Lipid Flippase Modulates Olfactory Receptor Expression and Odorant Sensitivity in Drosophila

Lipid flippase modulates olfactory receptor expression and odorant sensitivity in Drosophila

Tal Soo Haa,1, Ruohan Xiab,1, Haiying Zhangb, Xin Jinb,2, and Dean P. Smithb,3

aDepartment of Biomedical Science, Daegu University, Gyeongsan City 712-714, South Korea; and bDepartments of Pharmacology and Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111

Edited* by Dan L. Lindsley, University of California, San Diego, La Jolla, CA, and approved April 17, 2014 (received for review January 30, 2014) In Drosophila melanogaster, the male-specific pheromone cVA (11- affected in the mutants than other Orco-dependent olfactory neuron cis-vaccenyl acetate) functions as a sex-specific social cue. How- responses (compare Fig. 1C and Fig. S1C), so we focused our studies ever, our understanding of the molecular mechanisms underlying on these neurons. cVA pheromone transduction and its regulation are incomplete. To determine whether the severity of the cVA detection pheno- Using a genetic screen combined with an electrophysiological as- type is neuron-dependent, we expressed a farnesol-activated OR, say to monitor pheromone-evoked activity in the cVA-sensing Or83c, in Or67d neurons in lieu of the endogenous Or67d re- Or67d neurons, we identified an olfactory sensitivity factor encoded ceptor (9). Similar to basiconic neurons, there is a moderate re- by the dATP8B gene, the Drosophila homolog of mammalian duction in farnesol sensitivity in the mutant (Fig. 1 D and E) that ATP8B. dATP8B is expressed in all olfactory neurons that express is less severe than the dATP8B mutant cVA defects. This suggests Orco, the odorant receptor coreceptor, and the odorant responses that the severity of the response defects is receptor dependent in most Orco-expressing neurons are reduced. Or67d neurons are and not neuron dependent. severely affected, with strongly impaired cVA-induced responses We mapped the mutant locus to a single gene at position 87A and lacking spontaneous spiking in the mutants. The dATP8B on the right arm of the third chromosome (Fig. 2A). This gene locus encodes a member of the P4-type ATPase family thought encodes a member of the P4-type ATPase family similar to ver- to flip aminophospholipids such as phosphatidylserine and phos- tebrate ATP8B. All four mutants we recovered contain inacti- phatidylethanolamine from one membrane leaflet to the other. vating lesions in dATP8B (Fig. 2B). dATP8B1 (Z3-1778) has a 10-bp dATP8B protein is concentrated in the cilia of olfactory neuron deletion at the start of exon 17, which removes the splice ac- dendrites, the site of odorant transduction. Focusing on Or67d ceptor. This lesion is predicted to result in skipping exon 17 and neuron function, we show that Or67d receptors are mislocalized producing a frameshift mutation and premature termination (Fig. in dATP8B mutants and that cVA responses can be restored to 2B). dATP8B2 (Z3-3278) has a 56-bp deletion in exon 17 that is dATP8B mutants by misexpressing a wild-type dATP8B rescuing predicted to produce a frameshift mutation and a truncated prod- transgene, by expressing a vertebrate P4-type ATPase member in uct. dATP8B3 (Z3-3028) has a nonsense mutation in exon 8 that the pheromone-sensing neurons or by overexpressing Or67d re- 913 4 ceptor subunits. These findings reveal an unexpected role for lipid converts the Arg codon to a stop codon (CGA to TGA). dATP8B (Z3-4031) has a point mutation that introduces a premature stop translocation in olfactory receptor expression and sensitivity to 1160 5 volatile odorants. codon in exon 12 at Glu (CAA to TAA). A fifth allele (dATP8B ) was obtained that contains a piggyback transposable element 5 olfaction | aminophospholipid translocase integrated within the coding sequence of exon 11 (10). dATP8B mutants have cVA response defects that are indistinguishable from ethyl methanesulfonate alleles (Fig. 1A). n a forward genetic screen for mutants with defective responses Ito cVA (11-cis-vaccenyl acetate) pheromone, we recovered four mutants unresponsive to 1% cVA that also lacked normal Significance spontaneous action potentials in the absence of cVA (Fig. 1 A– C)(1–3). Approximately half of the Or67d neurons from these The work identifies dATP8B as a critical factor for proper four lines were insensitive to all concentrations of cVA, and odorant and 11-cis-vaccenyl acetate pheromone sensitivity. those that did respond had severely impaired cVA sensitivity These defects are attributed to a failure to translocate lipids, compared with