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• W1974135959 abstract "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 forN-linked glycosylation (N20I and N196I). We show that Asn20 is the sole site for glycosylation. The Rh1N20I protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1N20I; therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1N20I/NinaA interaction. We further show that Rh1N20I 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. 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 forN-linked glycosylation (N20I and N196I). We show that Asn20 is the sole site for glycosylation. The Rh1N20I protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1N20I; therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1N20I/NinaA interaction. We further show that Rh1N20I 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. endoplasmic reticulum rhodopsin endoglycosidase H autosomal dominant retinitis pigmentosa electroretinogram prolonged depolarizing afterpotential epitope tag The correct folding, assembly, and subcellular distribution of many glycoproteins is dependent on the proteins undergoingN-linked glycosylation within the endoplasmic reticulum (ER) (1Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (563) Google Scholar, 2Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar).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 (3Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar). Photoreceptors in both Drosophila and vertebrates utilize a G protein-coupled receptor, rhodopsin, for vision. The major rhodopsin in Drosophila, Rh1, like vertebrate rhodopsins, undergoesN-linked glycosylation during biosynthesis (4Heller J. Lawrence M.A. Biochemistry. 1970; 9: 864-869Crossref PubMed Scopus (84) Google Scholar, 5Kean E.L. Plantner J.J. Exp. Eye Res. 1976; 23: 89-104Crossref PubMed Scopus (12) Google Scholar, 6Fukuda M.N. Papermaster D.S. Hargrave P.A. J. Biol. Chem. 1979; 254: 8201-8207Abstract Full Text PDF PubMed Google Scholar, 7Papermaster D.S. Schneider B.G. McDevitt D. Cell Biology of the Eye. Academic Press, San Diego1982: 475-531Crossref Google Scholar, 8Fliesler S.J. Ohyama O. Muramatsu T. Furuta S. Proceedings of the Kagoshima International Symposium on Glycoconjugates in Medicine. Professional Postgraduate Services, Tokyo1988: 316-323Google Scholar, 9Huber A. Smith D.P. Zuker C.S. Paulsen R. J. Biol. Chem. 1990; 265: 17906-17910Abstract Full Text PDF PubMed Google Scholar, 10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Rh1 displays 22% amino acid identity with human rhodopsin (11O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M.L. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 12Zuker C.S. Cowman A.F. Rubin G.M. Cell. 1985; 40: 851-858Abstract Full Text PDF PubMed Scopus (331) Google Scholar). Since the initial finding that Drosophila Rh1 mutations lead to photoreceptor degeneration (13O'Tousa J.E. Leonard D.S. Pak W.L. J. Neurogenet. 1989; 6: 41-52Crossref PubMed Scopus (82) Google Scholar, 14Leonard D.S. Bowman V.D. Ready D.F. Pak W.L. J. Neurobiol. 1992; 23: 605-626Crossref PubMed Scopus (85) Google Scholar), over 90 distinct mutations in the human rhodopsin gene have been identified in patients with autosomal dominant retinitis pigmentosa (adRP) (15Dryja T. Li T. Hum. Mol. Genet. 1995; 4: 1739-1743Crossref PubMed Scopus (248) Google Scholar, 16Daiger S.P. Sullivan L.S. Rodriguez J.A. Behavioral Brain Sci. 1995; 18: 452-467Crossref Google Scholar, 17Rattner A. Sun H. Nathans J. Annu. Rev. Genet. 1999; 33: 89-131Crossref PubMed Scopus (197) Google Scholar, 18Van Soest S. Westerveld A. de Jong P.T. Bleeker-Wagemakers E.M. Bergen A.A. Survey Ophthalmol. 1999; 43: 321-334Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). 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 (16Daiger S.P. Sullivan L.S. Rodriguez J.A. Behavioral Brain Sci. 1995; 18: 452-467Crossref Google Scholar). Vertebrate rhodopsin has two consensus sites for N-linked glycosylation at N2 and N15 and is glycosylated at both sites (6Fukuda M.N. Papermaster D.S. Hargrave P.A. J. Biol. Chem. 1979; 254: 8201-8207Abstract Full Text PDF PubMed Google Scholar). 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 (16Daiger S.P. Sullivan L.S. Rodriguez J.A. Behavioral Brain Sci. 1995; 18: 452-467Crossref Google Scholar, 19Li Z.Y. Jacobson S.G. Milam A.H. Exp. Eye Res. 1994; 58: 397-408Crossref PubMed Scopus (101) Google Scholar, 20Papermaster D.S. Nat. Med. 1995; 1: 874-875Crossref PubMed Scopus (19) Google Scholar). 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 (21O'Tousa J.E. Visual Neurosci. 1992; 8: 385-390Crossref PubMed Scopus (48) Google Scholar, 22Brown G. Chen D.M. Christianson J.S. Lee R. Stark W.S. Visual Neurosci. 