Ubiquinol-binding site in the alternative oxidase: Mutagenesis reveals features important for substrate binding and inhibition

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in all plants, some fungi, green algae, bacteria and pathogenic protozoa. The lack of AOX in the mammalian host renders this protein an important potential therapeutic target in the treatment of pathogenic protozoan infections. Bioinformatic searches revealed that, within a putative ubiquinol-binding crevice in AOX, Gln242, Asn247, Tyr253, Ser256, His261 and Arg262 were highly conserved. To confirm that these amino-acid residues are important for ubiquinol-binding and hence activity substitution mutations were generated and characterised. Assessment of AOX activity in isolated Schizosaccharomyces pombe mitochondria revealed that mutation of either Gln242, Ser256, His261 and Arg262 resulted in >90% inhibition of antimycin A-insensitive respiration suggesting that hydroxyl, guanidino, imidazole groups, polar and charged residues in addition to the size of the amino-acid chain are important for ubiquinone-binding. Substitution of Asn247 with glutamine or Tyr253 with phenylalanine had little effect upon the respiratory rate indicating that these residues are not critical for AOX activity. However replacement of Tyr253 by alanine resulted in a 72% loss of activity suggesting that the benzoquinone group and not hydroxyl group is important for quinol binding. These results provide important new insights into the ubiquinol-binding site of the alternative oxidase, the identity of which maybe important for future rational drug design.     

Constitutive activity of Sauromatum guttatum alternative oxidase in Schizosaccharomyces pombe implicates residues in addition to conserved cysteines in alpha-keto acid activation

Activity of the plant mitochondrial alternative oxidase (AOX) can be regulated by organic acids, notably pyruvate. To date, only two well-conserved cysteine residues have been implicated in this process. We report the functional expression of two AOX isozymes (Sauromatum guttatum Sg-AOX and Arabidopsis thaliana At-AOX1a) in Schizosaccharomyces pombe. Comparison of the response of these two isozymes to pyruvate in isolated yeast mitochondria and disrupted mitochondrial membranes reveals that in contrast to At-AOX1a, Sg-AOX activity is insensitive to pyruvate and appears to be in a constitutively active state. As both of these isozymes conserve the two cysteines, we propose that such contrasting behaviour must be a direct result of differences in their amino acid sequence and have subsequently identified novel candidate residues.     

Induction of indole synthesis in the appendix of Sauromatum guttatum Schott

Induction of indole synthesis in young appendix sections ofSauromatum guttatum Schott was studied. The inducer, known ascalorigen, a compound synthesized in the mature staminate flowerbuds, is capable of triggering a rapid metabolic spurt in theappendix of the inflorescence. The induced indole-formationlasts for only a short period of time, and is similar to thenormal pattern of indole synthesis. Under the experimental conditions,indole was detectable after 7 to 8 hr of calorigen treatmentbut had disappeared about 35 hr later. The moment when indoleis present in maximal amount coincides with that of maximalheat production, i.e., about 27 hr after the application ofcalorigen. A simple and sensitive bioassay for detection ofcalorigen, based on the formation of indole, could be devised. Calorigen also causes, to a lesser extent, a temperature-riseof those young floral parts (male, female and sterile tissues)where indole is not detectable at any time. The time sequenceof the induced production of heat for the various floral partsmimics that of a natural metabolic flare-up. Extracts prepared from the mature staminate flowers of Arummaculatum L., Arum dioscoridis Sibth and Sm., and Dracunculusvulgaris Schott were all able to induce the formation of indolein the appendix of Sauromatum in a fashion similar to that causedby the staminate flower extract of this plant. (Received February 12, 1972; )     

Ultrastructural changes in the appendix of the Sauromatum guttatum inflorescence during anthesis