wild-type controls (Fig. 1 A and C). Comple- because mutants defective for dATP8B enzymatic activity do mentation analysis demonstrated that all four were alleles of not rescue, but a functional vertebrate homolog fully rescues a single locus we named dATP8B. The four mutant alleles were the pheromone detection defects. We identified abnormal lo- indistinguishable and have little or no variation in penetrance or calization of Or67d receptors as a likely cause for the pheno- NEUROSCIENCE mutant strength (Fig. 1A). types, and overexpression of Or67d can rescue 11-cis-vaccenyl To establish whether these mutants affect all or a subset of acetate sensitivity and loss of spontaneous spiking in the lipid odorant responses, we surveyed several of olfactory neuron classes flippase mutant. We suggest dATP8B is important for lipid that use different receptor classes (Figs. S1 and S2). Responses translocation in olfactory neurons, which in turn is important for proper subcellular trafficking of receptor subunits. to CO2, mediated by gustatory receptor family members (4, 5), and odorants detected by several variant ionotropic receptor Author contributions: D.P.S. designed research; T.S.H., R.X., H.Z., and X.J. performed re- family members (6) were not affected in the mutants (Fig. S2). search; T.S.H., R.X., and D.P.S. analyzed data; and T.S.H., R.X., and D.P.S. wrote the paper. By contrast, odorants detected by odorant receptor members The authors declare no conflict of interest. (ORs) that multimerize with the OR coreceptor Orco had re- *This Direct Submission article had a prearranged editor. duced odorant responses and action potential amplitudes that 1T.S.H. and R.X. contributed equally to this work. were 50% smaller in the mutants compared with wild-type con- 2Present address: Pfizer Inc., Analytical R&D, BioTherapeutics Pharmaceutical Sciences, trols (Fig. S1). Orco is a coreceptor thought to multimerize with Pearl River, NY 10965. OR receptor subunits to produce functional odorant-gated ion 3To whom correspondence should be addressed. E-mail: dean.smith@utsouthwestern. channels (7, 8). Orco mediates food odorant responses in most edu. basiconic sensilla neurons and cVA responses in Or67d-expressing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. trichoid neurons. The cVA response defects are more strongly 1073/pnas.1401938111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1401938111 PNAS | May 27, 2014 | vol. 111 | no. 21 | 7831–7836 Downloaded by guest on October 2, 2021 ATPases are encoded in the human genome, and six are present in the Drosophila genome, but the function of the majority of these proteins is unknown. To determine whether dATP8B is required in olfactory neurons or in the nonneuronal support cells that secrete the sensillum lymph and odorant binding proteins, we expressed a dATP8B cDNA in either the Or67d olfactory neurons or the associated support cells in the dATP8B5 mutant background. The cDNA fails to rescue when expressed in support cells (21) (Fig. 3A). dATP8B cDNA expressed in the Or67d neurons with Or67dGAL4 fully rescues the spontaneous activity and cVA sensitivity defects (22) (Figs. 1B and 3A). cDNAs that include exon7 also rescue the dATP8B5 mutant defects (Fig. 3C). Is the enzymatic activity of dATP8B required for olfactory function? dATP8B contains the conserved DGETN and DKTGT domains found in all P4-type ATPase family members (23) (Fig. 2C). The flipping mechanism for P4-type ATPases is thought to be analogous to that of SERCA1 (ATP2A1), a P-type ATPase that pumps calcium ions across membranes (24). Translocation involves a cycle of autophosphorylation and dephosphorylation on a conserved aspartate that converts the E1 phosphoaspartate, substrate-bound conformation to the E2 conformation that favors release of the substrate on the other side of the membrane and hydrolysis of the phosphoaspartate (25, 26). The conserved aspartic acid residue in the DKTGT domain is autophosphorylated and dephosphorylated during the substrate translocation cycle in related P-type ATPases, whereas the DGETN domain is important for the phosphatase activity of the enzyme (27, 28). We engineered Fig. 1. dATP8B mutants are defective for cVA responses and spontaneous activity in Or67d neurons. (A) Sample single sensillum recording traces to 1% a dATP8B mutant predicted to disrupt the phosphorylation cycle cVA from control (wild-type) and the five dATP8B mutant alleles. Df is Exelixis (29). Glutamate to glutamine substitutions in the dGETN do- 6162 (Bloomington stock 7641) deleted for the dATP8B locus. Bal is TM6B. (B) main in