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• W2043941101 abstract "In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. Here, we demonstrate a multifunctional role for calnexin as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration that is enhanced by light, suggesting that calnexin's function as a Ca2+ buffer is important for photoreceptor cell survival. Our results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration. In sensory neurons, successful maturation of signaling molecules and regulation of Ca2+ are essential for cell function and survival. Here, we demonstrate a multifunctional role for calnexin as both a molecular chaperone uniquely required for rhodopsin maturation and a regulator of Ca2+ that enters photoreceptor cells during light stimulation. Mutations in Drosophila calnexin lead to severe defects in rhodopsin (Rh1) expression, whereas other photoreceptor cell proteins are expressed normally. Mutations in calnexin also impair the ability of photoreceptor cells to control cytosolic Ca2+ levels following activation of the light-sensitive TRP channels. Finally, mutations in calnexin lead to retinal degeneration that is enhanced by light, suggesting that calnexin's function as a Ca2+ buffer is important for photoreceptor cell survival. Our results illustrate a critical role for calnexin in Rh1 maturation and Ca2+ regulation and provide genetic evidence that defects in calnexin lead to retinal degeneration. G protein-coupled receptors are synthesized on membrane-bound ribosomes and undergo translocation, modification, folding, oligomeric assembly, and quality control in the endoplasmic reticulum (ER) (Ellgaard and Helenius, 2003bEllgaard L. Helenius A. Quality control in the endoplasmic reticulum.Nat. Rev. Mol. Cell Biol. 2003; 4: 181-191Crossref PubMed Scopus (1593) Google Scholar). To deal with these complex and error-prone processes, the ER has evolved a system centered around the calnexin family of molecular chaperones, which promotes the proper folding and assembly of newly synthesized glycoproteins. Calnexin is a type I transmembrane protein that, like its soluble ER homolog calreticulin, interacts with the monoglucosylated glycan (Glc1Man7-9GlcNAc2) present on folding intermediates of glycoproteins. Nascent glycoproteins associate with calnexin or calreticulin via a cycle of binding and release. (Ellgaard and Helenius, 2003aEllgaard L. Helenius A. A Chaperone System For Glycoprotein Folding: The Calnexin/Calreticulin Cycle.in: Calreticulin. Kluwer Academic/Plenum Publishers, New York2003: 19-30Crossref Google Scholar, Molinari et al., 2004Molinari M. Eriksson K.K. Calanca V. Galli C. Cresswell P. Michalak M. Helenius A. Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.Mol. Cell. 2004; 13: 125-135Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, Schrag et al., 2001Schrag J.D. Bergeron J.J.M. Li Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. The structure of calnexin, an ER chaperone involved in quality control of protein folding.Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, Ware et al., 1995Ware F.E. Vassilakos A. Peterson P.A. Jackson M.R. Lehrman M.A. Williams D.B. The molecular chaperone calnexin binds Glc1Man9GlcNAc2 oligosaccharide as an initial step in recognizing unfolded glycoproteins.J. Biol. Chem. 1995; 270: 4697-4704Crossref PubMed Scopus (374) Google Scholar). This cycle is key for ER quality control, as it inhibits aggregation, prevents premature exit from the ER, and exposes glycoproteins to accessory enzymes and folding factors (Ellgaard and Helenius, 2003aEllgaard L. Helenius A. A Chaperone System For Glycoprotein Folding: The Calnexin/Calreticulin Cycle.in: Calreticulin. Kluwer Academic/Plenum Publishers, New York2003: 19-30Crossref Google Scholar). Despite considerable understanding of the calnexin/calreticulin cycle, little is known about the requirement for calnexin in protein processing in vivo. Rhodopsin is the prototypical member of the vast G protein-coupled receptor family. As in vertebrates, Drosophila rhodopsin (Rh1) initiates the phototransduction cascade by interacting with a heterotrimeric G protein, which then activates a distinct effector enzyme, namely phospholipase C (PLC-β). Activation of PLC leads to the opening of the cation-selective TRP and TRPL channels, resulting in a dramatic rise in intracellular Ca2+ (reviewed in Hardie and Raghu, 2001Hardie R.C. Raghu P. Visual transduction in Drosophila.Nature. 