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J. Biol. Chem., Vol. 275, Issue 32, 24752-24759, August 11, 2000

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Role of Asparagine-linked Oligosaccharides in Rhodopsin Maturation and Association with Its Molecular Chaperone, NinaA^*

Rebecca Webel, Indu Menon, Joseph E. O'Tousa^§, and Nansi Jo Colley^¶

From the  Department of Ophthalmology & Visual Science and the Department of Genetics, University of Wisconsin, Madison, Wisconsin 53706 and the ^§ Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

Received for publication, March 29, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites for N-linked glycosylation (N20I and N196I). We show that Asn²⁰ is the sole site for glycosylation. The Rh1^N20I protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1^N20I; therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1^N20I/NinaA interaction. We further show that Rh1^N20I interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The correct folding, assembly, and subcellular distribution of many glycoproteins is dependent on the proteins undergoing N-linked glycosylation within the endoplasmic reticulum (ER) (1, 2).¹ In eukaryotic cells, N-linked glycosylation occurs in the lumen of the ER and involves the transfer of a Glc₃Man₉GlcNAc₂ oligosaccharide unit from a dolichol donor to an asparagine of an NX(S/T) consensus sequence of a nascent polypeptide. Oligosaccharide processing and trimming are initiated in the ER prior to delivery to the Golgi apparatus, where further trimming and sequential addition of saccharides results in the protein becoming terminally glycosylated (3).

Photoreceptors in both Drosophila and vertebrates utilize a G protein-coupled receptor, rhodopsin, for vision. The major rhodopsin in Drosophila, Rh1, like vertebrate rhodopsins, undergoes N-linked glycosylation during biosynthesis (4-10). Rh1 displays 22% amino acid identity with human rhodopsin (11, 12). Since the initial finding that Drosophila Rh1 mutations lead to photoreceptor degeneration (13, 14), over 90 distinct mutations in the human rhodopsin gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP) (15-18). adRP is characterized by progressive retinal degeneration, often leading to blindness. Mutations at sites for N-linked glycosylation in rhodopsin have been identified in adRP patients (16). Vertebrate rhodopsin has two consensus sites for N-linked glycosylation at N2 and N15 and is glycosylated at both sites (6). Disease-causing point mutations in rhodopsin, T4K, N15S, and T17M, have been identified in patients with adRP, indicating that glycosylation defects act dominantly to cause retinal degeneration in humans (16, 19, 20). In Drosophila, the elimination of N-linked glycosylation of Rh1 leads to photoreceptor cell defects, indicating that rhodopsin glycosylation is critical for fly visual function (21-23).

Previous studies have generated conflicting data on the role of glycosylation in rhodopsin maturation. Studies using site-specific mutagenesis of consensus glycosylation sites in rhodopsin and expression in cell culture support a role for glycosylation in folding and maturation (24, 25). Another study indicates that transport defects can be overcome with vitamin A supplementation (11-cis retinal) (26), indicating that the maturation defect may not be a direct consequence of lack of glycosylation. Tunicamycin, which blocks the formation of precursor oligosaccharides required for N-linked glycosylation, has also been used to study the role of glycosylation in rhodopsin biosynthesis (25, 27, 28). In Rana pipiens, tunicamycin treatment causes nonglycosylated opsin to accumulate in the ER (27). In another frog, Xenopus laevis, the lack of opsin glycosylation disrupts normal outer segment disc assembly but has no effect on intracellular transport of rhodopsin (28). In addition, tunicamycin does not prevent rhodopsin transport in cell culture, indicating that glycosylation is not required for rhodopsin maturation (25). Further conflicting data stem from the analysis of the photoreceptor cells in a deceased 68-year-old patient with adRP, carrying the same point mutation that caused defects in rhodopsin maturation in cell culture. The photoreceptor cells do not display pathology indicative of rhodopsin transport defects (19). Although glycosylation is required for the folding and maturation of certain proteins, its role in rhodopsin folding and maturation requires further evaluation.

In general, N-linked oligosaccharides facilitate interactions with enzymes and chaperones in the ER to promote protein maturation. For example, chaperones such as calnexin and calreticulin bind N-linked oligosaccharides of proteins, as part of a pathway ensuring correct folding in the ER, and selective transport of properly folded proteins from the ER (1, 2, 29, 30). In Drosophila photoreceptor cells, NinaA, a type I membrane protein, is required as a molecular chaperone for Rh1 exit from the ER and transport through the secretory pathway (10, 31). NinaA is located predominantly within the ER but also colocalizes with Rh1 within vesicles located in distal compartments of the secretory pathway. NinaA forms a specific, stable complex with Rh1, and genetic dosage studies demonstrate a quantitative requirement for NinaA in Rh1 transport. These findings are consistent with NinaA functioning as a molecular chaperone for Rh1 rather than a folding catalyst (32).