SERCA1 impair the phosphatase activity of the enzyme, Spontaneous activity (spikes in the absence of cVA) is eliminated in the locking the enzyme in the phosphoaspartyl state (29, 30). We Or67d neurons from dATP8B1 mutants and restored by expressing a dATP8B produced transgenic flies expressing the equivalent mutation in cDNA with pOr67dGAL4 (Rescue). n ≥ 14 for each genotype. (C) Dose–response the DGETN domain, dATP8BE351Q. The mutant protein fails to curves for wild type and dATP8B1 mutants. dATP8B1 mutants have a pro- rescue the defects in the dATP8B mutant background (Fig. 3C). found loss of responsiveness to cVA compared with wild-type controls. n = These findings are consistent with a requirement for the enzymatic = 5 14 for WT, n 96 for dATP8B mutants. (D) Or83c misexpressed in Or67d activity of dATP8B in Or67d neurons. neurons instead of the endogenous Or67d receptor gene respond to farnesol (genotype w ; UAS Or83c ; Or67dGAL4). (Top) Responses of these Antiserum that recognizes the C terminus of the dATP8B transgenic flies to three concentrations of the Or83c ligand, farnesol (9). protein was used to identify its cellular and subcellular locali- (Middle) Responses are moderately reduced in these transgenic flies in the zation (Materials and Methods). The immune serum reacts with dATP8B mutant background (genotype w ; UAS Or83c ; Or67dGAL4,dATP8B5). the cilia of Drosophila olfactory neurons, and the protein is (Bottom) Wild-type Or67d neurons do not respond to farnesol. (E)Dose–represent in the cilia of Or67d neurons (Fig. 4 A, C,andD). sponse curves to farnesol for the three genotype in D. n = 6 for each genotype. dATP8B5 mutants lack staining (Fig. 4B), confirming the specificity of the antiserum and that dATP8B5 is a null allele. We further examined what other olfactory neurons express dATP8B. The dATP8B locus is predicted to encode several large, al- Although dATP8B expression levels vary among olfactory ternately spliced transcripts (11). To determine which tran- neuron cilia, we found that the protein colocalizes with GFP scripts were expressed in the antenna, we isolated dATP8B expressed with the Orco promoter (Fig. 4 E–G). Localization of cDNAs from reverse-transcribed mRNA purified from wild- dATP8B protein to the site of olfactory signal transduction in type antennas (Materials and Methods). Most of the dATP8B Orco-expressing olfactory cilia is consistent with the observed cDNAs lacked exon 7, corresponding closely to predicted tran- olfactory defects. script D (Fig. 2B) (11). This shorter splice variant is enriched in Could dATP8B function as a cVA flippase, explaining the antennas, whereas dATP8B transcripts with and without exon 7 particular sensitivity of cVA responses to the loss of this factor? are widely distributed in heads and bodies and throughout de- dATP8B might flip cVA lipids from outside to the inside of velopment (Fig. S3). The protein predicted for the major an- Or67d neurons, inactivating the cVA signal. Weak cVA exposure tenna transcript is a 10-transmembrane segment protein with to males in mixed cultures, for example, might result in desensi- amino acid sequence similar to human ATP8B and ATP8A iso- tization of the Or67d neurons, producing the dATP8B pheno- forms of the P4-type ATPase family (Fig. 2 C and D). P4-type type. To examine this possibility, we recorded responses from ATPases constitute a large, conserved family essential for creating virgin female dATP8B mutants isolated as larvae, raised in iso- and maintaining lipid asymmetry in membranes (12–15). In lation, and never exposed to cVA. Flies never exposed to cVA humans, ATP8B1 regulates the lipid composition of the canal- have the typical dATP8B phenotype, with no spontaneous ac- iculi cells that transport bile acids from the liver. ATP8B1 is tivity and blunted cVA sensitivity that is indistinguishable from thought to transfer phosphatidylserine (PS) from the outer to the flies raised in mixed cultures (Fig. 3D). This suggests that the inner leaflet of the plasma membrane, making these cells re- mutant phenotype does not arise from cVA desensitization. sistant to bile acids (16). Defects in ATP8B1 expression cause Orco and Snmp1 are both essential factors required for cVA progressive familial intrahepatic cholestasis type 1 (12, 17). detection. Snmp1 is a CD36 homolog essential for cVA detection Lesions in ATP8A1 are associated with abnormalities in the but not required for detection of most food odorants (1) (31). We developing nervous system (12, 16, 18–20). Sixteen P4-type tested whether the dendritic localization of dATP8B is affected