2001; 413: 186-193Crossref PubMed Scopus (393) Google Scholar, Pak and Leung, 2003Pak W. Leung H. Genetic approaches to visual transduction in Drosophila melanogaster.Receptors Channels. 2003; 9: 149-167Crossref PubMed Scopus (35) Google Scholar). To become functionally active, newly synthesized rhodospin must be precisely folded and successfully navigate the secretory pathway to the phototransducing compartment of the photoreceptor cells, the rhabdomeres (Colley et al., 1991Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. The cyclophilin homolog ninaA is required in the secretory pathway.Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (273) Google Scholar). Rhabdomeres consist of numerous tightly packed microvilli containing the phototransduction machinery. The mechanisms that regulate the folding and transport of rhodopsin are essential for photoreceptor cell function and survival, as defects in rhodopsin maturation lead to retinal degeneration in both Drosophila and vertebrates (Colley et al., 1991Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. The cyclophilin homolog ninaA is required in the secretory pathway.Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (273) Google Scholar, Colley et al., 1995Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.Proc. Natl. Acad. Sci. USA. 1995; 92: 3070-3074Crossref PubMed Scopus (199) Google Scholar, Kurada and O'Tousa, 1995Kurada P. O'Tousa J.E. Retinal degeneration caused by dominant rhodopsin mutations in Drosophila.Neuron. 1995; 14: 571-579Abstract Full Text PDF PubMed Scopus (107) Google Scholar, Pacione et al., 2003Pacione L.R. Szego M.J. Ikeda S. Nishina P.M. McInnes R.R. Progress toward understanding the genetic and biochemical mechansims of inherited photoreceptor degenerations.Annu. Rev. Neurosci. 2003; 26: 657-700Crossref PubMed Scopus (126) Google Scholar, Sung and Tai, 2000Sung C.H. Tai A.W. Rhodopsin trafficking and its role in retinal dystrophies.Int. Rev. Cytol. 2000; 195: 215-267Crossref PubMed Google Scholar, Webel et al., 2000Webel R. Menon I. O'Tousa J. Colley N.J. Role of asparagine-linked glycosylation sites in rhodopsin maturation and association with its molecular chaperone, NinaA.J. Biol. Chem. 2000; 275: 24752-24759Crossref PubMed Scopus (41) Google Scholar). Protein maturation defects characterized in blinding diseases have broad implications, as protein misfolding and aggregation are characteristic of a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease (Muchowski and Wacker, 2005Muchowski P.J. Wacker J.L. Modulation of Neurodegeneration by molecular chaperones.Nat. Rev. Neurosci. 2005; 6: 11-22Crossref PubMed Scopus (780) Google Scholar). Here, we investigate whether calnexin functions as a chaperone for Rh1 and whether mutations in calnexin lead to neurodegeneration. In addition to its role as a molecular chaperone, calnexin is thought to bind Ca2+ at two distinct sites. The first resides in the long N-terminal domain localized to the lumen of the ER. The crystal structure of vertebrate calnexin shows that this luminal domain consists of two distinct regions: a compact, globular domain and a proline-rich arm called the P domain (Figure 1A). The globular domain is thought to bind a single Ca2+ ion and is also involved in glucose binding (lectin domain) (Schrag et al., 2001Schrag J.D. Bergeron J.J.M. Li Y. Borisova S. Hahn M. Thomas D.Y. Cygler M. The structure of calnexin, an ER chaperone involved in quality control of protein folding.Mol. Cell. 2001; 8: 633-644Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Although less well defined, several lines of evidence suggest that calnexin may harbor a second Ca2+-binding domain within the highly charged C-terminal cytosolic tail (C domain). Interestingly, this cytosolic domain displays structural similarity to calreticulin's luminal C domain, but is positioned on the opposite, cytosolic side of the ER membrane (Tjoelker et al., 1994Tjoelker L.W. Seyfried C.E. Eddy R.L. Byers M.G. Shows T.B. Calderon J. Schreiber R.B. Gray P.W. Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5.Biochemistry. 1994; 33: 3229-3236Crossref PubMed Scopus (91) Google Scholar). Calreticulin's C domain displays low-affinity and high-capacity Ca2+ binding and is thought to buffer luminal Ca2+ (Baksh and Michalak, 1991Baksh S. Michalak M. Expression of calreticulin in Escherichia coli and identification of its Ca2+ binding domains.J. Biol. Chem. 1991; 266: 21458-21465Abstract Full Text PDF PubMed Google Scholar). While the in vivo role of calnexin's C domain is unknown, these Ca2+-binding properties would make it ideal for buffering high concentrations of cytosolic Ca2+. Precise spatial and temporal control over Ca2+ levels is essential for phototransduction in both vertebrates and invertebrates. Furthermore, prolonged elevation of cytosolic Ca2+ can be toxic, leading to cell death (Dolph et al., 1993Dolph P.J. Ranganathan R. Colley N.J. Hardy R.W. Socolich M. Zuker C.S. Arrestin function in inactivation of G protein-coupled receptor rhodopsin in vivo.Science. 1993; 260: 1910-1916Crossref PubMed Scopus (255) Google Scholar, Fain and Lisman, 1999Fain G.L. Lisman J.E. Light, Ca2+, and photoreceptor death: New evidence for the equivalent-light hypothesis from arrestin knockout mice.Invest. Ophthalmol. Vis. Sci. 1999; 40: 2770-2772PubMed Google Scholar, Wang et al., 2005Wang T. Xu H. Oberwinkler J. Gu Y. Hardie R.C. Montell C. Light activation, adaptation, and cell survival functions of the Na+/Ca2+ exchanger CalX.Neuron. 2005; 45: 367-378Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). In Drosophila, the light-sensitive TRP and TRPL channels mediate a massive Ca2+ influx into the rhabdomeres that is essential for amplification, rapid-response kinetics, and light adaptation (Hardie and Raghu, 2001Hardie R.C. Raghu P. Visual transduction in Drosophila.Nature. 2001; 413: 186-193Crossref PubMed Scopus (393) Google Scholar). Ca2+ can rise to about 1 mM in the rhabdomeres (Postma et al., 1999Postma M. Oberwinkler J. Stavenga D.G. Does Ca2+ reach millimolar concentrations after single photon absorption in Drosophila photoreceptor microvilli?.Biophys. J. 1999; 77: 1811-1823Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar) and is removed from the rhabdomeres by a combination of the Na+/Ca2+ exchanger (CalX) (Oberwinkler and Stavenga, 2000Oberwinkler J. Stavenga D.G. Calcium imaging demonstrates colocalization of calcium influx and extrusion in fly photoreceptors.Proc. Natl. Acad. Sci. USA. 2000; 97: 8578-8583Crossref PubMed Scopus (17) Google Scholar, Wang et al., 2005Wang T. Xu H. Oberwinkler J. Gu Y. Hardie R.C. Montell C. Light activation, adaptation, and cell survival functions of the Na+/Ca2+ exchanger CalX.Neuron. 2005; 45: 367-378Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and diffusion into the cell body, where Ca2+ rises to about 10 μM. The mechanisms controlling Ca2+ in the cell body are poorly understood but presumably include sequestration by the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) (Hardie, 1996aHardie R.C. Excitation of Drosophila photoreceptors by BAPTA and ionomycin: evidence for capacitative Ca2+ entry?.Cell Calcium. 1996; 20: 315-327Crossref PubMed Scopus (34) Google Scholar) and buffering by cytosolic Ca2+-binding proteins. Here, we explore the hypothesis that calnexin serves as a buffer for cytosolic Ca2+ following the acute Ca2+ rise during phototransduction and that it may thereby help to prevent Ca2+ toxicity and promote photoreceptor cell survival. In this report, we demonstrate a multifunctional role for calnexin as both a critical molecular chaperone for Rh1 biosynthesis and a regulator of cytosolic Ca2+. Mutations in calnexin cause retinal degeneration that is enhanced by light, suggesting that calnexin's role in Ca2+ removal may be important for photoreceptor cell survival. These results provide genetic evidence that failure in Rh1 maturation and Ca2+ overload, resulting from