Drosophila is an excellent model organism for analyzing the in vivo role of glycosylation in chaperone-mediated rhodopsin maturation. Mutant forms of Rh1 may be introduced into flies, and their function may be studied in their native photoreceptor cells, eliminating potential artifacts present in heterologous cell culture systems. We undertook the current study to investigate the role of rhodopsin glycosylation in Drosophila. Rh1 has two consensus sites for N-linked glycosylation at Asn²⁰ and Asn¹⁹⁶. Using site-directed mutagenesis, we made isoleucine substitutions at each of these two sites and expressed the mutant Rh1^N20I and Rh1^N196I forms in Drosophila. The transgenic animals expressing Rh1^N20I and Rh1^N196I were analyzed for rhodopsin maturation, interaction with NinaA, and retinal degeneration.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Drosophila Strains, Genetics, and Transgenic Animals-- The wild type strain used in these studies is Drosophila melanogaster w¹¹¹⁸. ninaA^P269 (33) is a null allele. ninaE is the structural gene for Rh1, and the ninaE^I17 strain is a null allele of Rh1 (11, 12). The Rh1^N20I and Rh1^N196I mutant forms of Rh1 were created by in vitro mutagenesis and P-element transformation as described in O'Tousa (21) and placed in a ninaE^I17 genetic background. We used transgenic flies expressing wild type Rh1 tagged with a 12-amino acid bov-epitope tag at the C terminus (Rh1-bov) (34). The bov-epitope corresponds to the C terminus of bovine rhodopsin and is recognized by the 1D4 antibody (35, 36). All stocks were constructed using standard balancer stocks (37).

Immunocytochemistry and Electron Microscopy-- Immunocytochemistry was carried out according to Colley et al. (10). One-day-old adult fly heads were fixed on ice in 3% paraformaldehyde in 100 mM sodium phosphate buffer containing 2 mM calcium chloride and protease inhibitors (pH 7.2-7.4). The tissue was infiltrated with 2.3 M sucrose and frozen in liquid nitrogen for cryoultramicrotomy (38-40). Ultrathin cryosections were obtained using a Reichert Ultracut-E equipped with a FC-4D cryo-attachment. Sections were indirectly immunolabeled with the 4C5 and 1D4 monoclonal antibodies directed to Rh1 and the bov-epitope tag, respectively (35, 36, 41). Primary antibody labeling was detected by fluorescein-conjugated goat anti-mouse IgG (Jackson ImmunoResearch). Nuclei were labeled with ToPro-3 nucleic acid stain (Molecular Probes, Inc.). Sections were viewed using a Bio-Rad MRC1024 laser scanning confocal microscope (Bio-Rad).

For electron microscopy, adult heads were fixed and processed according to a modification of the methods of Baumann and Walz (42) as described previously (10, 34). The fixed tissue was dehydrated in serial changes of ethanol followed by propylene oxide and embedded in Spurr's medium (Polysciences, Inc.). Ultrathin sections were obtained on a Reichert Ultracut E ultramicrotome, stained with 2% uranyl acetate and lead citrate, and viewed at 80kV on a Phillips 410 electron microscope. For all genotypes described, at least five individual heads were sectioned, and 100 ommatidia were observed from each eye.

Electroretinogram Analysis-- Electroretinograms (ERGs) were carried out on 3-5-day-old flies according to published procedures (43, 44).

SDS-Polyacrylamide Gel Electrophoresis and Immunoblotting-- Heads from one- or two-day-old flies were placed in cold sample buffer containing protease inhibitors. Samples were sonicated, separated by electrophoresis in 10% SDS-polyacrylamide gels (45), and electroblotted onto nitrocellulose filters (46). The nitrocellulose was incubated with either of the 4C5 and 1D4 mouse monoclonal antibodies directed to Rh1 or the bov-epitope tag, respectively (35, 36, 41), or a rabbit polyclonal antibody directed to NinaA (gift of A. Becker and C. S. Zuker) (10, 31, 47). The immunoreactive proteins were visualized using horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Jackson ImmunoResearch) followed by ECL detection (Amersham Pharmacia Biotech). Endoglycosidase H (endo H) (Roche Molecular Biochemicals) treatments were carried out overnight at 37 °C according to standard methods (10).