7832 | www.pnas.org/cgi/doi/10.1073/pnas.1401938111 Ha et al. Downloaded by guest on October 2, 2021 Fig. 2. The dATP8B locus encodes a P4-type ATPase. (A) Polytene chromosome region encoding the dATP8B locus (modified from ref. 43). Df3128 (longer thick bar) failed to complement cVA sensitivity in the dATP8B1 mutant allele. Smaller deficiencies further restricted the dATP8B locus to a small region of 87A, defined by Df7641 (small thick bar) that failed to complement dATP8B1, and Df7642 and Df7968 that complement dATP8B1.(B) Genomic structure of the dATP8B locus. Exons are denoted by bars, introns by lines. The gray bar denotes exon 7 that is absent from most dATP8B cDNAs isolated from antennal tissues. The position and molecular lesions of the five dATP8B alleles are depicted. (Scare bar, 1 kb.) (C) Predicted transmembrane structure of the dATP8B protein. Aspartate 597 is the first residue in the conserved sequence DKTGTLT and glutamate 351 in the conserved DGETN domain. The position of the engineered Glu to Gln mutant allele is depicted. Numbers represent relative location of the different mutant alleles and exon 7. (D) Structural similarity between dATP8B and human ATP8B1. dATP8B (Upper) and ATP8B1 are more than 40% identical, sharing well-conserved domains. The numbers below each domain represent sequence identity and similarity.

