OR83b Is Required for OR Trafficking
The major olfactory organ of Drosophila
is the third segment of the antenna, a cuticle-covered appendage that contains approximately 1,200 OSNs (A, left panel) [30
]. The surface of this organ is covered with porous sensory hairs, or sensilla, which are of three major morphological classes (basiconic, coeloconic, and trichoid), and house the dendrites of between one and four OSNs (A, middle panel) [31
]. OSN dendrites comprise a proximal inner segment and a distal-ciliated outer segment (A, right panel). There are 62 ORs in Drosophila,
and 37 of these are expressed in specific subpopulations of antennal OSNs that display characteristic odor response profiles [3,9,19–21,32–34]. OR83b is estimated to be co-expressed with these ORs in 70%–80% of antennal OSNs [12
OR83b Is Essential for OR Membrane Trafficking
Conventional ORs, such as OR22a/b, are concentrated in the outer dendritic segment, where they co-localize with OR83b (B) [22
]. OR83b is also abundant in the cell body in a perinuclear rim and is enriched in regions that co-localize precisely with the small fraction of OR22a/b detected in the cell body (B, arrowheads). In the absence of OR83b, OR22a/b is highly unstable and detected only at very low levels in the neuronal cell bodies (C) [22
]. Despite these dramatic effects on OR localization, OSN morphology and membrane organization appear normal in Or83b
mutants when visualized using the membrane marker mCD8:GFP (C).
We next investigated the distribution of mislocalized OR22a/b in Or83b mutants with respect to intracellular organelles. The Drosophila Golgi apparatus has a punctate distribution (D, top panel), which is unaffected in Or83b mutants. Mislocalized OR22a/b does not co-localize with this Golgi marker (D, bottom panel). We observed a similar lack of co-localization of OR22a/b with endosomal and lysosomal compartments labeled by a GFP:RAB7 fusion protein (unpublished data), suggesting that OR trafficking is impaired prior to arrival at the Golgi.
The ER was visualized using a KDEL antibody, which recognizes the major retention signal for soluble ER proteins. In wild-type OSNs, α-KDEL displays extremely faint perinuclear staining (E, top panels). In contrast, in Or83b mutants, brightly stained KDEL accumulations are observed (E, bottom panels). These accumulations are specific to neuronal cell bodies as determined by examining KDEL staining in antennae in which Or83b mutant neurons are also labeled with mCD8:GFP (unpublished data). In contrast to the fully penetrant defects in OR localization, these accumulations are detected in only a small fraction of OSNs (<20%), which suggests that they may be a secondary consequence of the failure in OR trafficking. Mislocalized OR22a/b partially overlaps with these accumulations when they occur in Or22a/b neurons, consistent with at least a fraction of ORs being retained in the ER in the absence of OR83b (E, arrowheads). These changes in ER organization could have indirect effects on the forward transport of other membrane proteins, which may account for the reduced levels of mCD8:GFP in Or83b mutant dendrites (C, bottom panel).
OR83b Is Continuously Required for OR Localization
To assess when OR83b is required for OR localization, we performed rescue experiments of Or83b
mutants using the TARGET (temporal and regional gene expression targeting) system to achieve temporal control of OR83b expression, specifically in Or22a/b
]. This technique combines cell-type specific induction of a UAS-Or83b
transgene by an Or22a-Gal4
driver line with temporal regulation through use of a ubiquitously expressed temperature-sensitive Gal80 (Gal80ts)
transgene. At the permissive temperature (18 °C), the GAL80 protein is active and represses induction of OR83b expression by inhibiting GAL4 (A, top row). At the restrictive temperature (29 °C), GAL80 is inactivated, permitting expression of OR83b in OR22a/b neurons (A, bottom row).