NinaA/Rh1 Affinity Chromatography-- Flies, 3 days old or younger, were subjected to affinity chromatography essentially as described previously (31) using approximately 3,000 heads for wild type, ninaE^I17, and Rh1^N196I, and approximately 6,000 heads for Rh1^N20I. Membranes were prepared by centrifugation at 100,000 × g for 60 min. The membrane pellet was resuspended in sodium phosphate buffer with protease inhibitors containing 1% n-dodecyl--D-maltoside, homogenized, and centrifuged at 150,000 × g for 60 min to remove insoluble material. The supernatant was loaded onto columns of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) conjugated to 4C5 mouse monoclonal antibody directed to Rh1. After exhaustive rinsing, Rh1 and its associated proteins were eluted with 5 ml of triethylamine (pH 11.2). The samples were dialyzed, concentrated, and suspended in sample buffer (45) before being subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting. All procedures were carried out on ice or at 4 °C. The eluted sample was divided such that 80% of the sample was used for detection of NinaA using a rabbit polyclonal antibody (gift of A. Becker and C. S. Zuker), and 10% of sample was used for detection of Rh1 using the 4C5 mouse monoclonal antibody.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Glycosylation Is Required for Expression and Transport of Rhodopsin-- Drosophila photoreceptor cells contain specialized regions of the plasma membrane, made up of numerous tightly packed microvilli, called rhabdomeres, that contain the rhodopsin photopigments and the other constituents of the phototransduction cascade (Fig. 1A). In Drosophila photoreceptor cells, Rh1 is core glycosylated in the ER, transported through the Golgi complex, and delivered to the rhabdomeres of the R1-6 photoreceptor cells where it functions in phototransduction (10, 48). Rh1 is a G protein-coupled receptor that has two potential sites for N-linked glycosylation, Asn²⁰ and Asn¹⁹⁶ (Fig. 1B) (11, 12, 49). We used site-directed mutagenesis to obtain single nucleotide alterations in the coding capacity from an asparagine to an isoleucine at positions 20 and 196 (see boxes in Fig. 1B). We then constructed transgenic flies expressing these mutant forms, Rh1^N20I and Rh1^N196I, to examine the in vivo role of glycosylation in Rh1 transport through the secretory pathway.

View larger version (122K): [in this window] [in a new window]   Fig. 1.   Rh1N20I and Rh1N196I mutants display defects in transport to the rhabdomeres. A, a schematic of a cross-section of the R1-7 photoreceptor cells. Each rhabdomere (R) is the photosensitive organelle, comprised of 60,000 microvilli, containing the rhodopsin photopigments and the other components of the phototransduction cascade. N, nucleus. The diagram is adapted from Ref. 67. B, the proposed secondary structure of Rh1 showing the location of the two potential glycosylation sites at positions 20 and 196 (49). The nucleotide sequences of the wild type genes and the origin of the mutants are indicated in the boxes. In both mutants, a single nucleotide change alters the coding capacity from an Asn to an Ile (N20I and N196I) (see boxes). C, a cross-section of a wild type eye (w1118) from a 1-day-old fly reveals that Rh1 immunolocalizes to the rhabdomeres of the R1-6 photoreceptor cells (arrows). In C-E, Rh1 is detected with the 4C5 antibody (green), and in C-F, the nuclei are detected with ToPro (blue). D, a cross-section of a 1-day-old Rh1N20I mutant reveals that Rh1N20I is detected in the rhabdomeres and that the rhabdomeres are severely reduced in size (arrows). In addition, a substantial amount of Rh1N20I immunolocalizes to the endoplasmic reticulum in a perinuclear fashion. E, an oblique cross-section of a 1-day-old Rh1N196I mutant shows that Rh1N196 is detected in the cell body, presumably reflecting an accumulation in the ER as well other compartments of the secretory pathway. Rh1N196 is also detected in the rhabdomeres (arrow). F, a cross-section of a 1-day-old Rh1N20I mutant expressing wild type Rh1 with the bov-epitope tag recognized by the 1D4 antibody. Wild type Rh1 is specifically detected with the 1D4 antibody in a perinuclear fashion and in the rhabdomeres (arrow). The rhabdomeres appear more continuous in E because the section is oblique.

In wild type flies, Rh1 localizes predominantly to the rhabdomeres of the R1-6 photoreceptor cells (Fig. 1C). In contrast, the photoreceptor cells expressing Rh1^N20I (Fig. 1D) and Rh1^N196I (Fig. 1E) display an altered distribution of Rh1. In both cases, substantial amounts of Rh1^N20I and Rh1^N196I mutant proteins are distributed in a perinuclear fashion, presumably reflecting their accumulation within the endoplasmic reticulum. Consistent with this interpretation, ultrastructural analysis of the mutant photoreceptors revealed large accumulations of ER and Golgi membranes (Fig. 2, B and C). In wild type photoreceptor cells, the cytoplasm displays limited amounts of ER and Golgi cisternae (Fig. 2A). The Golgi membranes were particularly prominent in the Rh1^N20I mutant (Fig. 2B), suggesting that some protein may be escaping the retention system of the ER. Although the ER is the primary site for quality control, the Golgi complex also participates (30).