Summary The ultrastructure of the epidermal and sub-epidermal cells of the appendix of the Sauromatum guttatum inflorescence reveals developmental changes during anthesis. These changes precede, and probably make possible, heat and odor production. Two days before D-day (the day of heat production and inflorescence-opening) the mitochondria of the epidermis divide; apparent division of the amyloplasts was observed at the same time. The presence of lipid bodies and peroxisomes in the epidermis was clearly evident. On D-day, the epidermis becomes a continuous layer in which the cell walls separating two adjacent cells disappear. At the same time, in the sub-epidermal cells, the mitochondria and the amyloplasts undergo division. The mitochondria become electron-dense, and their DNA is clearly visible. On that day, lipids as well as starch are being depleted. The peroxisomes change in structure every day, from D-2 to D-day. It has also been demonstrated by histochemical techniques that during anthesis the activity of cytochrome c oxidase (3,3-diaminobenzidine as a substrate) decreases whereas the activity of NADH dehydrogenase [tetrazolium salts: nitro-blue tetrazolium chloride (NBT) or neotetrazolium chloride (NT) in the presence of NADH], increases. Oxygen consumption of isolated mitochondria from the D-day appendix was inhibited in the presence of the two tetrazolium salts to a different degree: oxidation of NADH in the presence of NBT was the most sensitive to inhibition, more so than the oxidation of malate and succinate. NT was less effective as an inhibitor in the presence of those three respiratory substrates.     

Further studies of the glandular tissue of the Sauromatum guttatum (Araceae) appendix

Electron microscopic studies showed that the trans-Golgi network (trans indicates the polarity of cisternae within the Golgi apparatus; it is opposite to the cis-face that is adjacent to the rough endoplasmic reticulum) was involved in the processing of the osmiophilic material present in the appendix of the inflorescence of Sauromatum guttatum. This material accumulated in the rough endoplasmic reticulum and in special pockets of the plasma membrane prior to heat production. Associations between the endoplasmic reticulum and trans-Golgi network were observed. The Golgi apparatus was composed of 5–6 dictyosomes on one side and one or two somewhat detached cisternae on the other side. Various nonosmiophilic Golgi-derived vesicles were observed: small ones covered with spike-like material, large ones with a smooth surface, and irregularly shaped ones. These electron-translucent vesicles seemed to accumulate in specific localities at the plasma membrane surface in the vicinity of the osmiophilic material; they were not found when the aroma was released. During heat production, the Golgi structures shrank and the activity of the trans-Golgi network seemed to be reduced. At the same time, coated pits were seen at the plasma membrane surface. In some cells, hypertrophic Golgi apparatuses were seen with only 2–3 dictyosomes that contained granulated material in their lumens. Finally, the osmiophilic material was also found in the plasmodesmata.     

Exploring the molecular nature of alternative oxidase regulation and catalysis

Plant mitochondria contain a non-protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post-translational regulatory phenomena. Furthermore, we suggest a catalytic cycle for AOX that involves at least one transient protein-derived radical. The model is based on the reviewed information and on recent insights into the mechanisms of cytochrome c oxidase and the hydroxylase component of methane monooxygenase.     

Oxidation of Proline and Glutamate by Mitochondria of the Inflorescence of Voodoo Lily (Sauromatum guttatum)

In appendices of Sauromatum guttatum that are developing thermogenicity, mitochondria isolated from successive developmental stages of the inflorescence show an increase in the oxidation rates of proline and glutamate. A similar rise in the oxidation rates of these compounds is observed in mitochondria obtained from the spathe, a nonthermogenic organ of the inflorescence. Changes in oxidative metabolism were also observed in mitochondria isolated from sections of immature appendix treated with salicylic acid (SA) at 0.69 microgram per gram fresh weight indicating that they are induced by SA. At that concentration, however, SA has no effect on oxygen consumption by mitochondria in the presence of glutamate, proline, or malate. Furthermore, oxygen uptake by mitochondria in the presence of proline or glutamate is partially sensitive to salicylhydroxamic acid (SHAM) at concentrations greater than 2 millimolar when in the presence of 1 millimolar KCN. For NADH, succinate, and malate a high capacity of the alternative (cyanide-resistant) pathway is found that is completely sensitive to SHAM at 1.5 to 4 millimolar. The increase in the mitochondrial capacity to oxidize either amino acid is also found in four other Araceae species including both thermogenic and nonthermogenic ones. After anthesis, the rates of proline and glutamate oxidation decline.     