defects in calnexin, are responsible for retinal degeneration. By screening the Zuker collection of EMS-mutagenized Drosophila lines, we identified two independent mutants that displayed a severe reduction in Rh1 compared to wild-type (wt) (Figure 2A, lanes 1–3). We established that the two mutants were allelic, as the transheterozygotes displayed reduced levels of Rh1 (Figure 2A, lane 4). Furthermore, we established that both alleles were recessive (Figure 2A, lanes 8 and 9). To identify the mutant locus responsible for the phenotype, we first used deficiency mapping to narrow the cytogenetic location to 99A7 on the third chromosome, corresponding to four genes (Figures 1C and 2A, lanes 5 and 6). We sequenced this small region and identified mutations in the coding region of calnexin in both alleles (Brody et al., 2002Brody T. Stivers C. Nagle J. Odenwald W.F. Identification of novel Drosophila neural precursor genes using a differential embryonic head cDNA screen.Mech. Dev. 2002; 113: 41-59Crossref PubMed Scopus (45) Google Scholar, Christodoulou et al., 1997Christodoulou S. Lockyer A.E. Foster J.M. Hoheisel J.D. Roberts D.B. Nucleotide sequence of a Drosophila melanogaster cDNA encoding a calnexin homologue.Gene. 1997; 191: 143-148Crossref PubMed Scopus (17) Google Scholar). We found that one allele harbored a C to T transition at nucleotide position 544, causing a premature stop codon at glutamine182, and that the other allele harbored a G to A transition at nucleotide position 356, causing a premature stop codon at tryptophan119. Based on cytology, we designated the two alleles cnx99A1 (cnx1) and cnx99A2 (cnx2), respectively. Drosophila calnexin99A (Cnx) displays 49% amino acid identity with human calnexin (chromosome 5q), shown in Figure 1B. To provide further evidence that mutations in cnx were responsible for the severe reduction in Rh1, we introduced the wt cnx gene into the cnx mutants by using a duplication for 99A that was translocated to the X chromosome [Dp(3;1) B152] and confirmed that it restored the normal function (Figures 1C and 2A, lane 7). Consistent with the presence of the stop codons, the cnx mutants displayed severely reduced levels of cnx transcript (Figure 2B, lanes 1, 2, 5, and 6). In addition, the Cnx protein was absent in both of the cnx mutants (Figure 2C, lanes 2 and 3), while it was detected in wt flies (Figure 2C, lane 1). Lane 4 confirms that the mutants were allelic. Cnx protein was not detected in the cnx1 and cnx2 mutants when they were crossed to a deficiency (Df) that eliminated 99A (Df(3R)Ptp99A[R3]) (Figure 2C, lanes 5 and 6). These data supported the hypothesis that the mutations were in the cnx gene. Calnexin is thought to play a role in the folding of a large number of proteins. Consistent with this notion, cnx was ubiquitously expressed at all stages during development and in the adult (Figure 2B). cnx transcript was detected in wt embryos (Figure 2B, lane 9), larvae (Figure 2B, lane 8), and adult heads and bodies (Figure 2B, lanes 3 and 7, respectively). cnx transcript and Cnx protein were also detected in heads from flies lacking eyes (eya1) (Figure 2B, lane 4, and Figure 2C, lane 8), indicating that cnx expression was not restricted to the eyes. Despite the widespread expression pattern of cnx, it was not required for viability or fertility of the flies, as the mutants were homozygous viable. Although Rh1 required Cnx for its expression, Cnx expression was normal in Rh1 mutants (Figure 2C, lane 7). Although Rh1 protein levels were severely reduced in the cnx mutants (Figure 2A, lanes 2 and 3), Rh1 transcript levels were normal in both (see Figure S1 in the Supplemental Data available online). These data suggest that Cnx functions post-transcriptionally and are consistent with a role for Cnx as a chaperone in Rh1 biosynthesis. To investigate the role of Cnx in Rh1 biosynthesis, we assessed the kinetics of Rh1 maturation in the cnx1 mutant (Figure 3A). We utilized transgenic flies carrying the Rh1 gene under the control of a heat-shock promoter (hs) and tagged with an epitope corresponding to 12 amino acids at the C terminus of bovine rhodopsin (hs-Rh1-bov transgene) (Colley et al., 1995Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.Proc. Natl. Acad. Sci. USA. 1995; 92: 3070-3074Crossref PubMed Scopus (199) Google Scholar). The transgene was introduced into wt, cnx1, and ninaAP269 mutant flies. To initiate Rh1 biosynthesis, the transgenic flies were given a 1 hr heat-pulse at 37°C, shifted back to 22°C, and assayed at specific time points. We followed the fate of the heat-induced Rh1 protein by using a monoclonal antibody (1D4) directed to the epitope tag (bov). No Rh1 expression was detected in the flies prior to heat-pulse (Figure 3A, lane 1, no pulse). In wt flies, Rh1 was initially detected as immature high-MW forms that were converted to the mature low-MW form by 18 hr. In the cnx1 mutant, Rh1 was also initially detected as immature high-MW forms but was significantly reduced by 24 hr. By 48 hr, very little Rh1 was detected, suggesting that most of the Rh1 was degraded. The failure of Rh1 to mature in the cnx1 mutant was similar to the fate of Rh1 in the ninaA mutant (Figure 3A). We have previously shown that NinaA is a chaperone specifically required for Rh1 biosynthesis and maturation (Baker et al., 1994Baker E.K. Colley N.J. Zuker C.S. The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin.EMBO J. 1994; 13: 4886-4895Crossref PubMed Scopus (270) Google Scholar, Colley et al., 1991Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. The cyclophilin homolog ninaA is required in the secretory pathway.Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (273) Google Scholar). As in the cnx1 mutant, Rh1 was initially detected as immature high-MW forms in the ninaA mutant. In contrast to the cnx1 mutant, where most of the Rh1 was degraded, Rh1 accumulated in the immature high-MW form in the ninaA mutant. We also followed the subcellular localization of Rh1 in the pulse-chase experiments (Figure 3B). In wt flies, by 8 hr following the heat-pulse, Rh1 immunolocalized to the ER in a perinuclear fashion, was detected in a punctate pattern consistent with transport vesicles, and was detected in the rhabdomeres. By 12 hr, more Rh1 was detected in the rhabdomeres, and by 24 hr, mature Rh1 localized solely to the rhabdomeres. This represents the normal progression for Rh1 maturation and transport through the secretory pathway. In the cnx1 mutant, by 8 hr, Rh1 was detected predominantly in the ER. By 12 hr, Rh1 was still most noticeable in the ER. By 24 hr, Rh1 labeling was detected in both the ER and rhabdomeres, but was significantly fainter than wt. These results show that in the cnx mutant, while most Rh1 was degraded, some Rh1 successfully evaded the quality-control mechanisms and was transported to the rhabdomeres. In the ninaA mutants, at 8, 12, and 24 hr, Rh1 was detected primarily in the ER. A very small amount of Rh1 was detected in the rhabdomeres of ninaA mutants, again indicating that a small amount of Rh1 evaded the ER's quality-control system. It is possible that Cnx and NinaA are part of a protein-processing pathway, ensuring proper folding and quality control of Rh1 during biosynthesis. To gain insights into the epistatic relationship between the two chaperones, we created mutant flies that were defective in both cnx and ninaA. The ninaAP269;cnx1 double mutant displayed severely reduced levels of Rh1, comparable to those seen in the cnx1 mutant alone (Figure 3C, lanes 4 and 2, respectively). These data demonstrate that in the double mutant, Rh1 was effectively degraded (as in cnx, Figure 3C, lane 2) rather than accumulating in the ER (as in ninaA, Figure 3C, lane 3). Therefore, cnx is epistatic to ninaA, as the phenotype of the cnx mutation overrides the phenotype of the ninaA mutation in the double mutant. Because the two chaperones are required for Rh1 biosynthesis, we investigated the levels of NinaA protein in the cnx mutants. Figure 3D shows that the NinaA levels in cnx were indistinguishable from wt levels, suggesting that the defects in Rh1 were the result of a lack of Cnx, rather than a lack of NinaA. The association of Cnx with Rh1 was assessed by coimmunoaffinity experiments (Baker et al., 1994Baker