View larger version (175K): [in this window] [in a new window]   Fig. 2.   Rh1N20I and Rh1N196I mutants overproduce ER cisternae. Electron micrographs of cross-sectioned photoreceptor cells are 1 day old (flies are 1 day post-eclosion). A, wild type photoreceptors display normal morphology. Scale bar, 0.4 µm. B, photoreceptors expressing Rh1N20I display accumulations of rough ER cisternae and dilated Golgi. Scale bar, 0.37 µm. C, photoreceptors expressing Rh1N196I also display accumulations of ER and dilated Golgi. Scale bar, 0.58 µm. Inset, the Rh1N196I mutant, showing ER, transition vesicles and Golgi. Scale bar, 0.31 µm. D, photoreceptors expressing both Rh1N20I and wild type Rh1 display accumulations of ER and dilated Golgi. Scale bar, 0.43 µm. R, rhabdomere; G, Golgi; V, transition vesicles.

The quality control retention mechanisms are not 100% effective, because some of the Rh1^N20I and Rh1^N196I mutant rhodopsins avoid the retention system and are successfully transported to the rhabdomeres (Fig. 1, D and E). To investigate whether these rhodopsins are functional, we measured ERGs from 3-5-day-old flies. Fig. 3A shows an ERG trace of a wild type fly subjected to bright orange and blue light stimuli. Upon blue light stimulation, the trace fails to return to base line until application of an orange stimulus. This condition, known as the prolonged depolarizing afterpotential (PDA), is indicative of normal rhodopsin levels (44). An ERG trace of Rh1^N20I is shown in Fig. 3B. The reduced amplitude of the light response and the lack of the PDA demonstrate that the mutant flies express low levels of Rh1^N20I protein. However, the small amount of Rh1^N20I protein that is transported to the rhabdomeres is functional (21, 22). Rh1^N196I flies display an ERG response, demonstrating that Rh1^N196I is also functional (Fig. 3C). The larger amplitude of the ERG response suggests that more Rh1^N196I is successfully transported to the rhabdomeres compared with Rh1^N20I. However, the absence of the PDA in Rh1^N196I mutants reflects a reduction in functional Rh1^N196I protein relative to wild type.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Electroretinogram analysis. A, the ERG of a wild type fly. The blue light stimulus invokes the PDA; this is reversed by application of an orange light stimulus. B, the Rh1N20I ERG. The amplitudes of all light responses are diminished, and the PDA is lacking because of low levels of functional Rh1N20I protein. The amplitude of light response is larger than the ninaEI17 null allele (21, 22). C, the Rh1N196I ERG. The amplitudes of all light responses are greater than those seen in Rh1N20I, showing that Rh1N196I is functional and expressed to a greater level than Rh1N20I. The absence of the PDA reflects a reduction in Rh1N196I rhodopsin relative to the wild type fly. Electroretinograms were recorded from white-eyed flies using 5-s light stimuli. Orange light intensity was approximately 3 × 104 µW/cm2; blue light intensity was 5 × 105 µW/cm2.