by loss of Or67d, Snmp1, or Orco. We found normal localization Discussion Z0429 GAL4 of dATP8B in the cilia in Snmp1 and Or67d mutants, We present evidence that an aminophospholipid transferase but much of the dATP8B protein mislocalized to the olfactory plays a role in olfactory sensitivity and receptor trafficking in 2 neurons cell bodies in Orco mutants (Fig. S4). This suggests Orco-expressing olfactory neurons. Because we can rescue the Orco is important for localization of dATP8B to the cilia. mutant phenotype by overexpressing Or67d receptor subunits, Is the flippase required for localization of any cVA-sensitivity we think the most likely role for dATP8B is to facilitate the factors? Orco and Snmp1 localization are unaffected in dATP8B proper localization of Orco-associated tuning receptor subunits. mutants (Fig. 5D and Fig. S4). However, there is a reduction in How lipid translocation mediates this process remains a ques- Or67d in the dendritic cilia relative to the cell bodies in the tion. In yeast, the major phenotype associated with loss of P4- mutants. We quantified Or67d using GFP-tagged Or67d in lieu of ATPase function is abnormal transport vesicle budding from the untagged Or67d. We found a significant reduction in the fraction transgolgi network (19, 33, 34). Similar defects in cargo sorting of Or67d that localized to the cilia in dATP8B mutants compared have been observed in Caenorhabditis elegans Tat-1 mutants (35), with wild-type controls (Fig. 5 A–C). This suggests that receptor and other C. elegans P4-ATPase mutants are defective for trafficking may underlie part or all of the olfactory defects. phagocytosis of dead cells and regulation of ectosome pro- Is dATP8B protein specifically required, or is proper lipid duction (35–38). Proper lipid localization may be required for localization the essential aspect of the dATP8B mutant pheno- vesicle docking or receptor recycling leading to reduced receptor type?WeexpressedavertebrateP4-typeATPase,bovineATP8A2, levels in the cilia. Reduced receptor density could explain the in Or67d neurons in dATP8B5 mutant background (Fig. 5E). observed right-shift in the odorant and pheromone dose–re- This isoform is selective for PS over phosphatidylethanolamine sponse curves and the reduced maximal activation of Or67d NEUROSCIENCE and phosphatidylcholine, according to the ATPase activity of re- neurons. This could also explain the absent spontaneous activity, combinant protein in the presence of these different lipids (32). although it is puzzling that there is a complete loss of these Remarkably, expression of this vertebrate enzyme completely events and not reduced frequency. It will be of great interest in rescued the dATP8B5 mutant phenotype (Fig. 5F). This suggests the future to determine whether dATP8B is a competence factor lipid translocation is critical for olfactory neuron responsiveness important for receptor expression, or whether it actively regu- and not a specific requirement for dATP8B protein itself, for lates receptor density and odorant sensitivity through a lipid example as a receptor subunit. translocation mechanism. It will also be of interest to establish Because Or67d levels are reduced in the cilia, we tested whether whether PS or phosphatidylethanolamine or both are mis- this is the basis of the mutant phenotype by overexpressing localized in dATP8B mutant Or67d cilia, and to understand why Or67d subunits in these neurons to determine whether this could levels of Orco in the cilia seem to be unaffected. rescue these defects. We introduced an extra copy of UAS-Or67d Malaria-transmitting mosquitoes Anopheles gambiae and Aedes expressed under control of the strong ELAV neuronal promoter aegypti harbor genes with 69% and 77% amino acid identity with in the dATP8B5 mutant background. The Or67d transgene, to- dATP8B. If these homologs perform a similar role, they are poten- gether with the two endogenous Or67d genes, restored spontaneous tial targets to manipulate mosquito behavior. Finally, there are activity and normal cVA pheromone detection in the dATP8b four additional P4-type ATPase genes in the fly genome, and mutant background (Fig. 6). dATP8B splicing variants are widely expressed in the head and