OR83b Is Required to Maintain OR Localization in Adults and Has No Essential Developmental Function
In the first experiment, we cultured flies at 18 °C, collected adults, and aged these for 10 d at 18 °C. We then split these animals into two groups and incubated them for a further 2 d at either 18 °C or 29 °C before fixing and staining. Flies that had been maintained continuously at 18 °C do not express OR83b, and OR22a/b is absent from the cilia (B, top row). In contrast, OR83b expression is robustly induced in flies that had been transferred to 29 °C and OR22a/b is localized correctly to the sensory compartment (B, bottom row). This result indicates that late expression of OR83b is sufficient to promote OR22a/b localization and rules out a developmental role for OR83b in cilia morphogenesis or OR trafficking.
In a second experiment, we cultured flies and aged adults for 3 d at 29 °C, transferred them to 18 °C to switch off expression of OR83b, and fixed these animals immediately for 3, 6, or 9 d later (C). We observe a progressive decline in OR83b expression levels in the cell body, although protein perdures in the sensory cilia for several days after levels in the cell body become undetectable. OR22a/b shows an essentially parallel decline in the cilia, and we never detect OR22a/b in the sensory compartment in the absence of OR83b. We conclude that OR83b is essential to maintain OR localization and stability.
OR83b-Dependent and -Independent Trafficking Pathways in the Antenna
To examine the spatial requirements for OR localization, we first ectopically expressed one OR, OR43a, bearing an N-terminal GFP tag, throughout the antenna using Or83b-Gal4. GFP:OR43a is functional (see below) and in wild-type tissue localizes to cilia in all Or83b-expressing neurons (A, left panels). In contrast, in Or83b mutant antennae, GFP:OR43a is delocalized and destabilized, with only a weak signal detected in OSN cell bodies (A, right panel). OR83b is therefore essential for OR localization in all neurons in which it is detectably expressed, and no cell-type specific factors appear to be required for trafficking of individual ORs.
Spatial Requirements for OR83b in OR Localization
mutant adult flies retain some olfactory function [22
], and, given the heterogeneous expression of OR83b in the antenna (A), we wondered whether this reflects the function of OR83b-independent ORs. We investigated this in Or47b
neurons, which are situated in the lateral-distal region of the antenna where OR83b is expressed at the lowest levels. GFP:OR47b was selectively expressed in Or47b
neurons, and these were stained for OR83b and GFP. Although these neurons express only extremely low levels of OR83b (B, left panels, arrowheads), the localization of GFP:OR47b to the cilia remains dependent upon OR83b (B, compare left and right panels). This suggests that OR83b is likely to function with all ORs, regardless of its expression level.
OR genes represent an expanded lineage of the ancestral chemosensory family of gustatory receptor (GR) genes [37
]. GR genes are primarily expressed in gustatory neurons, but at least three are detected in antennal sensory neurons, including Gr21a,
which is expressed in the ab1C neurons that respond specifically to carbon dioxide (CO2
]. The CO2
response of ab1C neurons is independent of Or83b
]. Consistent with this physiological phenotype, no OR83b is present in Gr21a
neurons (C, left panel), and the ciliary localization of GFP:GR21a is unaffected in Or83b
mutants (C, compare left and right panels). However, GFP:GR21a fails to localize to cilia when misexpressed in Or83b
neurons (D), suggesting that factors selectively present in Gr21a
neurons are essential for its localization. Thus, although OR and GR protein families are related, ORs must have evolved distinct molecular properties that confer their absolute dependence upon OR83b.
OR-Independent Localization of OR83b to OSN Ciliated Dendrites
We tested whether there is a reciprocal requirement for conventional ORs for the ciliary accumulation of OR83b by examining OR83b localization in the absence of OR22a/b. Or22a/b
mutant neurons display no odor-evoked potentials to any odorant tested, indicating that no other ORs are likely to be expressed in these neurons [7
]. To distinguish the dendrites of Or22a/b
neurons from those of Or85b
neurons, which share the same sensillum [9
], we expressed an N-terminal GFP-tagged version of OR83b specifically in Or22a/b
neurons. GFP:OR83b is functional, as assayed by rescue of odor-evoked behavior in Or83b
mutant larvae (M. Louis, RB, and LBV, unpublished data), and its distribution in the cell body and dendrite of wild-type Or22a/b
neurons is identical to endogenous OR83b (, top row). This localization is unchanged Or22a/b
in mutant neurons (, bottom row), but levels of GFP:OR83b are reduced in both the cell body and the cilia. This indicates that OR83b is partly destabilized in the absence of a conventional OR, but that it can localize to sensory cilia independently of ORs.
