Effect of deletions within the leader peptide of pre-ornithine transcarbamylase on mitochondrial import

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N-terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15-19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.     

Targeting of pro-apoptotic TLR adaptor SARM to mitochondria: definition of the critical region and residues in the signal sequence

The fifth and the most well-conserved member of the TLR (Toll-like receptor) adaptor, SARM (sterile α- and HEAT/armadillo-motif-containing protein), has been reported to be an important mediator of apoptosis. However, the exact cellular localization of SARM with respect to its role is unclear. In the present study we show that SARM specifically co-localizes with mitochondria. Endogenous SARM is mainly found in the mitochondria. We demonstrate that the N-terminal 27 amino acids (S27) of SARM, which is hydrophobic and polybasic, acts as a mitochondria-targeting signal sequence, associating SARM to the mitochondria. The S27 peptide has an inherent ability to bind to lipids and mitochondria. This sequence effectively translocates the soluble EGFP (enhanced green fluorescence protein) reporter into the mitochondria. Positioning S27 downstream of the EGFP abrogates its mitochondria-targeting ability. Transmission electron microscopy confirms the ability of S27 to import EGFP into the mitochondria. Importantly, by mutagenesis study, we delineated the specificity of the mitochondria-targeting ability to the arginine residue at the 14th position. The R14A SARM mutant also showed reduced apoptotic potential when compared with the wild-type. Taken together, S27, which is a bona fide signal sequence that targets SARM to the mitochondria, explains the pro-apoptotic activity of SARM.     

Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.     

Conserved residues of the leader peptide are essential for cleavage by leader peptidase

Gene 8 of bacteriophage M13 codes for procoat, the precursor of its major coat protein. Gene 8 has been cloned into a plasmid and mutagenized. We have isolated mutants of this gene in which procoat is synthesized but is not processed to coat protein. We now describe mutants in the leader region of procoat, at positions -6, -3, and -1 with respect to the leader peptidase cleavage site. These positions are quite conserved among the leader peptides of various pre-proteins. Each of these mutant procoats is synthesized at a normal rate and inserts correctly into the plasma membrane, as judged by its accessibility to protease in intact spheroplasts. Procoat accumulates, largely in its transmembrane form, and is not cleaved to coat. In detergent extracts, the mutant procoats are very poor substrates for added leader peptidase. We conclude that these 3 residues are not conserved for insertion across the membrane but are part of an essential recognition site for the leader peptidase.     

Thermodynamic characterization of interactions between ornithine transcarbamylase leader peptide and phospholipid bilayer membranes

The interactions of the targeting sequence of the mitochondrial enzyme ornithine transcarbamylase with phospholipid bilayers of different molecular compositions have been studied by high-sensitivity heating and cooling differential scanning calorimetry, high-sensitivity isothermal titration calorimetry, fluorescence spectroscopy, and electron microscopy. These studies indicate that the leader peptide interacts strongly with dipalmitoylphosphatidylcholine (DPPC) bilayer membranes containing small mole percents of the anionic phospholipids dipalmitoylphosphatidylglycerol (DPPG) or brain phosphatidylserine (brain PS) but not with pure phosphatidylcholines. For the first time, the energetics of the leader peptide-membrane interaction have been measured directly by using calorimetric techniques. At 20 degrees C, the association of the peptide with the membrane is exothermic and characterized by an association constant of 2.3 X 10(6) M-1 in the case of phosphatidylglycerol-containing and 0.35 X 10(6) M-1 in the case of phosphatidylserine-containing phospholipid bilayers. In both cases, the enthalpy of association is -60 kcal/mol of peptide. Additional experiments using fluorescence techniques suggest that the peptide does not penetrate deeply into the hydrophobic core of the membrane. The addition of the leader peptide to DPPC/DPPG (5:1) or DPPC/brain PS (5:1) small sonicated vesicles results in vesicle fusion. The fusion process is dependent on peptide concentration and is maximal at the phase transition temperature of the vesicles and minimal at temperatures below the phase transition.     

Mitochondrial import of rat pre-ornithine transcarbamylase: accurate processing of the precursor form is not required for uptake into mitochondria, nor assembly into catalytically active enzyme

Mitochondrial uptake of the cytoplasmically synthesized precursor of the mammalian enzyme ornithine transcarbamylase is mediated by an N-terminal leader sequence of 32 amino acids. In the mitochondrial matrix, the precursor form is processed to the mature subunit by proteolytic removal of this pre-sequence and in the enzyme from rat liver it has been suggested that this occurs in a two-step process which involves an intermediate cleavage at residue 24. We show that deletion of residues 20-26 spanning this intermediate cleavage site prevents correct processing to the mature subunit but it does not prevent mitochondrial targeting and internalization or assembly of the incorrectly processed product into a catalytically active enzyme. The incorrectly processed enzyme, which is larger than the normal mature enzyme, is nevertheless more susceptible to proteolytic degradation in permanently transfected human cells than the correctly processed enzyme.     

