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TRP channels have emerged as key biological sensors in vision, taste, olfaction, hearing and touch. Despite their importance, virtually nothing is known about the folding and transport of TRP channels during biosynthesis. Here, we identify XPORT (exit protein of rhodopsin and TRP) as a critical chaperone for TRP and its G-protein coupled receptor (GPCR), rhodopsin (Rh1). XPORT is a resident ER and secretory pathway protein that interacts with TRP and Rh1, as well as with Hsp27 and Hsp90. XPORT promotes the targeting of TRP to the membrane in Drosophila S2 cells, a finding that provides a critical first step towards solving a longstanding problem in the successful heterologous expression of TRP. Mutations in xport result in defective transport of TRP and Rh1, leading to retinal degeneration. Our results identify XPORT as a novel molecular chaperone and provide a mechanistic link between TRP channels and their GPCRs during biosynthesis and transport.
Molecular chaperones ensure the appropriate folding, assembly, transport, targeting and quality control of newly synthesized proteins. Neurons have evolved complex and diverse mechanisms involving numerous families of chaperones to deal with these error-prone processes and the detrimental effects of protein aggregation (Buchberger et al., 2010; Tyedmers et al., 2010). Accumulation of misfolded proteins often leads to severe pathology and neurodegeneration. Hence, chaperones are the first line of defense against misfolded proteins and can effectively suppress certain forms of neurodegeneration (Bonini, 2002; Gibbs and Braun, 2008; Muchowski and Wacker, 2005).
TRP channels and their G-protein coupled receptor (GPCR), rhodopsin, are synthesized on membrane-bound ribosomes in the endoplasmic reticulum (ER) and must undergo precise folding and successful transport to the rhabdomeres to become functionally active. In Drosophila photoreceptors, the rhabdomeres are the photosensitive organelles containing rhodopsin and the other components of phototransduction. Rhabdomeres are comprised of numerous tightly packed microvilli and are functionally equivalent to the outer segments of the vertebrate rods and cones (Colley, 2010; Fain et al., 2010; Yau and Hardie, 2009). Phototransduction in Drosophila is a G-protein-coupled, phosphoinositide-mediated signaling cascade, initiated when light stimulated rhodopsin (Rh1) interacts with the heterotrimeric G-protein, DGq. In turn, Gqα activates the norpA (no receptor potential A) encoded PLCβ effector molecule, leading to the opening of the TRP and TRPL channels and the subsequent influx of sodium and calcium (Hardie and Postma, 2008; Hardie and Raghu, 2001; Katz and Minke, 2009; Wang and Montell, 2007). The precise mechanisms for gating the TRP and TRPL channels are unresolved but may involve PLC’s dual role in phosphoinositide (PIP2) depletion and proton release (Huang et al., 2010).
Since the initial discovery of the canonical TRP channel in Drosophila photoreceptors (Hardie and Minke, 1992; Montell and Rubin, 1989), TRP channels have emerged as key biological sensors, responding to a wide variety of sensory stimuli in almost every organism, tissue and cell-type. The TRP superfamily displays greater diversity than any other group of ion channels and is comprised of seven subfamilies that function in vision, taste, olfaction, hearing, touch, and the sensation of both pain and temperature (Clapham, 2003; Damann et al., 2008; Gallio et al., 2011). This diversity is reflected in the growing list of disorders involving TRP, including congenital stationary night blindness (Audo et al., 2009; Everett, 2011; van Genderen et al., 2009). Despite their importance, virtually nothing is known about the initial folding and targeting of TRP channels during their biosynthesis.
Photoreceptor cells utilize a wide array of folding factors, chaperones, and transport mechanisms for the biosynthesis of rhodopsin (Colley, 2010; Deretic, 2010; Deretic and Mazelova, 2009; Kosmaoglou et al., 2008). In the vertebrate retina, rhodopsin interacts with multiple ER chaperones including the ER degradation enhancing alpha-mannosidase-like 1 (EDEM1) protein and a DnaJ/Hsp40 chaperone (HSJ1B) (Chapple and Cheetham, 2003; Kosmaoglou et al., 2009). In Drosophila, Rh1 biosynthesis is also mediated by a variety of factors including both molecular chaperones and at least three Rab-GTPases, namely Rab1, Rab6, and Rab11 (Satoh et al., 1997; Satoh et al., 2005; Shetty et al., 1998). Additionally, myosin V and the Drosophila Rab11 interacting protein (dRip11) function in the transport of Rh1 (Li et al., 2007). Interestingly, Rab11 also functions in the transport of TRP (Satoh et al., 2005). Two integral membrane proteins, calnexin99A (Cnx) and NinaA, play critical and highly specific roles during Rh1 biosynthesis (Colley et al., 1991; Rosenbaum et al., 2006; Stamnes et al., 1991). Cnx is a molecular chaperone that interacts with folding intermediates of glycoproteins in the ER to ensure their proper folding and inhibit their aggregation or premature release (Ellgaard and Frickel, 2003). NinaA is a cyclophilin homolog that also functions as a chaperone for Rh1 (Colley et al., 1991; Schneuwly et al., 1989; Shieh et al., 1989; Stamnes et al., 1991). Mutations in cnx or ninaA lead to the accumulation of ER membranes in response to mislocalization of Rh1. Ultimately, these protein aggregations lead to severe reductions in Rh1 protein levels and retinal degeneration.