OR-Independent Localization of OR83b to OSN Ciliated Dendrites
OR83b Is Necessary and Sufficient to Mediate OR Localization to Ciliated Dendrites in Other Sensory Neurons
To ask whether OR83b is sufficient to promote OR localization, we ectopically expressed GFP:OR43a with and without OR83b in sensory neurons that do not normally express ORs. When GFP:OR43a is expressed alone in Gr21a
neurons, the protein fails to localize to the sensory compartment and is detected only in the cell body and inner dendritic segment (A, top row). In contrast, OR83b localizes to cilia, albeit extremely weakly, when expressed alone in these neurons (A, middle row, arrowhead). However, when these proteins are co-expressed, both GFP:OR43a and OR83b display clear localization to the ciliated outer segment (A, bottom row). Similar results are obtained by misexpression of ORs and OR83b in the ciliated mechanosensory neurons in the second antennal segment, which mediate the perception of sound [40
] (B). We conclude that OR83b is the only protein required to couple ORs to a transport pathway common to ciliated neurons.
OR83b Is Necessary and Sufficient to Mediate OR Localization to Ciliated Dendrites in Other Sensory Neurons
OR/OR83b Reconstitutes a Functional OR in Gr21a Neurons
We investigated whether ORs and OR83b form a functional odorant receptor in Gr21a
cilia, by ectopically expressing GFP:OR43a and OR83b in these neurons along with the calcium-sensitive fluorescent reporter G-CaMP [12
]. Odor-evoked activity was measured as changes in intracellular calcium concentration in the axon termini of Gr21a
neurons in the V glomerulus of the antennal lobe [38
In flies expressing only G-CaMP, robust responses are observed, as expected, to CO2
, but not to cyclohexanol, a known OR43a ligand [26
] (A, left column). When GFP:OR43a and OR83b are co-expressed in these neurons, significant calcium increases in response to cyclohexanol stimulation are now observed (A, right column). Significant responses of GFP:OR43a/OR83b in Gr21a
neurons are also observed with cyclohexanone, hexanol, benzaldehyde, isoamyl acetate, and geranyl acetate but not octanol, linalool, or caproic acid (B). These results are consistent with previous reports of the ligand specificity of Or43a
]. Thus, OR83b is the only factor required with OR43a to reconstitute a functional OR that is capable of recognizing ligands and stimulating neuronal signaling.
Expression of ORs and OR83b Reconstitutes a Functional OR in Gr21a Neurons
ORs and OR83b Form Heteromeric Complexes in the Sensory Cilia of OSNs
We next asked whether this functional odorant receptor is composed of a complex of OR and OR83b proteins that is present in vivo at the site of odor detection (). Previous in vitro efforts suggested that ORs can form both homomers and heteromers with OR83b, but no evidence was offered that these complexes are functional [29
]. To investigate the existence of OR/OR83b complexes in OSNs, we employed the protein-fragment complementation assay (PCA), using a yellow fluorescent protein (YFP) reporter [43
]. In this technique, complementary N-terminal and C-terminal fragments of YFP [YFP(1) and YFP(2)] are fused to two proteins suspected to interact. The two halves of YFP are not fluorescent alone and do not associate spontaneously, but the physical interaction of the proteins to which they are fused brings the YFP fragments into proximity where they can fold into an active form. The YFP fluorescent signal output therefore not only provides direct evidence for the existence of protein complexes in vivo but also information on their subcellular distribution [44
]. YFP fragments were fused to ORs via a flexible ten-amino-acid linker that has a fully extended length of about 40 Å. With this linker, complex formation can therefore be detected between directly interacting proteins or those that are within 80 Å of each other, which is approximately twice the diameter of the helical bundle of the rhodopsin monomer [45