Structural bases for inhibitor binding and catalysis in polyamine oxidase

Polyamine oxidase (PAO) carries out the FAD-dependent oxidation of the secondary amino groups of spermidine and spermine, a key reaction in the polyamine catabolism. The active site of PAO consists of a 30 A long U-shaped catalytic tunnel, whose innermost part is located in front of the flavin ring. To provide insight into the PAO substrate specificity and amine oxidation mechanism, we have investigated the crystal structure of maize PAO in the reduced state and in complex with three different inhibitors, guazatine, 1,8-diaminooctane, and N(1)-ethyl-N(11)-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm). In the reduced state, the conformation of the isoalloxazine ring and the surrounding residues is identical to that of the oxidized enzyme. Only Lys300 moves away from the flavin to compensate for the change in cofactor protonation occurring upon reduction. The structure of the PAO.inhibitor complexes reveals an exact match between the inhibitors and the PAO catalytic tunnel. Inhibitor binding does not involve any protein conformational change. Such lock-and-key binding occurs also in the complex with CHENSpm, which forms a covalent adduct with the flavin N5 atom. Comparison of the enzyme complexes hints at an "out-of-register" mechanism of inhibition, in which the inhibitor secondary amino groups are not properly aligned with respect to the flavin to allow oxidation. Except for the Glu62-Glu170 pair, no negatively charged residues are involved in the recognition of substrate and inhibitor amino groups, which is in contrast to other polyamine binding proteins. This feature may be exploited in the design of drugs specifically targeting PAO.     

Mutagenesis of the herpesvirus saimiri terminal repeat region reveals important elements for virus production

Deletion of the terminal repeats (TR) from herpesvirus saimiri (HVS) renders it unable to produce infectious virus or generate plaques. However, a TR-deleted HVS bacterial artificial chromosome can form replication compartments. Complementation of this mutant shows that one copy of the TR, plus the right junction of the genome with the TR, is sufficient for efficient plaque formation and generation of infectious virus. Within the TR unit, the region around the cleavage site of the genome appears both necessary and sufficient for virus production. Analysis of episomes from productive cells indicates a propensity to amplify TR numbers during the lytic cycle.     

Purification of a NAD(P) Reductase-Like Protein from the Thermogenic Appendix of the Sauromatum guttatum Inflorescence

A NAD(P) reductase-like protein with a molecular mass of 34.146 ± 34 Da was purified to homogeneity from the appendix of the inflorescence of the Sauromatum guttatum. On-line liquid chromatography/electrospray ionization-mass spectrometry was used to isolate and quantify the protein. For the identification of the protein, liquid chromatography/electrospray ionization-tandem mass spectrometry analysis of tryptic digests of the protein was carried out. The acquired mass spectra were used for database searching, which led to the identification of a single tryptic peptide. The 12 amino acid tryptic peptide (FLPSEFGNDVDR) was found to be identical to amino acid residues at the positions 108–120 of isoflavone reductase in the Arabidopsis genome. A BLAST search identified this sequence region as unique and specific to a class of NAD(P)-dependent reductases involved in phenylpropanoid biosynthesis. Edman degradation revealed that the protein was N-terminally blocked. The amount of the protein (termed RL, NAD(P) reductase-like protein) increased 60-fold from D-4 (4 days before inflorescence-opening, designated as D-day) to D-Day, and declined the following day, when heat-production ceased. When salicylic acid, the endogenous trigger of heat-production in the Sauromatum appendix, was applied to premature appendices, a fivefold decrease in the amount of RL was detected in the treated section relative to the non-treated section. About 40 % of RL was found in the cytoplasm. Another 30 % was detected in Percoll-purified mitochondria and the rest, about 30 % was associated with a low speed centrifugation pellet due to nuclei and amyloplast localization. RL was also found in other thermogenic plants and detected in Arabidopsis leaves. The function of RL in thermogenic and non-thermogenic plants requires further investigation.     