Defects in rhodopsin biosynthesis and trafficking cause retinal degeneration in both Drosophila and humans. For example, more than 25% of human autosomal dominant retinitis pigmentosa (adRP) cases result from mutations that disrupt the rhodopsin gene. A great majority of these mutations lead to misfolded rhodopsin that aggregates in the secretory pathway (Hartong et al., 2006). Aberrant protein processing and accumulation are also the culprits of numerous neurodegenerative diseases in the brain such as prion diseases, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease. There are likely many similarities between the cellular and molecular mechanisms underlying these disorders, making the Drosophila eye an invaluable model system for unravelling the complexity of neurodegenerative disorders as they relate to protein misfolding, aggregation, and trafficking (Bilen and Bonini, 2005; Colley, 2010).
One major group of chaperones that is utilized by all neurons in the face of cell stress and protein misfolding is the family of heat shock proteins (Hsps). Although initially identified as heat shock proteins, most of these chaperones are expressed constitutively and have indispensable functions in the folding of newly synthesized proteins, as well as in the refolding or elimination of misfolded proteins. Members of the Hsp27, Hsp40 (DnaJ), Hsp70 and Hsp90 families have been associated with human brain lesions corresponding to almost all neurodegenerative diseases (Muchowski and Wacker, 2005). Accordingly, these same Hsps are potent suppressors of neurodegeneration (Bonini, 2002; Stetler et al., 2009). Indeed, Hsp27, Hsp70, and Hsp90 have all been implicated as neuroprotective agents in the retina (Gorbatyuk et al., 2010; O’Reilly et al., 2010; Tam et al., 2010).
Here, we characterize XPORT (exit protein of rhodopsin and TRP), a novel molecular chaperone in Drosophila. Mutations in xport result in the accumulation of TRP and Rh1 in the secretory pathway and ultimately, lead to a severe light-enhanced retinal degeneration. XPORT, along with calnexin and NinaA, functions as part of a highly specialized pathway for rhodopsin biosynthesis. Furthermore, XPORT physically associates with TRP and Rh1, as well as with members of the Hsp family of molecular chaperones. Our results demonstrate a critical role for XPORT as a chaperone during the biosynthesis and transport of the TRP channel and its G-protein coupled receptor, Rh1.
By screening the Zuker collection of EMS-mutagenized Drosophila (Koundakjian et al., 2004) we identified a novel mutant, xport1, which displayed an abnormal electroretinogram (ERG) compared to wild-type (Figure 1A). The xport1 mutant had a transient response during prolonged light stimulation, which was indistinguishable in amplitude and time-course from the transient receptor potential (trp) phenotype observed in the trp343 null mutant. Photoreceptor cells that lack TRP protein are unable to sustain a steady-state current, via TRPL, due to reduced Ca2+ influx. More specifically, low levels of Ca2+ result in a failure of Ca2+ and PKC dependent inhibition of PLC. Consequently, uncontrolled PLC activity depletes its substrate (microvillar PIP2) leading to premature closure of TRPL channels (Gu et al., 2005; Hardie et al., 2001). Consistent with the transient light response, xport1 displayed a severe reduction in TRP protein levels compared to wild-type (Figure 1B). Interestingly, Rh1 protein levels were also severely reduced in the xport1 mutant (Figure 1C). The mutation in xport1 is recessive, as the heterozygotes were normal for both TRP and Rh1 protein (Figure 1B and 1C). Therefore, xport is required for the proper expression of both TRP and Rh1.
To identify the xport locus, we first narrowed the cytogenetic location by deficiency mapping to 92B3-92C1 on the third chromosome, corresponding to 26 loci spanning 145 kb of DNA (Figures 1B, 1C and S3A). We identified a C to T substitution at nucleotide position 145 within the coding region of CG4468, causing a premature stop codon at glutamine49 (Figure S3A, arrow).
To confirm that CG4468 was the xport locus, we restored wild-type function by introducing a wild-type copy of CG4468 into the genome of the xport1 mutant. TRP and Rh1 protein expression were restored to wild-type levels in the rescue line (Figure 1B and 1C). These data confirm that CG4468 is, indeed, the xport locus. In addition, we found that eye-specific expression of three independent CG4468 RNAi transgenes leads to a severe reduction in TRP by Western blot analysis (Figure S1).
We analyzed electrical responses to light in the xport1 mutants by whole cell voltage-clamp recordings of dissociated ommatidia. In wild-type photoreceptors, brief flashes elicited rapid macroscopic inward currents mediated by TRP and TRPL channels (Figure 1D). In xport1 mutants, response amplitudes were ~20-fold reduced (Figure 1E), consistent with a severe reduction in TRP and Rh1. Sensitivity was restored to wild-type levels in xport1 flies expressing the wild-type xport cDNA rescue construct (Figure 1D).
If TRP channel expression was completely eliminated in the xport1 mutants, then we would predict that the residual response in xport1 would be eliminated in a trpl302;xport1 double mutant. Instead, we found a residual response in the trpl302;xport1 double mutant (Figure 1F), but sensitivity was now reduced ~500-fold with respect to wild-type and 25-fold with respect to xport1 (Figure 1G).
To determine whether the residual response in trpl302;xport1 was mediated by TRP channels, we measured the reversal potential (Erev) of the light response. Erev in wild-type and rescue flies represented the mixed contribution of both TRP and TRPL channels and was approximately 11 mV (Figure S2A). As predicted, Erev in xport1 mutants was negatively shifted compared to wild-type and indistinguishable from that measured in trp343 mutants. However, Erev for the trpl302;xport1 double mutant was similar to that measured in trpl302 mutants, indicating that the residual response was mediated by TRP channels.
