desensitized by arrestins and is endocytosed to terminate further signaling activity (Figure 7). Because of this similarity, Drosophila rhodopsin-1 has been used as a prototype to study agonist-induced desensitization and internalization of vertebrate GPCRs [48].
In Drosophila photoreceptors, Arr2 promotes rhodopsin endocytosis and degradation when stable Rh1-Arr2 complexes are generated by loss of norpA or rdgC while Arr1 promotes rhodopsin endocytosis and recycling in the normal condition [14,49]. Although Arr1 was previously reported to localize in endosomes [49], we found that Arr2 was absent in the endosomal system (data not shown), indicating Arr2 dissociates from Rh1 near the rhabdomeric membrane. This is reminiscent of functional classification of vertebrate GPCRs: Class A and Class B [50][51][52]. Thus, it can be clearly surmised that Drosophila photoreceptors operate two separate mechanisms of Rh1 endocytosis: (1) Arr1 for quenching and recycling, and (2) Arr2 for quenching and degradation. Since Arr2 is several folds more abundant than Arr1 in Drosophila photoreceptor cells to ensure rapid quenching of rhodopsin signaling for visual sensitivity [53,54], the negative blockage by Osi21 may be evolved to counterbalance excessive Rh1 degradation as a result of Arr2 binding to activated Rh1. Therefore, it should be further investigated whether arrestins play roles in the decision between recycling and degradation for endosomal Rh1, and in the activation of cell death machinery.
Post-endocytic trafficking of GPCRs implicates in many human diseases [55]. Especially, stable rhodopsin-arrestin complexes in vertebrates also result in photoreceptor degeneration [56,57]. In addition, cytoplasmic accumulation of proteins often implicates various neurodegenerative disorders, including the accumulation of rhodopsin in retinitis pigmentosa [56] and the accumulation of polyQexpanded huntingtin in Huntington's disease [58]. Our results suggest that Osi21 regulation may underlie accumulation of disease-causing proteins in the endosomal compartment and that the elimination of Osi21 regulation may clean up this pathologic accumulation. Therefore, the identification and characterization of this specific cellular machinery may provide a therapeutic target for many GPCRrelated human diseases and neurodegenerative disorders.

Drosophila Strains and Genetics
Drosophila was grown on standard food in a 25uC incubator. Standard genetic schemes were used to generate flies with the genotypes described. The Canton-S and w 1118 fly were used as a wild-type strain, norpA P24 , rdgC 306 , trp 1 and rdgB 2 as loss-offunction strains of phototransduction. Genomic deficiencies listed in Table S1. Loss-of-function mutants used for complementation test listed in Table S2. A w norpA p24 eyFLP chromosome was made using meiotic recombination to subsequently generate die4 mosaic flies in the norpA p24 background. GMR-hid were used in combination with the w norpA p24 eyFLP chromosome to make photoreceptor cells exclusively homozygous for the die4 FRT chromosome [59]. The second and third chromosome inserts of the Rh1::GAL4 driver were derived from the Rh1::GAL4 line constructed by Tabuchi et al. [60] and used for driving expression of various UAS targets including the die4 knock-down construct, UAS::Osi21-RNAi and fluorescently subcellular markers, UAS::YFP-Rab5, UAS::YFP-Rab7 and UAS::GFP-Rab6. UAS::Rh1-GFP was constructed using the prh1::eGFP construct from Pichaud and Desplan [61]. All Drosophila stocks except Osi21 knockdown strain were obtained from the Bloomington Stock Center at Indiana University. The UA-S::Osi21-RNAi strain was obtained from Vienna Drosophila RNAi Center (VDRC, Vienna).

Generation of Transgenic Animals
Primers for Osi21 or ninaE (Rh1) were specifically designed for use in the Gateway system (Invitrogen, Inc., Carlsbad, CA). Exact primer sequences for the rescue experiment, the expression of GFP-tagged Osi21 or RFP-tagged Rh1 were listed in Table S3. Directional cloning into the pENTR TOPO vector and the destination vectors (Carnegie Institution of Washington) followed manufacturer's instruction. The pTW, pTWG, and pTWRvector were used as destination vectors for Rescue constructs, UA-S::Osi21-GFP, and UAS::Rh1-RFP, respectively. Plasmid isolation was performed from positive clones using the Qiagen Midi Kit (Valencia, CA). After injection, G0 flies were crossed with w; SM1/Sco; TM2/Sb balancer flies. The progeny from the cross were sorted for mini-w + eyes. Mini-w + was used as a marker to determine the presence of the transgene. Flies with the mini-w + eye (G1 generation) were subsequently crossed with w 1118 flies to map the location of the transgene. After mating with w 1118 flies, mini-w + flies were crossed to the driver stock. In all cases, mini-w + flies were crossed to w; Rh1::GAL4 to drive expression of the transgene.