In Vivo Formation and Distribution of OR/OR83b Complexes
Transgenic constructs encoding YFP(1):OR83b and YFP(2):OR83b fusion proteins were first expressed individually in Or83b mutant neurons (A, top and middle rows). Immunostaining reveals that YFP(1):OR83b and YFP(2):OR83b localize normally and are functional as they rescue the ciliated localization of OR22a/b, but neither protein alone displays detectable YFP fluorescence. When co-expressed, however, a strong YFP fluorescence signal is detected in these neurons, providing evidence for homomeric complex formation by OR83b (A, bottom row, and 7B). Two lines of evidence indicate that this homomerization reflects an intrinsic property of OR83b and does not depend upon the presence of conventional ORs: first, abundant YFP fluorescence is detected within both the cell body and sensory dendrites of these neurons even though OR22a/b protein is concentrated in the sensory compartment (A, bottom row). Second, YFP fluorescence is detected when these fusion proteins are expressed in Gr21a neurons, where no OR is expressed (C). We note that formation of homomers by OR83b does not preclude functional interaction with conventional ORs, as these complexes retain the ability to promote OR22a/b localization (A, bottom row).
To assess heteromeric interactions between ORs and OR83b, YFP(1)- and YFP(2)-tagged versions of OR43a were expressed with complementary YFP(1/2):OR83b fusions in Or83b mutant neurons. Both combinations of these fusion proteins produce a robust fluorescent signal in the sensory cilia, with discrete puncta of fluorescence also observed around the nucleus in the cell body and in the inner dendritic segment (D). Thus, OR83b forms heteromeric complexes with conventional ORs in vivo, and these are concentrated at the site of odor detection.
We investigated whether these heteromeric complexes are functional by expressing these fusion proteins along with G-CaMP in Or83b
mutant neurons and assessing odor-evoked calcium release at OSN axon terminals in the antennal lobe (F). In control animals that express YFP(2):OR83b alone, sparse glomerular activation patterns in response to two known OR43a ligands (cyclohexanol and benzaldehyde) are observed (F, left column). These are similar to activity patterns seen in wild-type animals [12
], indicating that YFP(2):OR83b is able to rescue odor-evoked responses of endogenous ORs. Co-expression of YFP(1):OR43a with YFP(2):OR83b results in odor-evoked calcium responses across broad domains of the antennal lobe (F, right column), and quantification reveals highly significant increases in individual glomerular response properties (F, far right column). These ectopic glomerular responses are due to the activity of YFP(1):OR43a in these neurons because a control odor that does not activate OR43a (ethyl-3-hydroxybutyrate) gives similar glomerular activation patterns in both genotypes (F, bottom row). Thus, the YFP(1):OR43a/YFP(2):OR83b heteromer is functional for odor-evoked neuronal signaling.
As a control for the specificity of these complexes, we generated YFP fragment fusions to GR21a. These localize throughout the cell body in Or83b neurons and the inner segment of the dendrite, although not in the outer segment, as shown for GFP:GR21a (D; unpublished data). When placed in complementary combinations with either YFP-tagged OR43a or OR83b, only an extremely faint fluorescent signal is detected in the cell body (E). Similar results are obtained when YFP(1):GR21a and YFP(2):OR83b are co-expressed in Gr21a neurons (unpublished data). This suggests that the fluorescence observed between different combinations of OR83b and OR43a results from the formation of specific receptor complexes, rather than the mere presence of complementing YFP-fusion proteins within the same membrane.