E.K. Colley N.J. Zuker C.S. The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin.EMBO J. 1994; 13: 4886-4895Crossref PubMed Scopus (270) Google Scholar). Cnx was isolated in a stable complex with Rh1 (Figure 3E, lane 2), but did not bind to or elute from the immunoaffinity column in the absence of Rh1 (extracts from the Rh1 null allele, ninaEI17) (Figure 3E, lane 3). These data indicated that Cnx and Rh1 physically associate in a protein complex, consistent with a role for Cnx as a molecular chaperone for Rh1. Examination of the cnx mutants revealed that they displayed an age-related retinal degeneration (Figure 4). Photoreceptor cells in 1-day-old cnx mutants displayed diminished rhabdomere size (Figure 4B) as compared to wt (Figure 4A). They also displayed accumulations of rough ER membranes, dilated Golgi, and various types of deposits (Figures 4B and 4C). These secretory pathway defects were consistent with a failure in Rh1 maturation. The identity of the deposits is unknown, but they may consist of degraded material within the cells. At 1 month, cnx mutants displayed a dramatic loss of rhabdomeres in the R1-6 photoreceptor cells, while the R7 and R8 photoreceptor cell rhabdomeres remained (Figures 4D and 4E, respectively). The Drosophila compound eye is made up of approximately 750 individual eye units called ommatidia. Each ommatidium contains eight photoreceptor cells (Figure 3B). Only R1-6 photoreceptors express Rh1, whereas R7 and R8 cells express a variety of different opsins (Rh3-6) (Chou et al., 1996Chou W.H. Hall K.J. Wilson D.B. Wideman C.L. Townson S.M. Chadwell L.V. Britt S.G. Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells.Neuron. 1996; 17: 1101-1115Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, Montell et al., 1987Montell C. Jones K. Zuker C. Rubin G. A second opsin gene expressed in the ultraviolet-sensitive R7 photoreceptor cells of Drosophila melanogaster.J. Neurosci. 1987; 7: 1558-1566Crossref PubMed Google Scholar, Papatsenko et al., 1997Papatsenko D. Sheng G. Desplan C. A new rhodopsin in R8 photoreceptors of Drosophila: evidence for coordinate expression with Rh3 in R7 cells.Development. 1997; 124: 1665-1673PubMed Google Scholar). The finding that only R1-6 rhabdomeres degenerated, and not R7 and R8, suggested that the opsins located in R7 and R8 were expressed normally. To confirm normal expression of the minor rhodopsins in R7 and R8 cells, we performed immunocytochemical analysis of Rh3, Rh4, and Rh5 opsins in the cnx mutants. We found all three opsins to be correctly localized to the rhabdomeres of the R7 and R8 cells (Figure 5), confirming that while Cnx was required by Rh1, it was not required by the R7 and R8 opsins. We examined whether ER processing of other photoreceptor proteins was defective in the cnx mutant. Immunocytochemical analysis revealed that another membrane protein, chaoptin, was present at its normal location in the rhabdomeres of the R1-8 photoreceptor cells in both cnx mutants (Figure 5D) and wt (Figure 5H). In addition, we performed immunoblotting analysis to confirm that a number of key phototransduction proteins, including the G protein α subunit (Gqα), the TRP and TRPL channels, PLC-β (norpA), arrestin 1 (Arr1), and arrestin 2 (Arr2) were all expressed at levels indistinguishable from wt (Figure 5I). These results demonstrated that while Cnx was required for processing of Rh1, it was not required for expression of these other photoreceptor cell proteins. To determine the expression pattern for Cnx, we generated polyclonal antibodies that recognized a 97 kDa band in wt flies that was not present in the mutants (Figure 2C). Cnx localized to the ER of all eight photoreceptor cells, often to ER cisternae that were tightly associated with the nuclear envelope (Figure 6). The labeling pattern for Cnx was compared to the ER proteins, InsP3R (inositol-1,3,5-trisphosphate receptor) and NinaA (Figure 6). All three proteins were expressed in the ER, but were absent from the rhabdomeres. Although the rhabdomeres of the central R7 photoreceptor cells were labeled by the InsP3R antibody, we previously showed this labeling to be nonspecific (Raghu et al., 2000Raghu P. Colley N.J. Webel R. James T. Hasan G. Danin M. Selinger Z. Hardie R.C. Normal Phototransduction in Drosophila Photoreceptors Lacking an InsP3 Receptor Gene.Mol. Cell. Neurosci. 