Targeting of proteins to mitochondria

A clear picture has emerged over the past years on how a 'classic' mitochondrial protein, like subunit IV of cytochrome c oxidase, might be targeted to mitochondria. The targeting and subsequent import process involves the commitment of the TOM (translocase in the outer mitochondrial membrane) receptor complex on the mitochondrial surface, a TIM (translocase in the inner mitochondrial membrane) translocation complex in the mitochondrial inner membrane, and assorted chaperones and processing enzymes within the organelle. Recent work suggests that while very many mitochondrial precursor proteins might follow this basic targeting pathway, a large number have further requirements if they are to be successfully imported. These include ribosome-associated factors and soluble factors in the cytosol, soluble factors in the mitochondrial intermembrane space, an additional TIM translocase in the inner membrane and a range of narrow specificity assembly factors in the inner membrane. This review is focused on the targeting of proteins up to the stage at which they enter the TOM complex in the outer membrane.     

Targeting critical regions in genomic DNA with AT-specific anticancer drugs

Cellular DNA is not a uniform target for DNA-reactive drugs. At the nucleotide level, drugs recognize and bind short motifs of a few base pairs. The location of drug adducts at the genomic level depends on how these short motifs are distributed in larger domains. This aspect, referred to as region specificity, may be critical for the biological outcome of drug action. Recent studies demonstrated that certain minor groove binding (MGB) drugs, such as bizelesin, produce region-specific lesions in cellular DNA. Bizelesin binds mainly T(A/T)(4)A sites, which are on average scarce, but occasionally cluster in distinct minisatellite regions (200-1000 bp of approximately 85-100% AT), herein referred to as AT islands. Bizelesin-targeted AT islands are likely to function as strong matrix attachment regions (MARs), domains that organize DNA loops on the nuclear matrix. Distortion of MAR-like AT islands may be a basis for the observed inhibition of new replicon initiation and the extreme lethality of bizelesin adducts (<10 adducts/cell for cell growth inhibition). Hence, long AT-islands represent a novel class of critical targets for anticancer drugs. The AT island paradigm illustrates the potential of the concept of regional targeting as an essential component of the rational design of new sequence-specific DNA-reactive drugs.     

The ornithine transcarbamylase leader peptide directs mitochondrial import through both its midportion structure and net positive charge

The cytoplasmically synthesized precursor of the mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), is targeted to mitochondria by its NH2-terminal leader peptide. We previously established through mutational analysis that the midportion of the OTC leader peptide is functionally required. In this article, we report that study of additional OTC precursors, altered in either a site-directed or random manner, reveals that (a) the midportion, but not the NH2-terminal half, is sufficient by itself to direct import, (b) the functional structure in the midportion is unlikely to be an amphiphilic alpha-helix, (c) the four arginines in the leader peptide contribute collectively to import function by conferring net positive charge, and (d) surprisingly, proteolytic processing of the leader peptide does not require the presence of a specific primary structure at the site of cleavage, in order to produce the mature OTC subunit.     