We also observe relatively weak fluorescence in sensory cilia when YFP(1):OR43a and YFP(2):OR43a fusions are co-expressed in wild-type Or83b
neurons (G, top panel), suggesting that OR43a might homomultimerize. To ask whether this fluorescence signal reflects indirect interactions between OR43a molecules within multimeric OR43a/OR83b complexes, we expressed these fusion proteins in Or83b
mutant neurons (G, bottom panel). Although the YFP(1/2):OR43a fusion proteins are detected in the cell body by immunostaining (G, bottom panel, right side), no intrinsic YFP fluorescence signal is detected (G, bottom panel, left side). These observations suggest that, in contrast to in vitro results [29
], conventional ORs are unable to associate directly in homomeric complexes in OSNs without OR83b and reinforce the specificity of the formation of OR83b homomers and OR43a/OR83b heteromers.
ORs and OR83b Adopt a Novel Membrane Topology
To define the regions that mediate the specific association of ORs with OR83b, we initiated a structure/function analysis of these receptors. This was initially constrained by the lack of knowledge of their membrane topology and structure, as Drosophila
ORs were identified bioinformatically by algorithms that searched for novel proteins with multiple TM domains [19
]. Although these reports all proposed a seven-TM domain structure for the identified sequences, there was no consensus on the placement of these TM segments. By analogy to vertebrate and Caenorhabditis elegans (C. elegans)
ORs, the predicted heptahelical structure of Drosophila
ORs has led to the general acceptance that these proteins represent members of the GPCR family, despite the fact that the insect proteins show no significant sequence similarity to any known GPCR (unpublished data). Indeed, phylogenetic analysis suggests that insect ORs define a distinct family that is no more related to mouse ORs than these are to ion channels (A). Given this apparent novelty in the primary structure of Drosophila
we analyzed their membrane topology using the HMMTOP algorithm [48
] and compared this with a representative sample of mouse ORs. Surprisingly, although the majority of sequences are predicted to contain seven TM domains for both organisms (B), the membrane orientation predictions of these families are distinct (C). Mouse ORs are predicted to have an extracellular N-terminus, which is consistent with the known structure of the GPCR superfamily. In contrast, Drosophila
ORs are predicted to have an intracellular N-terminus (C). Similar predictions are obtained for the ORs with two independent algorithms, TMHMM Server version 2.0 [49
] and TMpred [50
]), and in analysis of the GR protein family (unpublished data).
Bioinformatic Analysis Defines Drosophila ORs As a Novel Family of TM Proteins
To obtain experimental evidence that TM1 of Drosophila
ORs inserts into the membrane with the N-terminus intracellular, we first used the β-galactosidase β-gal fusion technique. This method takes advantage of the observation that β-gal is enzymatically active when present in the cytosolic compartment but not in extracytosolic compartments (luminal or extracellular) [51
]. By fusing β-gal to the C-terminus of a TM domain and assessing enzymatic activity, the cellular location of the enzyme and hence orientation of insertion of the TM domain can be determined (A, top row). This assay does not require that the resulting fusion proteins are trafficked to the cell surface but merely assays the orientation of protein insertion in the ER.
The N-Terminus of Drosophila ORs Is Intracellular
We generated constructs encoding either the N-terminal domain alone or the N-terminal domain and TM1 of OR83b fused at their C-termini to β-gal. As a control, we generated constructs in which a synthetic TM domain was placed between the fragments of OR83b and β-gal, which are predicted to give opposite results to the corresponding direct fusions to the enzyme (A, top row). These constructs were expressed in cultured Drosophila S2 cells, and β-gal activity was assessed by X-gal staining. β-gal was scored as active if 10%–20% of cells were blue, which corresponded to the transfection efficiency in these experiments, and inactive if <1% cells were blue. The results are consistent with an intracellular localization of the OR83b N-terminus (A, bottom row). Identical results were obtained with fusion constructs of the conventional OR, OR9a. In contrast, equivalent fusions with N-terminal fragments of the class A GPCR, Drosophila rhodopsin RH1, give results consistent with the extracellular location of the RH1 N-terminus. These results support the computational prediction that the N-terminus of Drosophila ORs is intracellular.