TRP and TRPL channels can also be distinguished by their sensitivity to La3+, which completely blocks TRP channels, while leaving TRPL channels unaffected. In wild-type and rescue flies, La3+ (50 μM) blocked approximately 80% of the light-induced current, leaving a residual response mediated by TRPL channels (Figure 1H, wild-type data not shown). In xport1 mutants, La3+ had no detectable blocking action, indicating that most of the response was mediated by TRPL channels (Figure 1I). In the trpl302;xport1 double mutant, the response was completely blocked by perfusion with La3+ (Figure 1J), again confirming that TRP channels mediated this residual response.
Because sensitivity to light in the trp343 mutant was much greater than in the xport1 mutant (Figures 1E and 1G), the near complete loss of TRP channels in xport1 can only partially account for the 20-fold observed reduction in sensitivity (5% of wild-type sensitivity). It seemed likely that the additional loss of sensitivity would be accounted for by the reduction in Rh1 content. To test this, we measured effective quantum efficiency (Q.E.), which should be proportional to Rh1 concentration, by counting quantum bumps in response to dim flashes such that ~50% of the flashes contained no effective photons and induced no response (Figure S2B and S2C, failures). In xport1 mutants, Q.E. was reduced on average by approximately 8-fold compared to wild-type (Figure S2D). However, bump amplitude (3.6 pA), although smaller than in wild-type flies, was indistinguishable from that measured in trp343 mutants (Figures S2E and S2F). This indicates that the loss of sensitivity in xport1 mutants can be fully accounted for by a drastic reduction in TRP channels combined with an ~8-fold reduction in visual pigment concentration. Both bump amplitude and Q.E. were fully rescued by expression of the wild type xport cDNA rescue construct in the xport1 mutant (Figures S2D–F).
Taken together, these data indicate an ~60-fold reduction in TRP channel activity (1.7% of wild-type levels) and imply an ~8-fold reduction in Rh1 content (12% of wild-type levels) in the xport1 mutant. These estimates are consistent with the levels of TRP and Rh1 detected by immunoblotting (Figure 1B and 1C). While XPORT is required for both TRP and Rh1, TRP and Rh1 expression are not dependent upon one another. TRP protein levels were wild-type in the ninaEI17 (Rh1) null mutant (Figure 1B) and Rh1 protein levels were wild-type in the trp343 null mutant (Figure 1C). Therefore, XPORT provides a novel biosynthetic link between TRP and its GPCR, Rh1.
The xport locus is comprised of 2 exons and 1 intron (Figure S3A). The 953 base pair transcript encodes a 116 amino acid protein (Figure 2A and S3A) that was detected as a 14 kD band in wild-type flies (Figure 2B). Consistent with the presence of a premature stop codon, XPORT protein was reduced in xport1 heterozygotes and completely absent in xport1 homozygotes as well as in flies harboring the xport1 allele in trans to Df(3R)BSC636 (Figure 2B). XPORT expression was completely restored in the rescue line (Figure 2B). Although TRP and Rh1 require XPORT protein for their expression, XPORT is expressed normally in both the trp and ninaE (Rh1) null mutants (Figure 2B).
The XPORT protein is predicted to be a Type II transmembrane protein with a single C-terminal transmembrane domain and a cytosolic N-terminal globular domain (Figure 2A, S3A and S3B). Consistent with this prediction, following centrifugation of a total cell homogenate from wild-type heads, XPORT was absent from the soluble fraction and exclusively present in the membrane pellet (Figure 2C). XPORT was solubilized by suspension of the membrane pellet in SDS. Following subsequent centrifugation, XPORT was detected entirely in the supernatant and was absent from the pellet, confirming that XPORT is an integral membrane protein.
XPORT is highly conserved among 12 Drosophila species as well as among other Diptera, including two mosquito genera, Anopheles and Culex. Drosophila XPORT is also conserved in the Jerdon’s jumping ant and honeybee (Hymenoptera) as well as in the red flour beetle (Coleoptera) (Figure S3C). While XPORT is highly conserved among insect species, there are currently no vertebrate counterparts in the NCBI database. Although XPORT lacks an obvious vertebrate homolog, it has a small recognizable motif that displays 46% amino acid identity and 62% similarity with the KH domain of a DnaJ-like protein from Chlamydomonas reinhardtii (Figure 2A). DnaJ proteins, also known as Hsp40s, are members of a large family of highly diverse co-chaperones that bind Hsp70 via a 70 amino acid J-domain, and assist in the folding and quality control of a vast array of client proteins (Kampinga and Craig, 2010). While DnaJ and DnaJ-like chaperones are defined by the presence of the J-domain, XPORT lacks this domain.
The KH domain is a nucleic acid recognition motif that binds single stranded RNA or DNA with low affinity. KH domains contain a “GXXG” loop that is key to nucleotide binding and this motif is also present in XPORT (Figure 2A). KH-domain proteins perform a wide variety of cellular functions and have been implicated in regulation of transcription and translation (Valverde et al., 2008). To distinguish between these two possibilities, we performed Northern blot analysis. The trp and ninaE (Rh1) transcript levels were indistinguishable from wild-type in the xport1 mutant (Figure 2D), indicating that XPORT functions post-transcriptionally for TRP and Rh1.