Putting bioactivation reactions to work: Targeting antioxidants to mitochondria

The goal of this research was to test the hypothesis that bioactivation reactions could be exploited to deliver and activate mitochondria-targeted antioxidant prodrugs. The concept that bioactivation reactions could be used for prodrug delivery and activation has received little attention. Most bioactivation reactions result in the conversion of the parent drug to a reactive electrophilic metabolite, but bioactivating enzymes that catalyze elimination or hydrolytic reactions may offer potential for targeted drug delivery. Because mitochondria are the major cellular source of reactive oxygen species, there is much interest in targeting antioxidants to mitochondria. Previous studies showed that the mitochondrial fatty acid β-oxidation pathway biotransforms a range of xenobiotic alkanoates, including ω-(phenyl)alkanoates and ω-(phenoxy)alkanoates. 5,6-Dichloro-4-thia-5-hexenoate, the desamino analog of S-(1,2-dichlorovinyl)-l-cysteine, is biotransformed by the fatty acid β-oxidation pathway. Hence, the prodrugs ω-(phenoxy)alkanoates, 3-(phenoxy)acrylates, and ω-(1-methyl-1H-imidazol-2-ylthio)alkanoates were expected to undergo biotransformation by the mitochondrial β-oxidation pathway to release phenolic antioxidants and the antioxidant methimazole (Roser et al., Bioorg. Med. Chem. 18 (2010) 1441-1448). The rates of biotransformation of ω-(phenoxy)alkanoates varied with the structure, and bulky substituents on the phenoxy moiety reduced rates of biotransformation; this was attributed to substrate limitations imposed by the medium-chain acyl-CoA dehydrogenase. Hence, 3-(2,6-dimethylphenoxy)acrylate was prepared; it was expected that, after conversion to its CoA thioester, 3-(2,6-dimethylphenoxy)acryloyl-CoA would be a substrate for enoyl-CoA hydratase. This expectation was correct: 3-(2,6-dimethylphenoxy)acrylate was an excellent substrate. ω-(1-Methyl-1H-imidazol-2-ylthio)alkanoates were also good substrates for the β-oxidation pathway. Significantly, 3-(2,6-dimethylphenoxy)propanoate, 3-(2,6-dimethylphenoxy)acrylate, and 3-(1-methyl-1H-imidazol-2-ylthio)propanoate were cytoprotective in a hypoxia-reoxygenation model in rat cardiomyocytes. These results demonstrate the feasibility of exploiting bioactivation reactions for targeted drug delivery.     

Regulatory regions and critical residues of NOD2 involved in muramyl dipeptide recognition

Multiple genetic variants of CARD15/NOD2 have been associated with susceptibility to Crohn's disease and Blau syndrome. NOD2 recognizes muramyl dipeptide (MDP) derived from bacterial peptidoglycan (PGN), but the molecular basis of recognition remains elusive. We performed systematic mutational analysis to gain insights into the function of NOD2 and molecular mechanisms of disease susceptibility. Using an archive of 519 mutations covering approximately 50% of the amino-acid residues of NOD2, the essential regulatory domains and specific residues of NOD2 involved in recognition of MDP were identified. The analysis revealed distinct roles for N-terminal and C-terminal leucine-rich repeats (LRRs) in the modulation of NOD2 activation and bacterial recognition. Within the C-terminal LRRs, variable residues predicted to form the beta-strand/betaturn structure were found to be essential for the response to MDP. In addition, we analyzed NOD1, a NOD2-related protein, revealing conserved and nonconserved amino-acid residues involved in PGN recognition. These results provide new insights into the molecular function and regulation of NOD2 and related NOD family proteins.     

Targeting antioxidants to mitochondria: a new therapeutic direction

Mitochondria play an important role in controlling the life and death of a cell. Consequently, mitochondrial dysfunction leads to a range of human diseases such as ischemia-reperfusion injury, sepsis, and diabetes. Although the molecular mechanisms responsible for mitochondria-mediated disease processes are not fully elucidated yet, the oxidative stress appears to be critical. Accordingly, strategies are being developed for the targeted delivery of antioxidants to mitochondria. In this review, we shall briefly discuss cellular reactive oxygen species metabolism and its role in pathophysiology; the currently existing antioxidants and possible reasons why they are not effective in ameliorating oxidative stress-mediated diseases; and recent developments in mitochondrially targeted antioxidants and their future promise for disease treatment.     

Effect of amino acid substitutions in conserved residues in the leader peptide on biosynthesis of the lantibiotic mutacin II

The lantibiotic mutacin II, produced by Streptococcus mutans T8, is a ribosomally synthesized peptide antibiotic that contains thioether amino acids such as lanthionine and methyllanthionine as a result of post-translational modifications. The mutacin II leader peptide sequence shares a number of identical amino acid residues with class AII lantibiotic leader peptides. To study the role of these conservative residues in the production of active antimicrobial mutacin, 15 mutations were generated by site-directed mutagenesis. The effects of these substitutions vary from no effect to complete block-out. Mutations G-1A, G-2A, I-4D, and L-7K completely blocked the production of mature mutacin. Other mutations (I-4V, L-7M, E-8D, S-11T/A, V-12I/A, and E-13D) had no detectable effect on mutacin production. The changes of Glu-8 to Lys, Val-12 to Leu, Glu-13 to Lys reduced the mutacin production level to about 75%, 50%, and 10% of the wild-type, respectively. Thus, our data indicated that some of these conserved residues are essential for the mutacin biosynthesis, whereas others are important for optimal biosynthesis rates.     