The glycosylation state allows us to distinguish between rhabdomeric and ER forms of Rh1 (10, 34). Wild type Rh1 is detected as the mature rhabdomeric (34-kDa) form (Fig. 4A, lane 1). In ninaA²⁶⁹ mutants, Rh1 does not exit the ER (10) and accumulates as a high molecular mass glycosylated ER form (Fig. 4A, lane 2). Treatment of ninaA²⁶⁹ extracts with endo H increases the electrophoretic mobility of the Rh1 opsin such that it now migrates with the mature form (Fig. 4B, lanes 3 and 4). Because endo H selectively cleaves immature high mannosyl oligosaccharide chains (3), this form of Rh1 must represent the glycosylated ER form. Rh1^N20I is detected as a 34-kDa form, and its expression is severely reduced relative to wild type (Fig. 4A, lane 3). These results are consistent with the smaller amplitude in the light response by ERG analysis (Fig. 3B) and the reduction in the size of the rhabdomeres (Fig. 2B).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Immunoblot analysis of glycosylation states of wild type and mutant forms of Rh1. A, Rh1 retained in the ER is detected in a high molecular mass form. Lane 1, Rh1 in wild type flies (WT) is detected in the mature 34-kDa form. Lane 2, ninaA mutant (ninaA269). Lane 3, Rh1N20I mutant. Lane 4, Rh1N20I expressed in a ninaA269 mutant background. Lane 5, Rh1N196I mutant. Lane 6, Rh1N196I expressed in ninaA269/+. The number of heads loaded in each lane is indicated at the top of the gel. The immunoblot was probed with 4C5 antibody directed to Rh1. B, Rh1N20I is not glycosylated. Head extracts either untreated () and treated with endo H (+). Lanes 1 and 2, wild type extracts (WT). Lanes 3 and 4, extracts from ninaA269 mutants. Lanes 5 and 6, extracts from ninaA269 mutants expressing Rh1N20I, showing that endo H digestion does not alter the mobility of Rh1N20I. Lanes 7 and 8, extracts from Rh1N196I mutants. Treatment with endo H increases the electrophoretic mobility of the immature, high molecular mass 40-kDa form of Rh1 (+ endo H). The immunoblot was probed with 4C5 antibody directed to Rh1. Extracts from 100 heads were used, and all flies were 1-2 days old. C, Rh1N20I prevents the maturation of wild type Rh1. Lane 1, wild type (WT) Rh1 carrying the epitope tag recognized by the 1D4 antibody (Rh1-bov). The transgene was expressed in flies that carry one wild type copy of ninaE (ninaEI17/+). Lane 2, ninaA, Rh1 with the epitope tag expressed in the ninaA269 mutant. Lane 3, Rh1N20I expressed with the wild type Rh1 carrying the epitope tag recognized by the 1D4 antibody. The apparent molecular mass of Rh1 is about 2 kDa larger because of the presence of the epitope tag (Rh1-bov). The number of heads loaded in each lane is indicated at the top of the gel. The immunoblot was probed with the 1D4 antibody directed to the epitope tag. The mutant Rh1N20I is not recognized by the 1D4 antibody.

We have shown above that Rh1^N20I displays defective transport (Fig. 1D), indicating that if Rh1^N20I was glycosylated, it should be present in the high molecular mass immature ER form. However, because the 34-kDa band may represent the mature form, already subjected to the normal process of carbohydrate removal, we examined the potential for the ER form of Rh1^N20I to be glycosylated. We expressed Rh1^N20I in ninaA²⁶⁹ mutants. The Rh1^N20I isolated from ninaA²⁶⁹ mutants is still detected as a 34-kDa band (Fig. 4A, lane 4). Endo H digestion does not alter the mobility of the Rh1^N20I expressed in ninaA²⁶⁹ mutants (Fig. 4B, lanes 5 and 6), demonstrating that unlike wild type Rh1, the immature, ER form of Rh1^N20I is not glycosylated. The Rh1^N20I mutant protein is not glycosylated at the Asn¹⁹⁶ site.

Analysis of the electrophoretic mobility of Rh1^N196I reveals that it is detected in a 40-kDa endo H-sensitive form (Fig. 4, A, lanes 5 and 6, and B, lanes 7 and 8). This form of Rh1^N196I must represent the normal glycosylated ER form. Although a ninaA²⁶⁹ mutant genetic background is necessary to maintain wild type rhodopsin in the high molecular mass ER form, this ER form was also detected in Rh1^N196I mutants (Fig. 4A, lanes 5 and 6). These data show that despite the capability for proper glycosylation, Rh1^N196I is not efficiently transported properly through the secretory pathway. These data are consistent with the localization of Rh1^N196I to the ER (Fig. 1E) and the accumulation of ER cisternae in the Rh1^N196I mutants (Fig. 2C). The 40-kDa molecular mass for Rh1^N196I indicates that there is no oligosaccharide chain loss. If Asn¹⁹⁶ can be glycosylated, we would predict that elimination of the Asn¹⁹⁶ site would reduce the number of glycan chains and hence lower the molecular mass of the immature form. This is not observed. Thus, the data are only consistent with a model in which Asn²⁰ is the sole site for in vivo glycosylation.

