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Lipid Flippase Modulates Olfactory Receptor Expression and Odorant Sensitivity in Drosophila

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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 neu- rons or in the nonneuronal support cells that secrete the sensil- lum 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 re- lated 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 far- nesol (genotype w ; UAS Or83c ; Or67dGAL4).
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