1994; 11: 619-628Crossref PubMed Scopus (15) Google Scholar, 23Katanosaka K. Tokunaga F. Kawamura S. Ozaki K. FEBS Lett. 1998; 424: 149-154Crossref PubMed Scopus (34) Google Scholar). 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 (24Sung C.H. Schneider B.G. Agarwal N. Papermaster D.S. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8840-8844Crossref PubMed Scopus (404) Google Scholar, 25Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (194) Google Scholar). Another study indicates that transport defects can be overcome with vitamin A supplementation (11-cisretinal) (26Li T. Sandberg M.A. Pawlyk B.S. Rosner B. Hayes K.C. Dryja T.P. Berson E.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11933-11938Crossref PubMed Scopus (163) Google Scholar), indicating that the maturation defect may not be a direct consequence of lack of glycosylation. Tunicamycin, which blocks the formation of precursor oligosaccharides required forN-linked glycosylation, has also been used to study the role of glycosylation in rhodopsin biosynthesis (25Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (194) Google Scholar, 27Fliesler S.J. Basinger S.F. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1116-1120Crossref PubMed Scopus (55) Google Scholar, 28Fliesler S.J. Rayborn M.E. Hollyfield J.G. J. Cell Biol. 1985; 100: 574-587Crossref PubMed Scopus (75) Google Scholar). In Rana pipiens, tunicamycin treatment causes nonglycosylated opsin to accumulate in the ER (27Fliesler S.J. Basinger S.F. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 1116-1120Crossref PubMed Scopus (55) Google Scholar). 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 (28Fliesler S.J. Rayborn M.E. Hollyfield J.G. J. Cell Biol. 1985; 100: 574-587Crossref PubMed Scopus (75) Google Scholar). In addition, tunicamycin does not prevent rhodopsin transport in cell culture, indicating that glycosylation is not required for rhodopsin maturation (25Kaushal S. Ridge K.D. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4024-4028Crossref PubMed Scopus (194) Google Scholar). 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 (19Li Z.Y. Jacobson S.G. Milam A.H. Exp. Eye Res. 1994; 58: 397-408Crossref PubMed Scopus (101) Google Scholar). 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 (1Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (563) Google Scholar, 2Helenius A. Trombetta E.S. Hebert D.N. Simons J.F. Trends Cell Biol. 1997; 7: 193-200Abstract Full Text PDF PubMed Scopus (345) Google Scholar, 29Fiedler K. Simons K. Cell. 1995; 81: 309-312Abstract Full Text PDF PubMed Scopus (276) Google Scholar, 30Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1066) Google Scholar). InDrosophila 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 (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 31Baker E.K. Colley N.J. Zuker C.S. EMBO J. 1994; 13: 4886-4895Crossref PubMed Scopus (276) Google Scholar). 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 (32Hartl F.U. Nature. 1996; 381: 571-579Crossref PubMed Scopus (3130) Google Scholar). Drosophila is an excellent model organism for analyzing thein 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 Rh1N20I and Rh1N196I forms inDrosophila. The transgenic animals expressing Rh1N20I and Rh1N196I were analyzed for rhodopsin maturation, interaction with NinaA, and retinal degeneration. The wild type strain used in these studies is Drosophila melanogaster w1118 . ninaAP269 (33Stephenson R.S. O' Tousa J. Scavarda N.J. Randall L.L. Pak W.L. Cosens D. Vince-Price D. Biology of Photoreceptors. Cambridge University Press, Cambridge, UK1983: 477-501Google Scholar) is a null allele. ninaE is the structural gene for Rh1, and theninaEI17 strain is a null allele of Rh1 (11O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M.L. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (383) Google Scholar,12Zuker C.S. Cowman A.F. Rubin G.M. Cell. 1985; 40: 851-858Abstract Full Text PDF PubMed Scopus (331) Google Scholar). The Rh1N20I and Rh1N196I mutant forms of Rh1 were created by in vitro mutagenesis and P-element transformation as described in O'Tousa (21O'Tousa J.E. Visual Neurosci. 1992; 8: 385-390Crossref PubMed Scopus (48) Google Scholar) and placed in aninaEI17 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) (34Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3070-3074Crossref PubMed Scopus (212) Google Scholar). The bov-epitope corresponds to the C terminus of bovine rhodopsin and is recognized by the 1D4 antibody (35MacKenzie D. Arendt A. Hargrave P. McDowell J.H. Molday R.S. Biochemistry. 1984; 23: 6544-6549Crossref PubMed Scopus (181) Google Scholar, 36Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (387) Google Scholar). All stocks were constructed using standard balancer stocks (37Lindsley D.L. Zimm G.G. The Genome of Drosophila melanogaster. Academic Press, San Diego1992Google Scholar). Immunocytochemistry was carried out according to Colley et al. (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar). 