Oleic Acid and Its Positional Isomer,cis-Vaccinic Acid,in the Appendix of Sauromatum guttatum during Anthesis

The fatty acid profiles of various organs of the thermogenic inflorescence of Sauromatum guttatum and of the sporophylls of thermogenic male cones of two cycad species (Encephalartos ferox and Dioon edule var edule and var angustifolium) were determined by gas chromatography. During anthesis, palmitate (16:0), oleate [18:1 (9)], cis-vaccinate [18:1 (11)], and linoleate [18:2 (9, 12)] were the most abundant fatty acids in the Sauromatum appendix. cis-Vaccinic acid, a positional isomer of oleic acid, was identified by comparing its retention time on a gas chromatography column and its mass spectrum to an authentic compound. The percentage of oleic acid from total fatty acids dropped from about 9 in the morning 3 d before heat production to 6 in the morning 2 d before heat production. At this time, the percentage of cis-vaccinic acid increased from 3 to 11%, and then remained at this level until the inflorescence dried and died. Palmitoleic acid [16:1 (9)], the common precursor of cis-vaccinic acid, is a minor component of total fatty acids. In six other organs of the Sauromatum inflorescence including thermogenic organs, such as male flowers and lower spadix, palmitate, oleate, and linoleate were prevalent but cis-vaccinate was not. The thermogenic male cones of the two cycad species were rich in palmitic, oleic, and linolenic acids. The level of cis-vaccinic acid in these organs was less than 0.5%.     

Purification and partial characterization of two biologically active compounds from the inflorescence of Sauromatum guttatum Schott (Araceae)

The active principle triggering the metabolic outburst of theappendices of certain arum lily species has been purified andpartially characterized. Although in the past it has been thoughtof as a single factor ("calorigen"), both paper chromatographyand DEAE-Sephadex ion exchange chromatography reveal that thereare two compounds (calorigen I and II) with calorigen activity.These can act alone or together, but show no synergism. Theyare low-molecular compounds which appear to be common constituentsof the inflorescence of Sauromatum guttatum Schott. CalorigenII is also present in the spadix of Arum maculatum L., Arumdioscoridis Sibth. & Sm., and Dracunculus vulgaris Schott.Calorigen I absorbs UV at 242, 267 and 278 nm, calorigen IIat 229 and 295 nm. No spectral changes are noted in alkalineenvironment, whereas at low pH-values there is a shift of theabsorption peaks of both compounds towards longer wavelengths.Calorigen II displays a strong blue fluorescence and has a maximalemission at 410 nm when activated at 295 nm. The possible mechanismof calorigen action is discussed. ¹ Present address: Department of Biochemistry, University ofWashington, Seattle, Washington 98195, U.S.A. (Received August 26, 1974; )     

Mutagenesis of prochlorothrix plastocyanin reveals additional features in photosystem I interactions

Three surface residues of plastocyanin from Prochlorothrix hollandica have been modified by site-directed mutagenesis. Changes have been made in methionine 33, located in the hydrophobic patch of the copper protein, and in arginine 86 and proline 53, both located in the eastern hydrophilic area. The reactivity toward photosystem I of single mutants M33N, P53A, P53E, R86Q, R86E, and the double mutant M33N/P14L has been studied by laser flash absorption spectroscopy. All the mutations yield increased reactivity of plastocyanin toward photosystem I as compared with wild type plastocyanin, thus indicating that in Prochlorothrix electron donation to photosystem I is not optimized. The most drastic increases in the intracomplex electron transfer rate are obtained with mutants in methionine 33, whereas replacing arginine 86 only modestly affects the plastocyanin-photosystem I equilibrium constant for complex formation. Mutations at position 53 also promote major changes in the association of plastocyanin with photosystem I, yielding a change from a mechanism involving complex formation to a simpler collisional interaction. Molecular dynamics calculations indicate that mutations at position 33 promote changes in the H-bond network around the copper center. The comparative kinetic analysis of the reactivity of Prochlorothrix plastocyanin mutants toward photosystem I from other cyanobacteria reveals that mutations M33N, P53A, and P53E result in enhanced general reactivity.     