Rh1^N20I Has Dominant Effects on Rh1 Maturation and Retinal Degeneration-- Prior work established that several mutations in Rh1 act dominantly to cause retinal degeneration and that the retinal degeneration results from the interference in the maturation of the wild type Rh1 by the mutant proteins (34, 50). To examine whether defects in glycosylation and transport of Rh1^N20I interfere with the transport of wild type Rh1, we examined the localization of wild type Rh1 in the presence of the Rh1^N20I mutants. We utilized transgenic flies expressing a wild type Rh1 gene tagged with a 12-amino acid epitope tag (Rh1-bov) corresponding to a sequence at the C terminus of bovine rhodopsin (35, 36). The 1D4 antibody, directed to the bov-epitope tag, does not detect Rh1 lacking the epitope tag, and the epitope does not interfere with the biosynthesis or function of Rh1 (34). The Rh1-bov transgene was expressed in a wild type, ninaA²⁶⁹, or Rh1^N20I mutant genetic background. The expression of the wild type Rh1 was specifically monitored using the 1D4 antibody. Fig. 1F shows that in the Rh1^N20I mutant background, there is an accumulation of wild type Rh1 within the perinuclear ER. In addition, the photoreceptors accumulate ER cisternae (Fig. 2D), a characteristic of the Rh1^N20I mutant. We confirmed the ER localization by examining the presence of immature forms of the wild type Rh1 protein by immunoblotting. When wild type Rh1 is expressed in flies that are genetically ninaE^I17 (not shown) or ninaE^I17/+, Rh1 is detected exclusively in the low molecular mass mature form (Fig. 4C, lane 1). As expected, in the ninaA²⁶⁹ mutants, Rh1 is detected in the high molecular mass immature ER form (Fig. 4C, lane 2). Consistent with transport defects and ER retention, some Rh1 is detected in the high molecular mass immature ER form in the Rh1^N20I mutant background (Fig. 4C, lane 3). Together, these data indicate that Rh1^N20I interferes with the maturation of wild type Rh1.

We also investigated retinal degeneration in Rh1^N20I and Rh1^N196I mutants by examining photoreceptors at 5 weeks of age. Although wild type photoreceptors at the same age retain prominent rhabdomeres in the R1-6 cells and lack vesicular accumulations (Fig. 5A), Rh1^N20I and Rh1^N196I photoreceptors show severe retinal degeneration. The R1-6 rhabdomeres are completely lost, with only the R7 cell rhabdomeres remaining (Fig. 5, B and C). The R7 cell does not express the Rh1 rhodopsin and therefore is not directly affected by these mutations. Fig. 5D shows the morphology of the photoreceptors expressing both Rh1^N20I and wild type Rh1. This micrograph shows that Rh1^N20I mutant acts dominantly to cause severe retinal defects including reduced rhabdomere size, internalized rhabdomeres, membrane accumulations, and progressive, late onset, retinal degeneration.

View larger version (175K): [in this window] [in a new window]   Fig. 5.   Rh1N20I and Rh1N196I mutants undergo retinal degeneration. Electron micrographs of cross-sectioned photoreceptor cells, 5 weeks post-eclosion. A, wild type photoreceptors display normal morphology. Scale bar, 0.9 µm. B, Rh1N20I photoreceptors lack rhabdomeres in the R1-6 cells. The R7 cell rhabdomere remains because Rh1 is not expressed in the R7 and R8 photoreceptor cells. Scale bar, 0.9 µm. C, Rh1N196I photoreceptors also lack rhabdomeres in the R1-6 cells. Scale bar, 0.9 µm. D, photoreceptor cells expressing both Rh1N20I and wild type Rh1 shows reduced rhabdomeres, internalized rhabdomeres, and membrane accumulations (arrows). Scale bar, 1.0 µm. R, rhabdomere.

NinaA Associates with Rh1^N20I and Rh1^N196I-- We have previously shown that the cyclophilin homolog, NinaA, is a resident of the secretory pathway and is required for the transport of Rh1 opsin from the ER (10). Rh1 maturation is tightly linked to the levels of NinaA, and NinaA and Rh1 physically associate in a biologically relevant and specific complex (31). NinaA functions as a molecular chaperone in the folding, transport, and/or stability of Rh1 opsin during biosynthesis (10, 31). We first determined that NinaA protein levels in Rh1^N20I, Rh1^N196I, and ninaE^I17 mutants are indistinguishable from wild type protein levels (Fig. 6A). These data confirm that although NinaA is required by Rh1 for its biosynthesis, NinaA protein levels are not tightly regulated by the presence, absence, or glycosylation state of Rh1.