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 2.3m sucrose and frozen in liquid nitrogen for cryoultramicrotomy (38Tokuyasu K.T. Histochem. J. 1989; 21: 163-171Crossref PubMed Scopus (359) Google Scholar, 39Tokuyasu K.T. J. Microsc. 1986; 143: 139-149Crossref PubMed Scopus (408) Google Scholar, 40Colley N.J. Tokuyasu K.T. Singer S.J. J. Cell Sci. 1990; 95: 11-22PubMed Google Scholar). 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 (35MacKenzie D. Arendt A. Hargrave P. McDowell J.H. Molday R.S. Biochemistry. 1984; 23: 6544-6549Crossref PubMed Scopus (181) Google Scholar,36Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (387) Google Scholar, 41de Couet H.G. Tanimura T. Eur. J. Cell Biol. 1987; 44: 50-56Google Scholar). 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 (42Baumann O. Walz B. Cell Tissue Res. 1989; 222: 511-522Google Scholar) as described previously (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 34Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3070-3074Crossref PubMed Scopus (212) Google Scholar). 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. Electroretinograms (ERGs) were carried out on 3–5-day-old flies according to published procedures (43Larrivee D.C. Conrad S.K. Stephenson R.S. Pak W.L. J. Gen. Physiol. 1981; 78: 521-545Crossref PubMed Scopus (70) Google Scholar, 44Stephenson R.S. O'Tousa J. Scavarda N.J. Randall L.L. Pak W.L. Symp. Soc. Exp. Biol. 1983; 36: 477-501PubMed Google Scholar). 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 (45Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207529) Google Scholar), and electroblotted onto nitrocellulose filters (46Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The nitrocellulose was incubated with either of the 4C5 and 1D4 mouse monoclonal antibodies directed to Rh1 or the bov-epitope tag, respectively (35MacKenzie D. Arendt A. Hargrave P. McDowell J.H. Molday R.S. Biochemistry. 1984; 23: 6544-6549Crossref PubMed Scopus (181) Google Scholar, 36Oprian D.D. Molday R.S. Kaufman R.J. Khorana H.G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8874-8878Crossref PubMed Scopus (387) Google Scholar, 41de Couet H.G. Tanimura T. Eur. J. Cell Biol. 1987; 44: 50-56Google Scholar), or a rabbit polyclonal antibody directed to NinaA (gift of A. Becker and C. S. Zuker) (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 31Baker E.K. Colley N.J. Zuker C.S. EMBO J. 1994; 13: 4886-4895Crossref PubMed Scopus (276) Google Scholar, 47Stamnes M.A. Shieh B.-H. Chuman L. Harris G.L. Zuker C.S. Cell. 1991; 65: 219-227Abstract Full Text PDF PubMed Scopus (221) Google Scholar). 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 (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar). Flies, 3 days old or younger, were subjected to affinity chromatography essentially as described previously (31Baker E.K. Colley N.J. Zuker C.S. EMBO J. 1994; 13: 4886-4895Crossref PubMed Scopus (276) Google Scholar) using approximately 3,000 heads for wild type, ninaEI17, and Rh1N196I, and approximately 6,000 heads for Rh1N20I. 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-β-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 (45Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207529) Google Scholar) 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. 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. 1 A). InDrosophila 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 (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 48Montell C. Annu. Rev. Cell Dev. Biol. 1999; 15: 231-268Crossref PubMed Scopus (252) Google Scholar). Rh1 is a G protein-coupled receptor that has two potential sites for N-linked glycosylation, Asn20 and Asn196 (Fig. 1 B) (11O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M.L. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (383) Google Scholar, 12Zuker C.S. Cowman A.F. Rubin G.M. Cell. 1985; 40: 851-858Abstract Full Text PDF PubMed Scopus (331) Google Scholar,49Britt S.G. Feiler R. Kirschfeld K. Zuker C.S. Neuron. 1993; 11: 29-39Abstract Full Text PDF PubMed Scopus (42) Google Scholar). 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. 1 B). We then constructed transgenic flies expressing these mutant forms, Rh1N20I and Rh1N196I, to examine the in vivo role of glycosylation in Rh1 transport through the secretory pathway. In wild type flies, Rh1 localizes predominantly to the rhabdomeres of the R1–6 photoreceptor cells (Fig. 1 C). In contrast, the photoreceptor cells expressing Rh1N20I (Fig. 1 D) and Rh1N196I (Fig. 1 E) display an altered distribution of Rh1. In both cases, substantial amounts of Rh1N20I and Rh1N196I 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. 