2000; 15: 429-445Crossref PubMed Scopus (112) Google Scholar). While Cnx protein was uniquely required by Rh1 in the R1-6 cells, it was detected in the ER of all eight photoreceptor cells (Figure 6). To assess whether the retinal degeneration observed in the cnx mutants was enhanced by light activation of the phototransduction cascade, we reared the cnx mutants for 1 month in constant darkness. These flies displayed a less severe retinal degeneration (Figure 4F) compared with cnx mutants grown for 1 month on a 12:12 light-dark cycle (Figures 4D and 4E). Therefore, activation of phototransduction by light enhanced the retinal degeneration in the cnx mutants. This result is contrasted to other known mutants defective in Rh1 maturation, such as ninaA, in which the retinal degeneration was light independent (Figures 4G and 4H) (Colley et al., 1991Colley N.J. Baker E.K. Stamnes M.A. Zuker C.S. The cyclophilin homolog ninaA is required in the secretory pathway.Cell. 1991; 67: 255-263Abstract Full Text PDF PubMed Scopus (273) Google Scholar, Colley et al., 1995Colley N.J. Cassill J.A. Baker E.K. Zuker C.S. Defective intracellular transport is the molecular basis of rhodopsin-dependent dominant retinal degeneration.Proc. Natl. Acad. Sci. USA. 1995; 92: 3070-3074Crossref PubMed Scopus (199) Google Scholar, Kurada and O'Tousa, 1995Kurada P. O'Tousa J.E. Retinal degeneration caused by dominant rhodopsin mutations in Drosophila.Neuron. 1995; 14: 571-579Abstract Full Text PDF PubMed Scopus (107) Google Scholar, Webel et al., 2000Webel R. Menon I. O'Tousa J. Colley N.J. Role of asparagine-linked glycosylation sites in rhodopsin maturation and association with its molecular chaperone, NinaA.J. Biol. Chem. 2000; 275: 24752-24759Crossref PubMed Scopus (41) Google Scholar). In addition, the ninaA mutants degenerated more slowly compared to the cnx mutants (Figures 4D, 4E, and 4G). The finding that light enhanced the retinal degeneration in the cnx mutant led us to investigate whether Ca2+ influx through the light-sensitive channels contributed to the retinal degeneration. Null mutations in the gene encoding the eye-enriched PLC (norpA) eliminate the light-induced Ca2+ influx. We generated norpA;cnx double mutants and found that norpA slowed down the onset and progression of the retinal degeneration in the cnx mutants (Figures 4I and 4J). The finding that the retinal degeneration in the cnx mutants was light enhanced and slowed by norpA, in combination with previous findings that calnexin binds Ca2+ (Tjoelker et al., 1994Tjoelker L.W. Seyfried C.E. Eddy R.L. Byers M.G. Shows T.B. Calderon J. Schreiber R.B. Gray P.W. Human, mouse, and rat calnexin cDNA cloning: identification of potential calcium binding motifs and gene localization to human chromosome 5.Biochemistry. 1994; 33: 3229-3236Crossref PubMed Scopus (91) Google Scholar), prompted us to determine whether Cnx played a role in modulating Ca2+ in photoreceptor cells. Current models of phototransduction suggest that virtually all aspects of excitation and adaptation are mediated within the microvilli. Because Cnx is located in the ER, we predicted that mutations in cnx would not affect the basic light responses, but might cause defects in Ca2+ buffering in the cell body. We first investigated the basic properties of the light-induced current (LIC) by using whole-cell patch-clamp recordings of photoreceptors from dissociated ommatidia to record the elementary responses (quantum bumps) representin" @default.
• W2043941101 created "2016-06-24" @default.
• W2043941101 creator A5023583649 @default.
• W2043941101 creator A5066860292 @default.
• W2043941101 creator A5072022340 @default.
• W2043941101 date "2006-01-01" @default.
• W2043941101 modified "2023-10-16" @default.
• W2043941101 title "Calnexin Is Essential for Rhodopsin Maturation, Ca2+ Regulation, and Photoreceptor Cell Survival" @default.
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