Certain Hsp70/DnaJ chaperone complexes, as well as calnexin, have been shown to specifically associate with ribosomes to ensure the proper folding of newly synthesized polypeptide chains as they exit the ribosome during translation (Craig et al., 2003; Delom and Chevet, 2006; Hundley et al., 2005; Jaiswal et al., 2011). Members of this ribosome-tethered chaperone network are conserved from yeast through humans and are thought to serve as the first line of defense against protein misfolding. Consistent with a role for XPORT in the early stages of TRP and Rh1 biosynthesis, XPORT protein was detected in the peri-nuclear ER in all 8 photoreceptor cells (Figure 2E, R8 cell not shown). XPORT’s labeling pattern was similar to that of the known chaperones, calnexin and NinaA (Figure S4). Therefore, XPORT may exhibit co-translational chaperone function at the early stages of TRP and Rh1 biosynthesis at the ribosome. XPORT has ideal predicted topology for positioning its KH and “GXXG” motifs on the cytosolic face of the ER, where ribosomes reside.
Just like TRP and Rh1, XPORT is eye-specific. By Northern blot analysis, the xport, ninaE (Rh1) and trp transcripts were detected in wild-type heads but were absent in bodies and in heads from flies lacking eyes (eya1) (Figure 2D). Furthermore, by immunocytochemistry, XPORT was detected exclusively in the photoreceptor cell bodies, but was not detected in the lamina, medulla, lobula, lobula plate or brain, compared to the synaptic protein, synapsin (Figure 2F).
XPORT not only localized to the perinuclear ER, but was also detected more extensively in the secretory pathway (Figure 2E) unlike the inositol 1,4,5-trisphosphate receptor (IP3R), which was highly restricted to the perinuclear ER (Figure S4). This makes XPORT ideally situated to function as a chaperone in the early as well as in the later stages of TRP and Rh1 biosynthesis.
In wild-type flies, the TRP channel specifically resides within the rhabdomere for its function in phototransduction (Figure 3A, top). In contrast, TRP protein was severely mislocalized in all eight photoreceptor cells in the xport1 mutant. It was detected throughout the secretory pathway with very little labeling in the rhabdomeres (Figure 3A, bottom). These data are consistent with the electrophysiological analyses showing that there is very little functional TRP (1.7%) present in the xport1 mutant (Figures 1D-G). Therefore, successful transport of TRP to the rhabdomeres of all eight photoreceptors requires XPORT.
We investigated the kinetics of Rh1 maturation in the xport1 mutant using transgenic flies harboring an epitope-tagged Rh1 under the control of a heat-inducible promoter (hsp70). In wild-type flies, Rh1 was initially synthesized as immature high-molecular weight (MW) glycosylated forms that were processed down to the mature form by 14 hours. By 24 hours, the vast majority of Rh1 was detected in the mature low-MW form (Figure 3B, top). In the xport1 mutant, Rh1 was also initially detected as immature high-MW forms that were partially processed to the mature form. In contrast to wild-type flies, in the xport1 mutant, Rh1 disappeared rapidly between 16–24 hours, indicating that Rh1 was degraded (Figure 3B, bottom). Therefore, XPORT is required for the proper maturation and stability of newly synthesized Rh1.
In wild-type flies, Rh1 was precisely localized to the rhabdomeres for its role in phototransduction (Figure 3C, top). In contrast, in the xport1 mutant, Rh1 was abnormally retained in the ER and secretory pathway with only some Rh1 present in the rhabdomeres (Figure 3C, bottom). This is consistent with the electrophysiological analyses demonstrating that there is a small amount of functional Rh1 (~12%) present in the xport1 mutant (Figure S2D). Therefore, like TRP, successful transport of Rh1 through the secretory pathway and efficient delivery of Rh1 to the rhabdomere also requires XPORT.
Consistent with XPORT residing in the secretory pathway of photoreceptor cells (Figure 2E), XPORT was detected in the perinuclear ER and secretory pathway of Drosophila S2 cells transfected with xport (Figure 3D). Likewise, in cells singly transfected with either trp or ninaE (Rh1), the proteins were detected in the secretory pathway in a perinuclear and/or punctate fashion (Figure 3D, −X). However, when trp or ninaE were co-expressed with xport, TRP and Rh1 proteins were now detected at the cell surface (Figure 3D, +X). These results were quantified by analyzing over a 100 cells for each condition (Table S1) and cell surface labeling of TRP was confirmed by co-localization with a plasma membrane marker, wheat germ agglutinin (WGA) (Figure 3D, bottom row). These data demonstrate that XPORT promotes the transport of TRP and Rh1 to the cell surface in S2 cells, consistent with a role for XPORT as a chaperone for TRP and Rh1.
In addition to the compound eye, Drosophila have two additional light-sensing organs: the adult ocelli and the larval Bolwig’s organ. Phototransduction in ocelli likely occurs via a signaling pathway very similar to the compound eye, utilizing the ocellar-specific opsin, Rh2, the G-protein (DGq), norpA-encoded PLC, TRP channels, arrestin1 (Arr1) and arrestin2 (Arr2). To investigate the potential role of XPORT in Drosophila ocelli, we examined the expression of XPORT and TRP in both wild-type and xport1 mutants. Figure 4 shows that XPORT and TRP were both expressed in wild-type ocelli. TRP protein was reduced in the xport1 mutant, while Arr1 was normal. These results suggest that XPORT is specifically required for TRP in the ocelli.