2 A). The Golgi membranes were particularly prominent in the Rh1N20I mutant (Fig.2 B), 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 (30Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1066) Google Scholar). The quality control retention mechanisms are not 100% effective, because some of the Rh1N20I and Rh1N196I 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.3 A 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 (44Stephenson R.S. O'Tousa J. Scavarda N.J. Randall L.L. Pak W.L. Symp. Soc. Exp. Biol. 1983; 36: 477-501PubMed Google Scholar). An ERG trace of Rh1N20I is shown in Fig. 3 B. The reduced amplitude of the light response and the lack of the PDA demonstrate that the mutant flies express low levels of Rh1N20I protein. However, the small amount of Rh1N20I protein that is transported to the rhabdomeres is functional (21O'Tousa J.E. Visual Neurosci. 1992; 8: 385-390Crossref PubMed Scopus (48) Google Scholar, 22Brown G. Chen D.M. Christianson J.S. Lee R. Stark W.S. Visual Neurosci. 1994; 11: 619-628Crossref PubMed Scopus (15) Google Scholar). Rh1N196I flies display an ERG response, demonstrating that Rh1N196I is also functional (Fig. 3 C). The larger amplitude of the ERG response suggests that more Rh1N196I is successfully transported to the rhabdomeres compared with Rh1N20I. However, the absence of the PDA in Rh1N196I mutants reflects a reduction in functional Rh1N196I protein relative to wild type. The glycosylation state allows us to distinguish between rhabdomeric and ER forms of Rh1 (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 34Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3070-3074Crossref PubMed Scopus (212) Google Scholar). Wild type Rh1 is detected as the mature rhabdomeric (34-kDa) form (Fig.4 A, lane 1). In ninaA269 mutants, Rh1 does not exit the ER (10Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (278) Google Scholar) and accumulates as a high molecular mass glycosylated ER form (Fig. 4 A, lane 2). Treatment ofninaA269 extracts with endo H increases the electrophoretic mobility of the Rh1 opsin such that it now migrates with the mature form (Fig. 4 B, lanes 3 and4). Because endo H selectively cleaves immature high mannosyl oligosaccharide chains (3Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar), this form of Rh1 must represent the glycosylated ER form. Rh1N20I is detected as a 34-kDa form, and its expression is severely reduced relative to wild type (Fig. 4 A, lane 3). These results are consistent with the smaller amplitude in the light response by ERG analysis (Fig.3 B) and the reduction in the size of the rhabdomeres (Fig.2 B). We have shown above that Rh1N20I displays defective transport (Fig. 1 D), indicating that if Rh1N20Iwas 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 Rh1N20I to be glycosylated. We expressed Rh1N20I in ninaA269 mutants. The Rh1N20I isolated from ninaA269mutants is still detected as a 34-kDa band (Fig. 4 A,lane 4). Endo H digestion does not alter the mobility of the Rh1N20I expressed in ninaA269mutants (Fig. 4 B, lanes 5 and 6), demonstrating that unlike wild type Rh1, the immature, ER form of Rh1N20I is not glycosylated. The Rh1N20I mutant protein is not glycosylated at the Asn196 site. Analysis of the electrophoretic mobility of Rh1N196Ireveals that it is detected in a 40-kDa endo H-sensitive form (Fig. 4, A, lanes 5 and 6, andB, lanes 7 and 8). This form of Rh1N196I 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 Rh1N196I mutants (Fig. 4 A, lanes 5and 6). These data show that despite the capability for proper glycosylation, Rh1N196I is not efficiently transported properly through the secretory pathway. These data are consistent with the localization of Rh1N196I to the ER (Fig. 1 E) and the accumulation of ER cisternae in the Rh1N196I mutants (Fig. 2 C). The 40-kDa molecular mass for Rh1N196I 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 forin vivo glycosylation. 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 (34Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3070-3074Crossref PubMed Scopus (212) Google Scholar, 50Kurada P. O'Tousa J.E. Neuron. 1995; 14: 571-579Abstract Full Text PDF PubMed Scopus (115) Google Scholar). To examine whether defects in glycosylation and transport of Rh1N20I interfere with the transport of wild type Rh1, we examined the localization of wild type Rh1 in the presence of the Rh1N20I mutants. We utilized transgenic" @default.
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• W1974135959 date "2000-08-01" @default.
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• W1974135959 title "Role of Asparagine-linked Oligosaccharides in Rhodopsin Maturation and Association with Its Molecular Chaperone, NinaA" @default.
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