Ha et al. PNAS | May 27, 2014 | vol. 111 | no. 21 | 7833 Downloaded by guest on October 2, 2021 from inducing activity during these studies. cVA was diluted in paraffin oil, and 30 μL was applied to a filter paper, inserted in a Pasteur pipette, and air was passed over the filter and presented as the stimulus. The purity of the cVA was confirmed both by NMR and by mass spectroscopy. Signals were amplified 1,000× and fed into a computer via a 16-bit analog-to- digital converter and analyzed offline with AUTOSPIKE software (USB- IDAC system; Syntech). Low cutoff filter setting was 200 Hz, and the high cutoff was 3 kHz. Action potentials were recorded by inserting a glass electrode in the base of a sensillum. Data analysis was performed according to de Bruyne et al. (41). Signals were recorded for 20 s or 30 s, starting 10 s before cVA stimulation. Action potentials were counted 1 s before cVA stimulation and for 1 s after cVA stimulation. All recordings were performed from sepa- rate sensilla, with a maximum of two sensilla recorded from any single fly.

RT-PCR Analysis. Dissected whole antennae, heads, or bodies were disrupted using a Polytron homogenizer in TRIzol (Invitrogen) as recommended by the manufacturer. Total RNA (1 μg) was used for RT reactions using SuperScript III according to the manufacturer. One twentieth of this material was used as template for individual PCR reactions. For the CG14741 isoform assays, primers were designed that spanned several exons, including the alternatively spliced exon 7. Primer sequences are 5′-CAATACCTATGCCAAAGCCCG and 5′-ATCCGCCAGTTCATCCGTAAGC. RT products were amplified with Accuprime Fig. 3. dATP8B is required for normal function in Or67d neurons. (A) (Invitrogen). The predicted products are 2.72 kb for genomic DNA, 1.67 kb dATP8B1 homozygous mutants (open circles) have defective cVA responses for long isoform variants, and 0.98 kb for the short isoforms lacking exon 7. compared with wild-type controls (open squares). Expression of a dATP8B cDNA regulated by the Or67d promoter (rescue Or67d, genotype w ; UAS Or67d /+ ; Or67dGAL4, dATP8B1/dATP8B1) restores dATP8B1 mutants to wild- type cVA sensitivity (closed squares), but expression of dATP8B in support cells of dATP8B1 mutants using the lush promoter (rescue LUSH, closed circles; genotype w ; UAS dATP8B/LUSH GAL4 ; dATP8B1) fails to rescue cVA sensitivity. (B) Sample traces showing rescue of normal spontaneous activity and 1% cVA sensitivity to Or67d neurons from dATP8B5 mutants by expressing a dATP8B cDNA with a single copy of Or67dGAL4.(C)cVAdose–response curves for wild-type (open boxes), dATP8B5 mutants (open circles), and transgenic flies expressing wild-type (filled boxes) or dATP8BE351Q mutant dATP8B cDNAs (open stars) in the dATP8B5 mutant background. A longer dATP8B cDNA, including exon 7 also rescues (filled triangles). n = 6–8. (D) dATP8B5 mutants never exposed to cVA show typical defects. (Upper) Responses of dATP8B5 females raised in mixed cultures to 300-ms pulses of air with 1%, 10%, or 100% cVA. (Lower)FemaledATP8B5 mutants isolated as larvae, raised in iso- lation, and never exposed to cVA before testing are not different from those raised in mixed culture.

body. It will be important to determine what roles these additional flippase enzymes play in Drosophila biology. Materials and Methods Fly Stocks. All animal work was done in strict accordance with University of Texas Southwestern Animal Resource Center policies and the American As- sociation for Accreditation for Laboratory Animal Studies guidelines, and the animal care and use program conforms to all current National Institutes of Health standards for the care and use of animals in research. Drosophila melanogaster strains were provided by the Bloomington Stock Center and raised on standard cornmeal agar molasses medium. dATP8B mutants were identified in a screen for cVA defective mutants (1). The wild-type strain used in this study was w1118. Or67d2 mutants (Or67dGAL4) were generously provided by B. J. Dickson (Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, VA) (22), the UAS GFP-tagged Or67d was provided by Richard Benton (University of Lausanne, Fig. 4. dATP8B protein is enriched in a subset of olfactory neuron cilia. Lausanne, Switzerland) (31), and UAS CD4tdGFP was from Chun Han (Cornell (A) Anti-dATP8B antiserum recognizes antigen localized in the cilia of University, Ithaca, NY) (39). To test for rescue of dATP8B mutant cVA wild-type olfactory neurons. (B) Anti-dATP8B antiserum does not react GAL4 responses by dATP8B cDNAs, we crossed w ; UAS-dATP8B cDNA ; Or67d , to dATP8B5 mutant antenna indicating the antiserum is specific. (C) 5 5 dATP8B flies to w ; UAS-dATP8B cDNA ; dATP8B and tested the F1 progeny. Colocalization of dATP8B in wild-type cilia expressing membrane-bound GFP Similarly, to test for rescue of cVA responses using engineered mutant forms of in Or67d neurons. Arrow depicts colocalization of the two markers. (D) E351Q dATP8B, UAS- dATP8B or bovine ATP8A2 was substituted for the wild-type Or67d neuron cilia from dATP8B mutants expressing a dATP8B cDNA with dATP8B cDNA in the cross. (Fig. S5 shows complete genotypes used in this work.) Or67dGAL4. Arrow depicts dATP8B in the cilia. (E) Frozen tissue section through the antenna of a transgenic fly expressing membrane-tethered Single Sensillum Recordings and Preparation of cVA. Extracellular electro- GFP expressed with the Orco promoter. (F) dATP8B protein localization in physiological recordings were carried out according to ref. 40. Flies (2–7d thesamesection.(G)MergedimageofE and F above. Although dATP8B old, males and females) were under a constant stream of charcoal-filtered levels vary among the cilia of different receptor neurons, all Orco-expressing air (36 mL/min, 22–25 °C) to prevent any potential environmental odors neurons express dATP8B.