Given that mutations in xport severely disrupt the biosynthesis of TRP and Rh1, we examined the expression of additional photoreceptor cell proteins in the xport1 mutant. Figure 5A shows that other proteins critical for phototransduction and rhabdomere stability, including the G-protein alpha subunit (Gqα), PLCβ (norpA), the TRPL channel, Arr1, Arr2, chaoptin and NinaA were all expressed at normal levels. We also investigated expression levels of proteins involved in calcium regulation and synaptic transmission. We determined that calnexin, the Na+/Ca2+ exchanger (CalX), the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), as well as the synaptic proteins, synapsin and syntaxin, were all expressed normally in the xport1 mutant (Figure S5). Finally, we determined that the minor opsins, Rh3, Rh4, and Rh5, were properly localized to their rhabdomeres in the R7 and R8 cells in the xport1 mutant (Figure 5B). These results demonstrate that XPORT is specifically required by TRP and Rh1 and is not required for expression of other photoreceptor cell proteins.
In larvae, Bolwig’s organ is comprised of two bilateral groups of 12 photoreceptor cells that each express one of two R8-cell-type opsins, Rh5 and Rh6 (Malpel et al., 2002). Phototransduction in Bolwig’s organ is thought to involve similar components as the adult visual cascades, including norpA-encoded PLC (Busto et al., 1999; Malpel et al., 2002). To investigate the potential role of XPORT in Bolwig’s organ, we examined the expression of XPORT, TRP, TRPL, Rh1 and Rh5 in both wild-type and xport1 mutants. Figure 5C shows that XPORT was expressed in Bolwig’s organ. Interestingly, TRP was not detected in Bolwig’s organ, indicating that it is not used for larval phototransduction. In contrast, TRPL was detected in Bolwigs organ (Figure 5D), suggesting that it may function as the primary channel in the larvae. Consistent with previous reports, Rh1 was not detected in Bolwig’s organ (Figure 5C) whereas Rh5 was detected (Figure 5D) (Busto et al., 1999; Kaneko et al., 1997; Malpel et al., 2002; Sawin-McCormack et al., 1995). Interestingly, both Rh5 and TRPL are expressed normally in the xport mutant indicating that, as in the adult, XPORT is not required for Rh5 or TRPL expression in Bolwig’s organ (Figure 5D). Therefore, although XPORT is expressed in Bolwig’s organ, it does not appear to be required for visual processes in the larvae.
TRP and Rh1 are key components of phototransduction and mutations that alter the function of either of these proteins lead to severe retinal pathology and retinal degeneration. Consistent with the finding that XPORT is essential for TRP and Rh1 protein expression, xport1 mutants displayed an early-onset retinal degeneration. In 1-day-old xport1 mutants grown on a 12:12 light-dark cycle, the rhabdomeres were diminished in size (Figure 6B) compared to wild-type flies (Figure 6A). In agreement with this reduction in rhabdomere size, the membrane surface area in the xport1 mutant, as measured by whole-cell capacitance, was almost halved indicating a significant reduction in microvillar surface area (Figure S6). Furthermore, the photoreceptor cells displayed extensive ER membrane accumulations and dilated Golgi (Figure 7A), consistent with aggregation of TRP and Rh1 in the secretory pathway. At 2 weeks, the xport1 mutant photoreceptor cells were severely degenerated. The rhabdomeres of all eight photoreceptors were vastly reduced and many were completely missing (Figure 6C and 6D). To assess whether the retinal degeneration was enhanced by light stimulation of phototransduction, we reared the xport1 mutant for 2 weeks in constant darkness. Dark-reared flies still showed ER membrane accumulations and dilated Golgi (Figure 7D), but now exhibited nearly normal rhabdomere morphology (Figure 6E). Therefore activation of phototransduction by light enhances the retinal degeneration in xport1 mutants. The retinal pathology was fully rescued by the expression of wild-type XPORT in the xport1 mutant (Figure 6F).
The molecular mechanisms underlying retinal degeneration are diverse and have been well studied in the Drosophila visual system. Two well-characterized mechanisms involve either 1) accumulation of Rh1 in the secretory pathway due to defective folding/trafficking or 2) unregulated Ca2+ levels due to defective phototransduction (Colley, 2010; Rosenbaum et al., 2006; Wang and Montell, 2007). The finding that light significantly enhanced the retinal degeneration in the xport1 mutant is contrasted to other known mutants defective in Rh1 maturation, for which the retinal degeneration is light-independent (Colley et al., 1991; Colley et al., 1995; Kurada and O’Tousa, 1995; Webel et al., 2000). However, the xport1 mutant is unique in that it displays defects in both protein trafficking and TRP channel function. Loss of TRP channel expression can lead to a retinal degeneration unrelated to protein trafficking (Wang and Montell, 2007). In this instance, the retinal degeneration is light-dependent and is triggered by defects in calcium influx through the light-sensitive TRP channels. Given that the retinal degeneration in xport1 is light-enhanced, we investigated the relative contribution of protein trafficking defects versus the lack of TRP channel function to the overall retinal degeneration. To accomplish this, we took advantage of two retinal degeneration mutants, ninaE318 and trp343.
The ninaE318 mutant exhibited a severe reduction in Rh1 and displayed defects in Rh1 transport through the secretory pathway (Figures S7A and S7C). However, TRP protein levels were wild-type in ninaE318 (Figure S7B). Therefore, ninaE318 exhibits a retinal degeneration that is due solely to defects in protein trafficking. In contrast, the trp343 mutant was null for TRP protein (Figure S7B) but Rh1 levels were wild-type and Rh1 specifically localized to the rhabdomeres (Figures S7A and S7C). Therefore, trp343 mutants undergo a retinal degeneration that is independent of protein trafficking defects. The xport1 mutant displays a combination of protein accumulation in the secretory pathway (like ninaE318) and a severe reduction in TRP protein (like trp343).