View larger version (13K): [in this window] [in a new window]   Fig. 6.   Rh1N20I and Rh1N196I associate with the molecular chaperone NinaA. A, NinaA protein levels are normal in the Rh1N20I and Rh1N196I mutants. Immunoblot probed with an antibody directed to NinaA. Lane 1, NinaA protein in wild type flies (WT). Lane 2, NinaA protein in ninaEI17 flies (null for Rh1). Lane 3, ninaA269 null allele does not express NinaA. Lane 4, NinaA protein in mutant flies expressing Rh1N20I. Lane 5, NinaA is not present in the ninaA269 mutant flies expressing Rh1N20I. Lane 6, NinaA protein in mutant flies expressing the Rh1N196I. Extracts from 20 heads were loaded in each lane. All flies were 0-2 days old. B and C, Rh1-NinaA complexes isolated by immunoaffinity chromatography. The bound fraction was eluted and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting for Rh1 protein (4C5 mouse monoclonal antibody) (B) and NinaA protein (rabbit polyclonal antibody) (C). B, immunoblot probed for Rh1. Lane 1, elution of bound fractions from wild type fly extracts (WT) reveals that Rh1 binds to the immunoaffinity column. Lane 2, Rh1 is not bound to the column when extracts from ninaEI17 flies are applied (flies carrying a deletion in the endogenous Rh1 gene). Lane 3, Rh1N20I binds to the column. Because Rh1N20I protein levels are reduced, twice the number of heads were used. Lane 4, Rh1N196I binds to the column. The IgG light chain of the 4C5 antibody is detected by the secondary antibody used in the immunoblot. C, immunoblot probed for NinaA. Lane 1, elution of bound fractions from wild type fly extracts (WT) reveals that NinaA binds to the immunoaffinity column and co-elutes with Rh1 as shown in an earlier study (31). Lane 2, NinaA is not bound to the column when extracts from ninaEI17 flies are applied. Lane 3, NinaA co-elutes with Rh1N20I. Lane 4, NinaA co-elutes with the Rh1N196I. The mouse IgG light chain is not detected by the rabbit polyclonal antibody directed to NinaA.

The in vivo association of Rh1^N20I and Rh1^N196I with NinaA was assessed by co-immunoaffinity experiments (31). As previously documented, NinaA is readily isolated in a complex with wild type Rh1 (Fig. 6, B, lane 1, and C, lane 1) but does not bind to or elute from the immunoaffinity column in the absence of Rh1 (extracts from the Rh1 null allele, ninaE^I17) (Fig. 6, B, lane 2, and C, lane 2) (31). We also detect NinaA association with Rh1^N20I, even though this mutant form of Rh1 is not glycosylated (Fig. 6, B, lane 3, and C, lane 3). These data indicate that NinaA does not require the presence of N-linked oligosaccharides to interact with Rh1. Additionally, NinaA associates with Rh1^N196I (Fig. 6, B, lane 4, and C, lane 4). These data indicate that the observed defects in transport of these mutant forms of Rh1 is not due to a failure to associate with the NinaA chaperone.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

N-Linked oligosaccharides carry out numerous functions during protein biosynthesis including protein folding, preventing the aggregation of folding intermediates by rendering them more soluble and stable, and assisting in chaperone binding. (1, 2, 29). However, the role of glycosylation in rhodopsin folding and maturation has been controversial. In the present study, we evaluated whether the acquisition of N-linked oligosaccharides is required for the maturation of Rh1 and interaction with its molecular chaperone NinaA. We constructed transgenic Drosophila expressing Rh1^N20I and Rh1^N196I mutant forms of rhodopsin by making isoleucine substitutions at the two consensus glycosylation sites. Isoleucine was chosen because it has a nonreactive and nonpolar side chain and is not expected to have a major role in altering secondary structure. This substitution has been used in other studies to eliminate glycosylation sites (51, 52).

Using these mutants we showed that in vivo Rh1^N20I is the sole site for glycosylation. Nonglycosylated Rh1^N20I accumulates in the secretory pathway leading to a buildup of ER and dilation of the Golgi complex. The presence of large dilated Golgi complexes in the photoreceptor cells is consistent with the notion that some of the Rh1^N20I exits the ER, but it is not processed properly in the Golgi complex. Similar changes in ER and Golgi membranes have been observed when there is an accumulation of proteins in the incorrect compartments. For example, this has been observed in Chinese hamster ovary cells overexpressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (53, 54), in sec mutants in yeast (55, 56), and in mutations causing defects in Rh1 transport in Drosophila (10, 31, 34, 50). Immunoblot and ERG analyses in conjunction with the small rhabdomere size indicate that Rh1^N20I protein levels are severely reduced. Thus, the Rh1^N20I retained in the secretory pathway compartments is presumably degraded.

Although the levels of Rh1^N196I mutant protein are higher than Rh1^N20I, the Rh1^N196I mutant protein also accumulates in the secretory pathway. Retention is not due to a defect in glycosylation, because the Rh1^N196I mutant protein is glycosylated. Previous studies have shown that certain mutations in Drosophila Rh1 as well as in vertebrate rhodopsins lead to defects in rhodopsin folding and maturation (24, 25, 34, 50, 57). One speculation for a mechanism leading to the misfolding and retention of the Rh1^N196I mutant is its proximity to a necessary disulfide bond between Cys¹²³ and Cys²⁰⁰. The corresponding disulfide bond between Cys¹¹⁰/Cys¹⁸⁷ is essential for maturation of bovine rhodopsin (58-60). Both the Rh1^N20I and Rh1^N196I mutant proteins that are successfully transported to the rhabdomeres are competent to carry out phototransduction.