7834 | www.pnas.org/cgi/doi/10.1073/pnas.1401938111 Ha et al. Downloaded by guest on October 2, 2021 DNA Sequencing. PCR was used to isolate genomic DNA from dATP8B mutants for sequencing analysis. The entire CG14741 locus was sequenced for each mutant to unambiguously define the relevant lesions. Two independent PCR reactions were performed for each genomic fragment and sequenced to eliminate potential PCR artifacts. Sequencing was performed by the sequencing core facility at University of Texas Southwestern using Applied Biosystems Inc. (ABI) Big Dye Terminator 3.1 chemistry, and analyzed on ABI capillary instruments.

Generation of dATP8B Antiserum. A peptide corresponding to the C-terminal 160 amino acids of the dATP8B isoform D (amino acids 1322–1482) with an N-terminal 6-histidine tag was produced in bacteria using pET28a (Novagen). The polypeptide was purified by nickel chromatography (Qiagen), eluted, and dialyzed extensively with water and lyophilized. Purified polypeptide (1 mg) was mixed in 1 mL PBS and 1 mL complete Fruends adjuvant, emulsified, and 0.2 mL was injected s.c. into rabbits prescreened for lack of antibodies

Fig. 6. Overexpression of Or67d rescues dATP8B mutant defects. (A) Sample traces from flies overexpressing Or67d in the olfactory neurons with ELAV Gal4 (genotype ELAV Gal4/ + ; UAS Or67d/+ ;dATP8B5.Note restoration of spontaneous activity and cVA responses. (B) Quantifica- tion of spontaneous activity in flies overexpressing Or67d. n = 8foreach genotype. (C)Dose–response curves for wild-type (open squares), dATP8B5 (open circles), and dATP8B5 mutants overexpressing GFP-tagged Or67d with ELAV Gal4 (filled circles). n = 8 for each genotype and time point.

that cross-react with Drosophila tissue sections. Three boosts were performed as above every 3 wk but using incomplete Fruends adjuvant. Serum was used without further purification at 1:300–1:1,000 for immunofluorescence experiments.