Like the xport1 mutant, the ninaE318 mutant displayed considerable ER membrane accumulations and dilated Golgi at 1-day-old (Figure 7B). In contrast, the trp343 null mutant showed no sign of secretory pathway membrane accumulations (Figure 7C). The secretory pathway defects were light-independent, as the membrane accumulations were still present in 1-day-old xport1 and ninaE318 mutants that had been reared in constant darkness (Figures 7D and 7E).
At two weeks, trp343 displayed a severe retinal degeneration (Figure 7H) that was comparable to that observed in the xport1 mutant (Figure 6C and 6D). In contrast, the ninaE318 mutant exhibited milder pathology at two weeks (Figure 7G). As was shown for xport1 (Figure 6E), the retinal degeneration was significantly attenuated in trp343 mutants reared in constant darkness for 2 weeks (Figure 7J). Taken together, these results indicate that the retinal degeneration in the xport1 mutant is due to the combined detrimental effects of protein aggregation in the secretory pathway and misregulation of Ca2+ levels in the absence of TRP. More specifically, the light-independent membrane accumulations in xport1 are likely the result of defects in TRP and Rh1 trafficking, while the light-enhanced retinal degeneration is likely due to the near complete loss of TRP channels in the rhabdomere.
Given that two other chaperones, namely NinaA and calnexin, are also essential for Rh1 maturation and trafficking (Colley et al., 1991; Rosenbaum et al., 2006), XPORT may play a critical role in a conserved protein-processing pathway with these chaperones. To investigate the temporal sequence of calnexin, NinaA and XPORT chaperone activity for Rh1, we conducted genetic epistatic analyses by generating double mutant flies. In all three single mutants, Rh1 was severely reduced (Figure 8A). However, in the ninaAP269 mutant, a substantial amount of Rh1 was detected in the immature high MW form. The ninaAP269; calnexin1 double mutant displayed severely reduced levels of Rh1 in the mature low molecular weight form, a phenotype characteristic of the calnexin1 mutation alone (Figure 8A). These results demonstrate that calnexin functions upstream of NinaA in Rh1 biosynthesis. Consistent with this finding, calnexin was entirely digested by both endoglycosidase H (Endo H) and peptide N-glycosidase F (PNGase F) (Figure S4). Endo H selectively cleaves high mannosyl residues on glycoproteins that have not yet been processed in the Golgi and thus Endo H sensitivity implicates glycoproteins as ER residents. Therefore, calnexin is restricted to the ER. NinaA, however, was only partially digested by Endo H and fully digested by PNGase F (Figure S4). These results support previous findings that while some NinaA protein resides in the ER, NinaA is also retained by Rh1 as it travels through the distal compartments of the secretory pathway. Accordingly, NinaA is not entirely restricted to the ER (Colley et al., 1991). XPORT was insensitive to both enzymes, indicating that it is not glycosylated (Figure S4). Hence, for XPORT, Endo H sensitivity was not informative. To evaluate the epistatic relationship between xport and ninaA, we generated a ninaAP269; xport1 double mutant and again, examined Rh1 expression. The ninaAP269;xport1 double mutant displayed severely reduced levels of Rh1 with most of the Rh1 present in the immature high molecular weight form (Figure 8A). This phenotype is characteristic of the ninaAP269 mutation alone and suggests that NinaA functions upstream of XPORT in Rh1 biosynthesis. Taken together, these data suggest that calnexin, NinaA, and XPORT function in a coordinated pathway ensuring the proper folding, quality control and maturation of Rh1 during biosynthesis. We propose that calnexin functions upstream of NinaA which, in turn, functions upstream of XPORT during Rh1 biosynthesis (Figure 8B). Interestingly, neither calnexin nor NinaA are required for the biosynthesis of the TRP channel, as TRP protein is expressed normally in the cnx and ninaA mutants (Figure S8).
Consistent with XPORT’s function as a chaperone for TRP and Rh1, XPORT physically associates with both TRP and Rh1. Rh1 was isolated in a stable complex with XPORT and this association was specific, as Rh1 did not bind to or elute from the XPORT antibody column in the absence of XPORT protein (Figure 8C). TRP was also isolated in a stable complex with XPORT (Figure 8C). Further support for the specificity of these interactions was obtained by investigating several other photoreceptor cell proteins. Like all neurons, photoreceptors are polarized and, therefore, protein trafficking occurs in two directions: to the rhabdomeres and to the synapse. We investigated whether XPORT was required for the transport of the synaptic vesicle proteins synapsin and syntaxin. Neither protein interacted with XPORT, as both were found entirely in the unbound fraction in both wild-type and xport1 mutant tissue (Figure 8C). We also assessed the interaction between XPORT and two other chaperones involved in Rh1 biosynthesis, calnexin and NinaA. Neither calnexin nor NinaA interacted with XPORT, as both proteins were detected entirely in the unbound fraction in both wild-type and xport1 mutant tissues (Figure 8C). That XPORT does not associate with synapsin, syntaxin, NinaA or calnexin is consistent with the finding that these proteins do not require XPORT for their biosynthesis, as they were all expressed at wild-type levels in the xport1 mutant (Figure 5A and S5). Furthermore, these results support the notion that calnexin, NinaA and XPORT sequentially interact with Rh1 during its biosynthesis in a step-wise fashion, as opposed to functioning as components of a macromolecular chaperone complex.