We wished to determine whether OR N-termini reside intracellularly in the context of the full-length proteins in OSNs, and we therefore developed a novel method to probe protein topology in vivo based upon the YFP PCA. We generated transgenes encoding cytosolic topology-sensor proteins that comprise YFP fragments fused to a leucine zipper dimerization domain (referred to here as ZIP) (YFP(1):ZIP, YFP(2):ZIP) and corresponding OR83b fusion proteins bearing YFP fragments and the same leucine zipper sequence at their N-termini (YFP(1):ZIP:OR83b, YFP(2):ZIP:OR83b). YFP(1/2):ZIP:OR83b fusions are functional as assessed by rescue of OR22a/b localization (unpublished data).
When the complementary YFP(1/2):ZIP cytosolic sensors are expressed in OSNs, the ZIP domains promote their association and we detect YFP fluorescence concentrated in the nucleus (B), probably reflecting the tendency of small cytoplasmic proteins to translocate to this compartment. When these sensors are co-expressed with the complementary YFP(1/2):ZIP:OR83b proteins, fluorescence is observed in the puncta in the cell body and throughout sensory cilia (C). As the reconstitution of YFP fluorescence can occur only if both YFP fragments are present on the same side of the membrane, these results demonstrate unambiguously that the OR83b N-terminus is located in the cytoplasm. Similar results are obtained with combinations of YFP(1/2):ZIP and YFP(1/2):ZIP:OR43a fusions (D). We also note that the heteromeric OR43a/OR83b complexes observed in OSNs by the PCA (D) could only have been observed if the N-terminal YFP tags are topologically equivalent. Thus, both OR83b and conventional OR N-termini are located intracellularly in vivo, and the association of ORs with cytosolic topology sensors causes these sensor proteins to be relocalized to ciliated dendrites.
We generated equivalent extracytosolic topology sensors bearing the signal sequence from mammalian calreticulin at their N-termini that targets these proteins to the secretory pathway (SS:YFP(1):ZIP, SS:YFP(2):ZIP) [52
]). In OSNs expressing these sensors, we detect α YFP immunoreactivity in perinuclear membranes consistent with their targeting to the ER lumen (unpublished data). When co-expressed in complementary combinations with either YFP(1/2):ZIP:OR83b or YFP(1/2):ZIP:OR43a, we do not observe reconstitution of intrinsic YFP fluorescence (unpublished data), which is consistent with these extracytosolic sensors being on the opposite side of the membrane of the OR N-termini. Interpretation of this result must be tempered, however, by the fact that the combination of SS:YFP(1):ZIP and SS:YFP(2):ZIP fails to fluoresce either in the intracellular sorting pathway or extracellularly (unpublished data).
To examine OR topology beyond the location of the N-terminus, we performed OR83b antibody epitope-staining experiments, which probe topology by comparing antibody access in permeabilized and non-permeabilized conditions. OSN dendrites proved to be inaccessible to labeling without compromising cell permeability. We find that the larval salivary gland, a secretory tissue that is easily accessible to whole-mount staining, appears to support cell-surface expression of ectopically expressed GFP:OR83b, with the intrinsic GFP fluorescent signal detected in membranes along the cell boundaries and within cytoplasmic vesicles (A and B) similar to the pan-membrane localization of this protein in OSNs (). To determine whether GFP:OR83b is on the cell surface, we stained these cells with antibodies against GFP and the OR83b α-EC2 antibody, which recognizes an epitope within the computationally predicted second extracellular loop, either in the presence or absence of detergent. Under permeabilized conditions, both antibodies detect the entire pool of protein (B, top row). In contrast, when the cells are unpermeabilized, the GFP antibody fails to show staining, while the OR83b α-EC2 antibody labels the cell boundary, representing the fraction of protein within the plasma membrane (B, middle row). This staining is specific, as it is not observed in unpermeabilized salivary glands that do not express GFP:OR83b (B, bottom row). These observations demonstrate that GFP:OR83b is present on the surface of these cells, confirm that the N-terminal GFP tag is located intracellularly, and indicate that the EC2 epitope is exposed on the extracellular face of the membrane.