Immunocytochemistry and Image Quantification. Rabbit anti-dATP8B serum was heat-inactivated at 50 °C for 30 min and used without further purification. Alexa 543-conjugated goat anti-rabbit antibodies (Invitrogen Molecular Probes) were used at 1:500 dilutions. Anti-bovine ATP8A2 was reported previously (32) and was used at 1:300–1:1,000 dilutions. Images were captured on a Zeiss LSM 510 confocal microscope. For image quantification, 20 images of frozen tissue sections from flies expressing UAS GFP- tagged Or67d in wild-type or mutant background (genotypes: wild type, w; UAS GFPOr67d; Or67dGAL4,w;UASGFPOr67d;Or67dGAL4,dATP8B5)were stained with anti-GFP antiserum (Life Technologies, A-11122). For Orco quantification, flies expressing GFP under control of the pOr67d GAL4 (to identify at1 neurons) either in the dATP8B5 mutant background or dATP8B5 heterozygous background (carrying TM6 as the wild-type control) were stained with rabbit anti-Orco antiserum (gift of Leslie Vosshall, The Rockefeller University, New York). GFP and Orco signal intensities were quantified using a Zeiss LSM510 confocal microscope and NIH ImageJ (downloaded from http://rsbweb.nih.gov/ij/download.html). Images were obtained using identical settings between genotypes. A coworker blin- ded to the genotypes then analyzed the images. Pixel values were obtained for identical area rectangles (for cilia) or circles (cell bodies) between genotypes. Background was subtracted for each value (back- NEUROSCIENCE ground was determined by collecting pixels from an identical area in the same image with no fluorescence signal). The ratio of cilia and cell body of was calculated, and statistical analysis was performed using Student’s t test.

E351Q Fig. 5. Or67d levels are reduced in dATP8B mutant cilia. (A)Exampleof Generation of Bovine ATP8A2 and dATP8B Transgenes. dATP8BE351Q was GFP-tagged Or67 expressed in wild-type dATP8B background (genotype, generated by introducing the GAG to CAG mutation in two PCR primers and GAL4 w: UAS GFPOr67d ; Or67d /+). (B) Typical example of GFP-tagged amplifying the regions upstream and downstream, spanning unique re- Or67dinthedATP8B mutant background (genotype w;UASGFPOr67d; striction sites in the dATP8B cDNA (Aat2 upstream and Sfi1 downstream). GAL4 5 5 Or67d , dATP8B /dATP8B ).(C) Quantification of cilia to cell body These products were mixed and amplified to produce a 920-bp fragment ratio GFP-tagged Or67d in wild type and dATP8B mutants. Genotypes that was confirmed for the correct sequence and cloned into the Aat2-Sfi are significantly different at P < 0.01 by Student’s 2-tailed t test. n = 20 digested wild-type cDNA and cloned into pUASt (42) for production of for each bar. (D) Quantification of cilia to cell body signal ratio for Orco transgenic animals. Bovine ATP8A2 (ATPase II) was the gift of Xiao-Song Xie in Or67d neurons. Error bars represent SEM. n = 20 for each. (E) Bovine and was amplified, cloned into PCR2.1, sequenced and digested with Xho1 5 ATP8A2 expressed under control of the Or67d promoter in the dATP8B and Xba1, and cloned into pUASt. mutant background localizes to the proximal dendrites of the Or67d GAL4 5 5 neurons (genotype w ; UAS ATP8A2 ; Or67d ,dATP8B/dATP8B ). (F) ACKNOWLEDGMENTS. We thank Xiao-Song Xie for the gift of the bovine – 5 Graph depicts the cVA dose response curves for dATP8B mutants ATPaseA2 isoform clone and anti-peptide antibodies (32), Leslie Vosshall for expressing bovine ATP8A2 compared with wild-type controls. n ≥ 10 for anti-Orco antiserum, Richard Benton and Barry Dickson for fly stocks, Fran- each time point. cesca Bissman for generating transgenic flies, Charles Zuker for the Zuker

Ha et al. PNAS | May 27, 2014 | vol. 111 | no. 21 | 7835 Downloaded by guest on October 2, 2021 mutant collection (2), and Coral Warr and Konrad Zinsmaier for sharing and National Research Foundation of Korea Grant (NRF) funded by the results before publication. This work was supported by National Institutes Ministry of Education Grant NRF 2010-0024652 and Ministry of Science, ICT of Health (NIH) Grant DC002539 and NIH Grant R01 DCD011751 (to D.P.S.) and Future Planning Grant 2009-0066616 (to T.S.H.).

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