Although XPORT was not detected in a complex with calnexin and NinaA, it is possible that XPORT functions as part of an Hsp complex. Given that XPORT displays amino acid identity with a DnaJ-like protein, we first investigated whether the Hsp70 protein was present in a complex with XPORT, TRP and Rh1. Indeed, Hsp70 was detected in the bound fraction of wild-type tissue, but it was also detected in the bound fraction of the xport1 mutant tissue (Figure 8C). Due to the binding of Hsp70 in the absence of XPORT, we were unable to determine whether Hsp70 was truly part of the XPORT complex. To further investigate the potential interaction between XPORT and the Hsp family, we examined whether Hsp90 or Hsp27 were present in the complex. Hsp90 and Hsp27 represent two other highly conserved chaperones that function, together with Hsp70, to promote protein folding and prevent protein aggregation. Indeed, both Hsp90 and Hsp27 were specifically isolated in a stable complex with XPORT, with no binding detected in the xport mutant (Figure 8C). These results suggest that XPORT may serve as a chaperone in conjunction with the Hsp family.
Despite almost 20 years of extensive investigation into both native and heterologously expressed TRP channels, the fundamental mechanisms underlying TRP channel biosynthesis, trafficking, and gating remain elusive. An enduring obstacle in the Drosophila visual field has been that expression of Drosophila TRP in heterologous systems has either failed to yield active channels or the currents produced have failed to recapitulate the native properties of TRP channels in vivo (reviewed in Hardie, 2003; Minke and Parnas, 2006). The expression of mammalian TRP channels has also proven problematic, with the same isoform often differing in properties from one cell line to another. These difficulties are likely compounded by variations in the intracellular folding, trafficking and signaling components that exist between native cells and heterologous expression systems. There are likely many molecular factors necessary for the proper localization, activation and modulation of TRP channels, and these factors could be missing or differentially expressed from one cell type to another.
One challenge in heterologous expression systems is the defective targeting of TRP to the plasma membrane. Here, we show that XPORT is necessary for promoting the targeting of TRP to the plasma membrane. Once it has reached the membrane, TRP will likely require additional factors for its function and stability. Therefore, co-expression with XPORT and other proteins may be necessary for the successful heterologous expression of functional Drosophila TRP, an achievement that will have major implications for future studies on the kinetics and gating of this channel.
XPORT forms a stable complex with TRP and Rh1 as well as with Hsp27 and Hsp90. Hsp27 has been implicated in a variety of cellular processes, including the trafficking of steroid receptors to the plasma membrane (Razandi et al., 2010). Hsp27 forms large oligomeric complexes that are essential for its chaperone activity and has been shown to associate with chaperones from the DnaJ, Hsp70 and Hsp90 families (Nakamoto and Vigh, 2007; Nardai et al., 1996; Schnaider et al., 2000). Therefore, XPORT may function as part of a macromolecular complex with members of the Hsp family during TRP and Rh1 biosynthesis.
Hsp70 and Hsp90 are ubiquitous and highly conserved molecular chaperones that function to fold a wide array of client proteins with the help of numerous co-chaperones (Pearl and Prodromou, 2006; Pratt and Toft, 2003; Taipale et al.; Young et al., 2003). For example, DnaJ proteins function as co-chaperones for Hsp70, directing it to distinct locations in the cell and determining, in part, the identity of the client protein to be folded (Hennessy et al., 2005; Kampinga and Craig, 2010; Qiu et al., 2006; Young et al., 2003). DnaJ proteins are highly heterogeneous chaperones that contain a variety of motifs, in addition to the J-domain, that give them each unique structure and function (Hennessy et al., 2005; Kampinga and Craig, 2010; Qiu et al., 2006). For example, the KH domain in the Chlamydomonas DnaJ-like protein is unique among the DnaJ family and may serve to link Hsp70 activity to nucleotide binding. Therefore, while XPORT is not a DnaJ protein, its KH motif may serve to couple XPORT’s chaperone activity to the ribosome at the earliest stages of protein biosynthesis.
Just as the highly diverse DnaJ proteins offer functional specificity to Hsp70, a large number of proteins have been shown to cooperatively bind Hsp90. In many cases, the Hsp70 and Hsp90 chaperone complexes function together as a single macromolecular chaperone system. The list of cofactors and co-chaperones that bind to either Hsp70 or Hsp90 as part of this multi-chaperone machinery continues to grow (Pratt and Toft, 2003; Schumacher et al., 1996; Young et al., 2003). While many of these co-chaperones are soluble cytosolic proteins, a select few bind the cytoskeleton or are localized to a variety of membrane systems including the ER, mitochondria, plasma membrane, clathrin-coated vesicles or synaptic vesicles. Consequently, these chaperones recruit cytosolic Hsp70/Hsp90 complexes to specific locations in the cell (Young et al., 2003). Given its predicted topology as a Type II transmembrane protein, XPORT’s N-terminal globular domain is conveniently positioned at the cytosolic face of the ER membrane, where it could interact with the soluble Hsp chaperone machinery as well as with the polypeptide exit site of the ribosome machinery. In addition to its potential function at the ER/ribosome interface, XPORT is also more broadly detected throughout the secretory pathway. Therefore, XPORT may also function as a chaperone during later stages of TRP and Rh1 biosynthesis.
XPORT is key for cell viability as mutations in xport lead to a severe light-enhanced retinal degeneration. The retinal degeneration in the xport1 mutant is due to the combined detrimental effects of having improperly processed TRP and Rh1 accumulating in the secretory pathway and misregulation of calcium in the absence of TRP. It is possible that GPCRs and TRP channels in other cell-types and species may also require corresponding XPORT-like proteins for their biosynthesis. With the discovery of TRPM1 channels in ON-bipolar cells and the DAG-sensitive TRPC6/7 channels in intrinsically photosensitive retinal ganglion cells (ipRGCs), our findings may be relevant for understanding the mechanisms of TRP channel biosynthesis and trafficking in the vertebrate retina.