Deep Pseudopupil Analysis
The deep pseudopupil (DPP) was visualized in red-eyed flies which shows a bright trapezoidal structure when a white light illuminates the retina from the back of the head [62]. The flies of each genotype were collected daily and raised under the appropriate light condition. The flies were also scored daily for the presence of the deep pseudopupil. During degeneration, deep pseudopupils become increasingly diffused before being completely lost. The deep pseudopupil was scored as negative as soon as its trapezoidal shape became indistinct. The percentage of flies that retained their deep pseudopupils for a given day was calculated. In figure 1, total 20 flies were analyzed for each genotype tested. In figure 2, three replicates with a total of 100 flies were analyzed for rdgC 306 , trp 1 and rdgB 2 with or without die4 to determine the average percentage of deep pseudopupil -positive flies and the standard error for each day. Whole-mount Preparation of Drosophila Ommatidia and Fluorescence Microscopy Detailed procedures were also described previously [63]. Flies eclosed within 6 h were sacrificed with or without light treatment. For the whole-mount ommatidia isolation, fly heads were removed from bodies. A sagittal cut was made on the fly head creating two halves. The brain and proboscis were then removed. The eye was placed on a microscope slide containing 16 PBS. For Lysotracker (Invitrogen, Inc., Carlsbad, CA) staining, fly eyes were preincubated in ,1 mM Lysotracker for 90 min and washed 3 times in 16 PBS for 30 min. Then, 2% paraformaldehyde in 16PBS was used as a fixative and treated for 30 min followed by twice wash in 16 PBS. Residual pigments in fly retina were eliminated in 0.1% Triton X-100 (Sigma, MO) for 4 h, then washed twice for 10 min. Each ommatidia were removed using a sharp platinum needle from fly retina. Usually, a large piece of retina was then teased apart and mounted using mounting medium (Vector Labratories, Inc., Burlingame, CA). The FV500 confocal laser scanning microscope (Olympus Optical, Japan) was used for examining individual ommatidium. Optical images were acquired with an 6100 objective. Confocal images were analyzed with ImageJ software (NIH, MD) for quantitative analysis. For quantitative measurement of endosome/lysosome, mean size, number and total area of each vesicle in a photoreceptor cell were calculated with the Analyze Particle function. Measured values were normalized with the known distance option of imageJ. Statistical significances were calculated with two-tailed t tests using Prism 5.01 software. For colocalization analysis, Pearson's correlation coefficient (Rr) was calculated with Intensity colocalization analysis function of imageJ. The values for Rr range from 1 (perfect correlation) to 21 (perfect exclusion). Thus, a value close to 1 indicates reliable colocalization.

Electron Micrograph
Retinal degeneration was examined with electron microscopy using retinal tissue sections. Fly eyes were prepared for electron microscopy using procedures described by Washburrn and O'Tousa (1992). Electron microscopy sections were ,80-100 nm thick, stained first in 5% uranyl acetate in 50% EtOH and then in Reynold's lead citrate. The Hitachi H600 electron microscope was used to take electron micrographs. The micrographs shown in all figures are taken from ommatidia crosssectioned at a depth of R1-R6 photoreceptor nuclei to present a similar view of various genotypes.

Western Blot Analysis
Usually, 2-5 fly heads were homogenized in buffer A (20 mM Tris-HCl(pH 7.5), 100 mM NaCl, 5 mM MgCl2, 10% sucrose, 1% glycerol, 1 mM EDTA, 1% CHAPS and Complete Protease Inhibitor Cocktail) with pellet pestle. The homogenate was centrifuged at 4uC and 14,000 rpm for 2 min. The supernatant was separated on 12% SDS polyacrylamide gel and then transferred to PVDF membrane at 100 V for 1 hour. The membrane was blocked by 5% nonfat milk in TBST (TBS with 0.5% Tween 20) for 60 min. After blocking, the membrane was incubated with the mouse anti-rhodopsin antibody 4C5 (diluted 1:5000, Developmental Studies Hybridoma Bank) for 60 min at room temperature or overnight at 4uC. The polyclonal rabbit anti-arrestin2 antibody (Genscript, NY) is diluted 1:30,000. The antimouse or rabbit IgG HRP conjugated antibody was diluted 1:5000 in TBST containing 5% skin milk and the membrane was washed with TBST for 30 min. The blotted membrane was detected with a homemade ECL solution for 1 min, and then exposed to X-ray film. The detected bands were quantified using the Quantity One (Bio-Rad).

Time Course Analysis of Rhodopsin Endocytosis and Degradation
Newly eclosed norpA P24 and norpA P24 ; Osi21-RNAi flies with UAS::Rh1-RFP under control of hs::Gal4 were kept in complete darkness for 24 h and then subjected to heat-shock for one hour in the 37uC incubator three times at six-hour interval. These flies were kept in the 25uC incubator for another day and then moved under 2900 lux light. Whole mount photoreceptors were examined by confocal microscopy at 24 h intervals for 96 h. At each time point, approximately 30 photoreceptor cells from at least five individuals were scored and categorized as follows: Type I (most Rh1-RFP localizes in the rhabdomere), Type II (Rh1-RFP localizes equally in the rhabdomere and the cytoplasm), Type III (most Rh1-RFP localizes in the cytoplasm) and Type IV (most Rh1-RFP disappears due to degradation). The Kolmogorov-Smirnov test for equality distribution was performed using the STATA software package.

Supporting Information
Figure S1 Identification of the mutation responsible for the die4 phenotype. (A) Genes deleted in the EXEL6028 genomic deficiency: 11 genes deleted in the Exel6028 genomic deficiency were identified based on Release 5.1 of the Drosophila genome. To identify a gene responsible for the die4 phenotype, a complementation test was performed using existing fly mutant stocks. Only the Osi21 loss-offunction mutant uncomplemented the die4 phenotype. (B) Sequencing strategy: The die4 chromosome is homozygous lethal. Therefore, the GFP balancer was used to distinguish the homozygous die4 animal from the balanced heterozygotes. Fluorescently labeling the animal indicates the animal has the GFP balancer. Non-fluorescent homozygous die4 embryos were selected for sequencing analysis. Table S1 Genomic deficiencies for deficiency mapping. Note: Df(2L)Exel8005 failed to cover the lethality in the die4 chromosome. However, Df(2L)Exel6028 failed to cover the suppressive phenotype of die4, indicating lethality caused by a gene in 22B2 to B8 region is not responsible for the suppressive phenotype of die4.   Text S1 Method for Iodixanol density gradient analysis. (DOCX)