Mutagenesis reveals a specific role for Cox17p in copper transport to cytochrome oxidase

The provision of copper to cytochrome oxidase is one of the requisite steps in the assembly of the holoenzyme. Several proteins are involved in this process including Cox17p, Sco1p, and Cox11p. Cox17p, an 8-kDa protein, is the only molecule thought to be involved in shuttling copper from the cytoplasm into mitochondria. Given the small size of Cox17p, we have taken a random and site-directed mutagenesis approach to studying structure-function relationships in Cox17p. Mutations have been generated in 70% of the Cox17p amino acid residues, with only a small subset leading to a detectable respiration-deficient phenotype. We have characterized the respiration-deficient cox17 mutants and found in addition to the expected cytochrome oxidase deficiency, a specific lack of Cox2p and the presence of a misassembled cytochrome oxidase in a subset of mutants. These results suggest that Cox17p is involved upstream of Sco1p in delivering copper specifically to subunit 2 of cytochrome oxidase and predict the existence of a subunit 1-specific copper chaperone.     

Homolog-scanning mutagenesis reveals poliovirus receptor residues important for virus binding and replication.

Poliovirus initiates infection of primate cells by binding to the poliovirus receptor, Pvr. Mouse cells do not bind poliovirus but express a Pvr homolog, Mph, that does not function as a poliovirus receptor. Previous work has shown that the first immunoglobulin-like domain of the Pvr protein contains the virus binding site. To further identify sequences of Pvr important for its interaction with poliovirus, stable cell lines expressing mutated Pvr molecules were examined for their abilities to bind virus and support virus replication. Substitution of the amino-terminal domain of Mph with that of Pvr yields a molecule that can function as a poliovirus receptor. Cells expressing this chimeric receptor have normal binding affinity for poliovirus, yet the kinetics of virus replication are delayed. Results of virus alteration assays indicate that this chimeric receptor is defective in converting native virus to 135S altered particles. This defect is not observed with cells expressing receptor recombinants that include Pvr domains 1 and 2. Because altered particles are believed to be an intermediate in poliovirus entry, these findings suggest that Pvr domains 2 and 3 participate in early stages of infection. Additional mutants were made by substituting variant Mph residues for the corresponding residues in Pvr. The results were interpreted by using a model of Pvr predicted from the known structures of other immunoglobulin-like V-type domains. Analysis of stable cell lines expressing the mutant proteins revealed that virus binding is influenced by mutations in the predicted C'-C" loop, the C" beta-strand, the C"-D loop, and the D-E loop. Mutations in homologous regions of the immunoglobulin-like CD4 molecule alter its interaction with gp120 of human immunodeficiency virus type 1. Cells expressing Pvr mutations on the predicted C" edge do not develop cytopathic effect during poliovirus infection, suggesting that poliovirus-induced cytopathic effect may be induced by the virus-receptor interaction.     

Mutagenesis of bracelet cyclotide hyen D reveals functionally and structurally critical residues for membrane binding and cytotoxicity