In the ON-bipolar cells, the GPCR, mGluR6 (metabotrophic glutamate receptor 6) is coupled to TRPM1. Mutations in humans that lead to a loss of TRPM1 cause congenital stationary night blindness (Audo et al., 2009; Morgans et al.; van Genderen et al., 2009). Likewise, melanopsin and the DAG-sensitive TRPC6/7 channels expressed in ipRGCs may function together in a phototransduction cascade (Sekaran et al., 2007). The TRP channels that function in vision represent members of an extensive TRP superfamily, which now contains at least 29 unique isoforms. TRP channels are expressed in a wide variety of tissues and cell types outside of the retina and, accordingly, function in the sensory transduction of taste, smell, hearing, and touch, in addition to sight. Therefore, identification and characterization of the critical molecular factors that are required for the proper folding, assembly, and transport of TRP channels to the membrane will have implications for a wide variety of sensory systems. XPORT represents a critical first step towards obtaining mechanistic insights into TRP channel biosynthesis.
Genomic DNA was isolated from xport1 and bw;st using the DNeasy® Blood and Tissue Kit (Qiagen Inc., Valencia, CA). We prioritized the candidate genes based on those that would most likely play a role in TRP and Rh1 biosynthesis and signaling. Primer pairs spanning 18 loci between 92B3-92C1 were designed based on their GenBank sequence accession numbers. We sequenced the mRNA, introns and exons of each locus and determined that 17 out of 18 loci were wild-type compared to the parental strain, with the exception of silent mutations. In the 18th gene, we identified the xport mutation.
Electroretinograms (ERGs) and whole-cell photoreceptor recordings from dissociated ommatidia were carried out on newly eclosed adult flies. Further details of the experimental procedures are provided in the Supplemental Experimental Procedures.
Total RNA was prepared from the heads and bodies of 0–7 day old flies using TRI Reagent® Solution followed by TURBO DNA-free™, according to the manufacturer’s instructions (Ambion, Austin, TX). Poly(A)+ RNA was obtained using the Poly(A)Purist™ mRNA isolation kit (Ambion, Austin, TX). mRNA was run on a denaturing 1% agarose gel and transferred to BrightStar®-Plus nylon membrane (Ambion, Austin, TX). Northern blots were processed using the North2South® Chemiluminescent Hybridization and Detection kit, according to the manufacturer’s instructions (Pierce, Rockford, IL). For probe production see Supplemental Experimental Procedures.
All procedures were carried out on 1-day-old fly heads, prior to retinal degeneration, unless otherwise specified. For Western blotting, proteins were separated by electrophoresis and transferred to nitrocellulose membranes as previously described (Colley et al., 1991). For immunocytochemistry, fixation and sucrose infiltration (or O.C.T embedding) of fly heads was carried out as previously described (Colley et al., 1991). For each experiment, at least five individual heads were sectioned and between 50–100 ommatidia were observed per eye. For antibodies and microscope details see Supplemental Experimental Procedures.
Adult heads were xed and processed according to a modi cation of the methods of Baumann and Walz, as previously described (Colley et al., 1991; Colley et al., 1995). Ultrathin sections were viewed at 80 kV on a Phillips CM120 electron microscope. For all genotypes described, at least three individual heads were sectioned and 50–100 ommatidia were observed per eye.
The DNA constructs were transfected into S2 cells using the Effectene® Transfection Reagent (Qiagen Inc., Valencia, CA). Following a seven day copper induction, cells were fixed in 2% formaldehyde in PBS for 10 minutes and blocked with 1% BSA, 0.1% Triton in PBS for 30 minutes. For quantification of cell surface labeling, cells were observed with transmitted light. For vector identities, DNA concentrations and additional antibody and reagent information see Supplemental Experimental Procedures.
We thank Drs. W. Baehr, L. Levin, K. Moses, A. Polans, L. Puglielli, G. Wistow, C.S. Zuker and the reviewers for valuable discussions and comments on the manuscript. The authors thank A. Gajeski, B. Larsen, A. Muller, E. Pirie, E. Solberg and M. Sookochoff for their expert technical assistance, as well as B. Krieber and Dr. B. Ganetzky for assistance with fly stocks. Dr. J. O’Tousa provided the pGaSpeR expression vector and Dr. A. Huber provided the trp-pMT/V5 construct. We thank the following people for contributing antibodies to the study: Dr. M. Ramaswami, Dr. C. Montell, Dr. C.S. Zuker, A. Becker, M. Welsh and Dr. P. Robinson. We acknowledge Dr. D. Wassarman and R. Katzenberger for generous assistance with the S2 cell transfections. We thank R. Kalil, L. Rodenkirch and M. Hendrickson of the W.M. Keck Laboratory for Biological Imaging and B. August and R. Massey of the UW-Med. School Electron Microscope Facility. We are grateful to C. Vang for his assistance with the computer graphics. Finally, Dr. C.S. Zuker generously provided us with the opportunity to screen the EMS-generated alleles from the Zuker Collection. This work was supported by funding from NIH EY008768 (NJC), NIH AG321762 (EER), NIH/NIGMS T32GM007507 (Neuroscience Training Program, EER), BBSRC-BB/G006865/1 (RCH, CHL), as well as from the Retina Research Foundation and the RRF/Walter H. Helmerich Research Chair (NJC) and the Research to Prevent Blindness (RPB) foundation (Department of Ophthalmology & Vis. Sci., NJC).
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