Identification of residues in DmsD for twin-arginine leader peptide binding, defined through random and bioinformatics-directed mutagenesis

The twin-arginine translocase (Tat) system is used for the targeting and translocation of folded proteins across the cell membrane of most bacteria. Substrates of this system contain a conserved "twin-arginine" (RR) motif within their signal/leader peptide sequence. Many Tat substrates have their own system-specific chaperone called redox enzyme maturation proteins (REMPs). Here, we study the binding of DmsD, the REMP for dimethyl sulfoxide reductase in Escherichia coli, toward the RR-containing leader peptide of the catalytic subunit DmsA. We have used a multipronged approach targeted at the amino acid sequence of DmsD to define residues and regions important for recognition of the DmsA leader sequence. Residues identified through bioinformatics and THEMATICS analysis were mutated using site-directed mutagenesis. These DmsD residue variants were purified and screened with an in vitro dot-blot far-Western assay to analyze the binding to the DmsA leader sequence. Degenerative polymerase chain reaction was also used to produce a bank of random DmsD amino acid mutants, which were then screened by an in vivo bacterial two-hybrid assay. Using this hybrid method, each DmsD variant was classified into one of three groups based on their degree of interaction with the DmsA leader (none, weak, and moderate). The data from both the in vitro and in vivo analyses were then applied to a model structure of DmsD based on the crystal structure of the Salmonella typhimurium homologue. Our results illustrate the positions of important DmsD residues involved in binding the DmsA leader peptide and identify a "hot pocket" of residues important for leader binding on the structure of DmsD.     

Targeting of glutamine synthetase to the mitochondria of transgenic tabacco

Two transgenic tobacco lines were genetically engineered to contain chimaeric genes encoding the glutamine synthetase (GS) polypeptide of Phaseolus vulgaris (French bean), expressed from the cauliflower mosaic virus 35S promoter. One (MIT-1) contained two copies of a construct including the first 60 amino acids of the Nicotiana plumbaginifolia -F₁ ATPase to target the GS polypeptide to the mitochondrion. The other (CYT-4) contained a single copy of a cytosolic GS construct. Leaves of in vitro plantlets expressed the constructs and contained a novel GS polypeptide, which assembled into active GS isoenzymes constituting about 25% of the total GS activity. In in vitro plantlets of MIT-1, but not CYT-4, the novel polypeptide was found to be associated with the mitochondria. Moreover in MIT-1, the size of the novel polypeptide was not that predicted of the precursor (44.9 kDa) but was about 39 kDa, the same size as the authentic GS polypeptide in CYT-4. These results are consistent with the precursor being imported into the mitochondria and cleaved near the fusion junction between the two sequences. These experiments have therefore shown that the presequence of the -F₁ ATPase has successfully targeted the GS polypeptide to the mitochondria of transgenic tobacco where it has assembled into an active isoenzyme. However, in fully regenerated plants growing photoautotrophically in growth-room conditions, although the constructs were still expressed, the polypeptide did not accumulate to the same levels as in in vitro plantlets and new isoenzyme activities were now barely detectable. Moreover in leaves of the mature MIT-1 plants, the polypeptide was found to be associated with the insoluble fraction of the mitochondria. The results of these experiments are discussed.     

Newly processed ornithine transcarbamylase subunits are assembled to trimers in rat liver mitochondria

We have characterized further the biogenesis in vitro of ornithine transcarbamylase, a homotrimeric mitochondrial matrix enzyme synthesized in the cytoplasm as a larger precursor. When cell-free translation mixtures containing the ornithine transcarbamylase precursor (40 kDa) were chromatographed on Bio-Gel P-200 columns, all of the precursor eluted as aggregates or complexes with molecular weights greater than 200 kDa. None of the precursor bound to a ligand affinity column containing delta-N-(phosphonoacetyl)-L-ornithine (delta-PALO), a transition-state analog and competitive inhibitor of carbamyl phosphate binding, which recognizes native ornithine transcarbamylase. In contrast, a significant portion of the labeled mature-sized subunits, formed when intact mitochondria processed the precursor, bound specifically to the delta-PALO column, were eluted by carbamyl phosphate, and chromatographed on a Bio-Gel P-300 column with a mobility identical to that of native, trimeric ornithine transcarbamylase. No such binding to delta-PALO was observed for the mature-sized monomer or dimer, or for the intermediate-sized ornithine transcarbamylase polypeptide. Moreover, processing by a mitochondrial matrix fraction failed to yield trimeric enzyme, despite producing ample amounts of mature-sized monomer. We conclude that delta-PALO recognizes only trimeric ornithine transcarbamylase composed of mature-sized subunits and that such trimers can be assembled in vitro by intact mitochondria following translocation and proteolytic processing.     