Cyclotides have a wide range of bioactivities relevant for agricultural and pharmaceutical applications. This large family of naturally occurring macrocyclic peptides is divided into three subfamilies, with the bracelet subfamily being the largest and comprising the most potent cyclotides reported to date. However, attempts to harness the natural bioactivities of bracelet cyclotides and engineer-optimized analogs have been hindered by a lack of understanding of the structural and functional role of their constituent residues, which has been challenging because bracelet cyclotides are difficult to produce synthetically. We recently established a facile strategy to make the I11L mutant of cyclotide hyen D that is as active as the parent peptide, enabling the subsequent production of a series of variants. In the current study, we report an alanine mutagenesis structure-activity study of [I11L] hyen D to probe the role of individual residues on peptide folding using analytical chromatography, on molecular function using surface plasmon resonance, and on therapeutic potential using cytotoxicity assays. We found that Glu-6 and Thr-15 are critical for maintaining the structure of bracelet cyclotides and that hydrophobic residues in loops 2 and 3 are essential for membrane binding and cytotoxic activity, findings that are distinct from the structural and functional characteristics determined for other cyclotide subfamilies. In conclusion, this is the first report of a mutagenesis scan conducted on a bracelet cyclotide, offering insights into their function and supporting future efforts to engineer bracelet cyclotides for biotechnological applications.     

ACE2 X-ray structures reveal a large hinge-bending motion important for inhibitor binding and catalysis

The angiotensin-converting enzyme (ACE)-related carboxypeptidase, ACE2, is a type I integral membrane protein of 805 amino acids that contains one HEXXH + E zinc-binding consensus sequence. ACE2 has been implicated in the regulation of heart function and also as a functional receptor for the coronavirus that causes the severe acute respiratory syndrome (SARS). To gain further insights into this enzyme, the first crystal structures of the native and inhibitor-bound forms of the ACE2 extracellular domains were solved to 2.2- and 3.0-A resolution, respectively. Comparison of these structures revealed a large inhibitor-dependent hinge-bending movement of one catalytic subdomain relative to the other ( approximately 16 degrees ) that brings important residues into position for catalysis. The potent inhibitor MLN-4760 ((S,S)-2-[1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol4-yl]-ethylamino]-4-methylpentanoic acid) makes key binding interactions within the active site and offers insights regarding the action of residues involved in catalysis and substrate specificity. A few active site residue substitutions in ACE2 relative to ACE appear to eliminate the S(2)' substrate-binding subsite and account for the observed reactivity change from the peptidyl dipeptidase activity of ACE to the carboxypeptidase activity of ACE2.     

Mutagenesis reveals a role for epidermal growth factor receptor extracellular subdomain IV in ligand binding.

The extracellular domain of the epidermal growth factor (EGF) receptor (EGFR) comprises four subdomains (I-IV) and mediates binding of several different polypeptide ligands, including EGF, transforming growth factor-alpha, and heparin-binding EGF. Previous studies have predominantly implicated subdomain III in ligand binding. To investigate a possible role for sequences in subdomain IV, we constructed several mutant EGFRs in which clusters of charged or aromatic amino acids were replaced with alanine. Analysis of stably transfected Chinese hamster ovary cells expressing mutant EGFRs confirmed that they were present on the cell surface at levels approaching that of the wild-type receptor. Although tyrosine phosphorylation of most mutants was markedly induced by EGF, a cluster mutation (mt25) containing four alanine substitutions in the span of residues 521-527 failed to respond. EGF-induced tyrosine phosphorylation of an alternative mutant (DeltaEN) with amino acids 518-589 deleted was also greatly diminished. Larger doses of EGF or heparin-binding EGF induced only weak tyrosine phosphorylation of mt25, whereas the response to transforming growth factor-alpha was undetectable. These results suggest that mt25 might be defective with respect to either ligand binding or receptor dimerization. Quantitative analyses showed that binding of (125)I-EGF to mt25 and DeltaEN was reduced to near background levels, whereas binding of EGF to other cluster mutants was reduced 60-70% compared with wild-type levels. Among the mutants, only mt25 and DeltaEN failed to form homodimers or to transphosphorylate HER2/Neu in response to EGF treatment. Collectively, our results are the first to provide direct evidence that discrete subdomain IV residues are required for normal binding of EGF family ligands. Significantly, they were obtained with the full-length receptor in vivo, rather than a soluble truncated receptor, which has been frequently used for structure/function studies of the EGFR extracellular region.     

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying “microzones” of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.