In addition to glycosylation, Rh1 also requires the cyclophilin homolog NinaA as a molecular chaperone for its exit from the ER and proper transport through the secretory pathway (10). In contrast to two other chaperones, calnexin and calreticulin, which utilize oligosaccharides as the predominant mechanism for interaction with their glycoproteins (2), the NinaA association with Rh1 is not dependent on Rh1 glycosylation. Therefore, defective transport of nonglycosylated Rh1 cannot be explained by a failure to associate with the NinaA chaperone. However, association of nonglycosylated Rh1 with NinaA may not ensure a functional interaction leading to Rh1 maturation. We have previously shown that certain mutant alleles of NinaA associate with Rh1 but fail to promote Rh1 maturation (31). Alternatively, oligosaccharide binding proteins such as calnexin or calreticulin may be required for Rh1 maturation.

Rh1^N20I and Rh1^N196I mutant flies undergo an age-related retinal degeneration, resulting in the complete loss of rhabdomeres and clear signs of cellular degeneration by 5 weeks of age. The cellular signaling events that occur between defects in Rh1 transport and retinal degeneration are not known, but defects in Rh1 transport and a reduction in Rh1 protein are known to lead to retinal degeneration in Drosophila (13, 14, 34, 50, 61).

Mutations in glycosylation of Rh1 act dominantly to cause retinal degeneration. The mechanisms by which the mutant rhodopsin proteins cause dominant retinal degeneration have been the topic of intense investigation because of its prevalence in human adRP (16-18). We have previously shown that adRP-like dominance occurs in Drosophila and that the retinal degeneration results from the interference in the maturation of the wild type Rh1 by the mutant proteins (34, 50, 61). We have proposed that rhodopsin-protein complexes may be prevented from maturing if they contain one or more defective molecules. If these complexes contain multiple rhodopsin molecules, the presence of mutant rhodopsins could confer a profound inhibitory effect on the maturation of the whole complex. Four of the mutations that we identified in Drosophila correspond to identical mutations identified in families with adRP (34), suggesting that similar mechanisms of retinal degeneration occur in both flies and humans. Many adRP mutations in vertebrate rhodopsin are thought to lead to retinal degeneration by producing misfolded rhodopsins that are not transported properly through the secretory pathway (62-66). The identification of the T4K, N15S, and T17M point mutations in rhodopsin in adRP patients indicates that glycosylation defects act dominantly to cause retinal degeneration. Here, we show that Rh1^N20I is retained in the secretory pathway and also interferes with the transport and maturation of wild type Rh1, triggering severe retinal defects and retinal degeneration. These findings provide a potential mechanism for how defects in rhodopsin glycosylation lead to retinal degeneration in adRP.

	ACKNOWLEDGEMENTS

We thank D. Bownds, S. Britt, S. Fliesler, L. Levin, G. Panganiban, D. Papermaster, and A. Polans for valuable discussions and comments on the manuscript, P. K. Kurada for assistance in construction of the Rh1^N196I stock, and C. S. Zuker for generously providing reagents and fly stocks, including the antibody directed to NinaA. We also thank T. James, A. Kaupanger, J. Newman, and S. Seidel for technical assistance and B. Krieber and B. Ganetzky for Drosophila maintenance support. We are grateful to J. Loeffelholz for excellent assistance with the computer graphics. S. Carroll and S. Paddock generously provided expertise and confocal microscope, and R. Bromberg provided excellent assistance in the electron microscopy facility.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant EY08768, by the Retina Research Foundation, the Howard Hughes Medical Institute, the Foundation Fighting Blindness, Fight-For-Sight, and the Research to Prevent Blindness foundation (to N. J. C.), and by National Institutes of Health Grant EY06808 (to J. E. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Ophthalmology & Visual Science and the Dept. of Genetics, University of Wisconsin, Madison, WI 53706. Tel.: 608-265-5398; Fax: 608-265-6021; E-mail: njcolley@facstaff.wisc.edu.

Published, JBC Papers in Press, May 15, 2000, DOI 10.1074/jbc.M002668200

	ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; Rh1, rhodopsin; endo H, endoglycosidase H; adRP, autosomal dominant retinitis pigmentosa; ERG, electroretinogram; PDA, prolonged depolarizing afterpotential; bov, epitope tag.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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