Targeting of cloned firefly luciferase to yeast mitochondria

The firefly luciferase gene (luc) was fused to a 5' fragment of the 70-kDa protein gene (70K) from yeast. The fragment codes for the N-terminal putative signal sequence which targets and anchors the 70-kDa protein to the cytoplasmic side of the outer membrane in mitochondria. Two versions of the fusion gene, 70K[232]::luc and 70K[93]::luc (containing 292 and 93 5' codons from 70K, respectively), were constructed in a bacterial expression plasmid. Both the genes were expressed in Escherichia coli, and in both cases, bioluminescence activity was associated with the expression. The 70K[93]::luc gene was transferred to a yeast-bacteria shuttle vector used to transform Saccharomyces cerevisiae cells. As a control, the same strain was transformed with a plasmid including the original luc. With both transformants, bioluminescence activity was detected in intact cells and crude extracts. Upon growth on a nonfermentable carbon source and fractionation, the product of the fusion gene was associated mostly with mitochondria. In the control transformant, the product of luc was more delocalized. However, a significant amount remained associated with isolated mitochondria. No such spontaneous association of purified luciferase with wild-type mitochondria was observed in vitro. Trypsin treatment of mitochondria isolated from both transformed strains indicated that the fusion protein is anchored to the outer membrane and exposed to the medium while the unfused luciferase retained with the mitochondria is occluded in a compartment unaccessible to trypsin and released in the presence of detergent. The fusion protein retained the major catalytic properties of the parent firefly luciferase, as determined in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Import of the malate dehydrogenase precursor by mitochondria. Cleavage within leader peptide by matrix protease leads to formation of intermediate-sized form

The mitochondrial matrix enzyme malate dehydrogenase (MDH) is synthesized on cytoplasmic polysomes as a larger precursor (pMDH) with an NH2-terminal leader peptide of 24 amino acids. Import of in vitro synthesized MDH into mitochondria results in formation of the mature-sized subunit. We report here that the conversion of pMDH to mMDH occurs via two distinct cleavage events within the leader peptide. First, pMDH is cleaved to an intermediate form (iMDH) of MDH. Conversion of the precursor to the intermediate form is catalyzed by a protease localized to the mitochondrial matrix. The cleavage of pMDH to iMDH involves the removal of 15 amino acids from the NH2 terminus of the pMDH leader peptide. The iMDH is subsequently cleaved, also by a matrix protease, to mature MDH in a reaction which is O-phenanthroline-sensitive. Cleavage to iMDH and to mature MDH occurs prior to completion of translocation of the MDH polypeptide chain into the mitochondrial matrix.     

Targeting of tail-anchored proteins to yeast mitochondria in vivo.

Tail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.     

Targeting of Mycobacterium tuberculosis heparin-binding hemagglutinin to mitochondria in macrophages

Mycobacterium tuberculosis heparin-binding hemagglutinin (HBHA), a virulence factor involved in extrapulmonary dissemination and a strong diagnostic antigen against tuberculosis, is both surface-associated and secreted. The role of HBHA in macrophages during M. tuberculosis infection, however, is less well known. Here, we show that recombinant HBHA produced by Mycobacterium smegmatis effectively induces apoptosis in murine macrophages. DNA fragmentation, nuclear condensation, caspase activation, and poly (ADP-ribose) polymerase cleavage were observed in apoptotic macrophages treated with HBHA. Enhanced reactive oxygen species (ROS) production and Bax activation were essential for HBHA-induced apoptosis, as evidenced by a restoration of the viability of macrophages pretreated with N-acetylcysteine, a potent ROS scavenger, or transfected with Bax siRNA. HBHA is targeted to the mitochondrial compartment of HBHA-treated and M. tuberculosis-infected macrophages. Dissipation of the mitochondrial transmembrane potential (ΔΨ(m)) and depletion of cytochrome c also occurred in both macrophages and isolated mitochondria treated with HBHA. Disruption of HBHA gene led to the restoration of ΔΨ(m) impairment in infected macrophages, resulting in reduced apoptosis. Taken together, our data suggest that HBHA may act as a strong pathogenic factor to cause apoptosis of professional phagocytes infected with M. tuberculosis.