Probing OR83b Topology by Antibody Epitope Staining
We next used antibodies against the OR83b N-terminus and epitopes within the computationally predicted second and third intracellular loops (IC2 and IC3) and performed a similar set of experiments. Unlike α-EC2, these three antibodies display staining only when the cells are permeabilized (C), supporting an intracellular location of these epitopes. Identical results are obtained when using untagged OR83b (unpublished data), indicating that the GFP tag does not influence protein topology.
Because topology mapping by epitope access with detergents has inherent limitations (e.g., [53
]), we performed immunoelectron microscopy (immunoEM) in cross sections of wild-type sensilla to ask where the α-EC2 epitope lies relative to the dendritic membrane. Horizontal sections reveal multiple dendritic branches within the sensillum lymph by conventional EM (D). For immunoEM, we prepared ultrathin plastic sections and stained these with the OR83b α-EC2 antibody and a secondary antibody conjugated to 5 nm gold. To permit antibody access, more gentle fixation procedures are used and these somewhat distort dendritic membrane morphology (E). Nevertheless, gold particles are detected specifically along the dendritic membranes, consistent with the expected membrane localization of OR83b (E). Moreover, these gold particles show a striking bias in their distribution, with 87.5% of gold particles (n
= 471) found outside the boundaries of the ciliary membranes (F). Together with the immunofluorescence analysis in the salivary gland, this result indicates an extracellular location for the EC2 epitope.
Together, these data support the bioinformatic prediction that the EC2 epitope of OR83b is extracellular, while the N-terminus and the IC2 and IC3 epitopes are intracellular. Although we have obtained multiple lines of evidence for the topology of the OR N-terminus, we note that the exact number and precise placement of TM segments in the Drosophila ORs remain to be proven. One prediction of the model presented in the snake plots illustrated in the figures is that the C-terminus is extracellular. Unfortunately, we have not been able to test this experimentally, because, unlike the N-terminal GFP tag, fusion of GFP or the smaller Myc tag to the OR83b C-terminus destroys protein function (unpublished data).
ORs and OR83b Associate via Conserved Cytoplasmic C-Terminal Domains
To examine the domains that mediate OR/OR83b association, we used this new topology model to design a chimeric receptor [OR83b(1–170):OR43a(159–376)] with a breakpoint in EC2 such that the protein comprises predicted TM1–TM3 of OR83b and TM4–TM7 of OR43a. This chimera localizes to cilia in wild-type antennae, but fails to localize in Or83b mutants (A). In Gr21a neurons, the chimera localizes to ciliated dendrites only when OR83b is co-expressed (A). This chimera therefore displays the localization properties of OR43a, suggesting that the C-terminal region of OR43a is sufficient to couple to OR83b-dependent transport to olfactory cilia.
OR83b and ORs Associate via Conserved C-Terminal Domains
OR protein sequences are extremely divergent but show the strongest homology within this C-terminal region. Given its functional dependence on OR83b, we asked whether any of the computationally predicted cytoplasmic loops within this fragment of OR43a (IC2, IC3) physically interact with any cytoplasmic regions of OR83b (N-term, IC1, IC2, IC3) in a yeast two-hybrid assay. Although this technique analyzes OR interactions without the structural information that might be provided by OR TM domains, this approach has been successfully used to define cytoplasmic associations of many types of polytopic membrane protein (e.g., [54
]). We observe interactions between IC3 of OR43a and IC3 of OR83b but not any other combination (B). OR83b IC3 also interacts with an equivalent region of OR22a, but not of GR21a, demonstrating that this is a conserved interaction interface specific to the OR family (B).
These experiments defining OR/OR83b interactions also provide further evidence that conventional ORs adopt the same topology as OR83b. First, the membrane insertion orientation of the OR83b:OR43a chimera is determined by the N-terminus of OR83b, but this fusion protein retains the localization properties of OR43a (A). Second, we observe direct physical interactions between loops of OR83b and ORs that are predicted to be topologically equivalent (B).