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1.
The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4 degrees C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4 degrees C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.  相似文献   

2.
3.
The food-borne pathogen Listeria monocytogenes proliferates at refrigeration temperatures, rendering refrigeration ineffective in the preservation of Listeria-contaminated foods. The uptake and intracellular accumulation of the potent compatible solutes glycine betaine and carnitine has been shown to be a key mediator of the pathogen's cold-tolerant phenotype. To date, three compatible solute systems are known to operate in L. monocytogenes: glycine betaine porter I (BetL), glycine betaine porter II (Gbu), and the carnitine transporter OpuC. We investigated the specificity of each transporter towards each compatible solute at 4°C by examining mutant derivatives of L. monocytogenes 10403S that possess each of the transporters in isolation. Kinetic and steady-state compatible solute accumulation data together with growth rate experiments demonstrated that under cold stress glycine betaine transport is primarily mediated by Gbu and that Gbu-mediated betaine uptake results in significant growth stimulation of chill-stressed cells. BetL and OpuC can serve as minor porters for the uptake of betaine, and their action is capable of providing a small degree of cryotolerance. Under cold stress, carnitine transport occurs primarily through OpuC and results in a high level of cryoprotection. Weak carnitine transport occurs via Gbu and BetL, conferring correspondingly weak cryoprotection. No other transporter in L. monocytogenes 10403S appears to be involved in transport of either compatible solute at 4°C, since a triple mutant strain yielded neither transport nor accumulation of glycine betaine or carnitine and could not be rescued by either osmolyte when grown at that temperature.  相似文献   

4.
The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41-49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.  相似文献   

5.
Primary carnitine deficiency is a disorder of fatty acid oxidation caused by mutations in the Na+-dependent carnitine/organic cation transporter OCTN2. Studies with tyrosyl group-modifying reagents support the involvement of tyrosine residues in Na+ binding by sodium-coupled transporters. Here we report two new patients with carnitine deficiency caused by mutations affecting tyrosyl residues (Y447C and Y449D) close to a residue (Glu-452) previously shown to affect sodium stimulation of carnitine transport. Kinetic analysis indicated that the Y449D substitution, when expressed in Chinese hamster ovary cells, increased the concentration of sodium required to half-maximally stimulate carnitine transport from 14.8 +/- 1.8 to 34.9 +/- 5.8 mM (p<0.05), whereas Y447C completely abolished carnitine transport. Substitution of these tyrosine residues with phenylalanine restored normal carnitine transport in Y449F but resulted in markedly impaired carnitine transport by Y447F. This was associated with an increase in the concentration of sodium required to half-maximally stimulate carnitine transport to 57.8 +/- 7.4 mM (p<0.01 versus normal OCTN2). The Y447F and Y449D mutant transporters retained their ability to transport the organic cation tetraethylammonium indicating that their effect on carnitine transport was specific and likely associated with the impaired sodium stimulation of carnitine transport. By contrast, the Y447C natural mutation abolished the transport of organic cations in addition to carnitine. Confocal microscopy of OCTN2 transporters tagged with green fluorescent protein indicated that the Y447C mutant transporters failed to reach the plasma membrane, whereas Y447F, Y449D, and Y449F had normal membrane localization. These natural mutations identify tyrosine residues possibly involved in coupling the sodium electrochemical gradient to transmembrane solute transfer in the sodium-dependent co-transporter OCTN2.  相似文献   

6.
The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41–49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.  相似文献   

7.
Primary carnitine deficiency is an autosomal recessive disorder caused by defective carnitine transport which impairs fatty acid oxidation and manifests as nonketotic hypoglycemia or skeletal or heart myopathy. Here we report the functional characterization of this transporter in human fibroblasts. Carnitine enters normal cells by saturable and unsaturable routes, the latter corresponding to Na+-independent uptake. Saturable carnitine transport was absent in cells from patients with primary carnitine deficiency. In control cells, saturable carnitine transport was energized by the electrochemical gradient of Na+. Carnitine uptake was not inhibited by amino acid substrates of transport systems A, ASC, and X-AG, but was inhibited competitively (in potency order) by butyrobetaine > carnitine > palmitoylcarnitine = acetylcarnitine > betaine. Carnitine uptake was also noncompetitively inhibited by verapamil and quinidine, inhibitors of the multidrug resistance family of membrane transporters, suggesting that the carnitine transporter may share a functional motif with this class of transporters. A high-affinity carnitine transporter was present in kidney 293 cells, but not in HepG2 liver cells, whose carnitine transporter had a Km in the millimolar range. These result indicate the presence of multiple types of carnitine transporters in human cells.  相似文献   

8.
Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the sodium-dependent carnitine cotransporter OCTN2. The majority of natural mutations identified in this and other Na(+)/solute symporters introduce premature termination codons or impair insertion of the mutant transporter on the plasma membrane. Here we report that a missense mutation (E452K) identified in one patient with primary carnitine deficiency did not affect membrane targeting, as assessed with confocal microscopy of transporters tagged with the green fluorescent protein, but reduced carnitine transport by impairing sodium stimulation of carnitine transport. The natural mutation increased the concentration of sodium required to half-maximally stimulate carnitine transport (K(Na)) from the physiological value of 11.6 to 187 mm. Substitution of Glu(452) with glutamine (E452Q), aspartate (E452D), or alanine (E452A) caused intermediate increases in the K(Na). Carnitine transport decreased exponentially with increased K(Na). The E452K mutation is the first natural mutation in a mammalian cotransporter affecting sodium-coupled solute transfer and identifies a novel domain of the OCTN2 cotransporter involved in transmembrane sodium/solute transfer.  相似文献   

9.
Natural-abundance (13)C-nuclear magnetic resonance was used to probe the intracellular organic solute content of the moderately halophilic bacterium Tetragenococcus halophila. When grown in complex growth media supplemented or not with NaCl, T. halophila accumulates glycine betaine and carnitine. Unlike other moderate halophiles, T. halophila was not able to produce potent osmoprotectants (such as ectoines and glycine betaine) through de novo synthesis when cultured in defined medium under hyperosmotic constraint. Addition of 2 mM carnitine, glycine betaine, or choline to defined medium improved growth parameters, not only at high salinity (up to 2.5 M NaCl) but also in media lacking NaCl. These compounds were taken up when available in the surrounding medium. The transport activity occurred at low and high salinities and seems to be constitutive. Glycine betaine and carnitine were accumulated by T. halophila in an unmodified form, while exogenously provided choline led to an intracellular accumulation of glycine betaine. This is the first evidence of the existence of a choline-glycine betaine pathway in a lactic acid bacterium. An assay showed that the compatible solutes strikingly repressed the accumulation of glutamate and slightly increased the intracellular potassium level only at high salinity. Interestingly, osmoprotectant-treated cells were able to maintain the intracellular sodium concentration at a relatively constant level (200 to 300 nmol/mg [dry weight]), independent of the NaCl concentration of the medium. In contrast, in the absence of osmoprotectant, the intracellular sodium content increased sharply from 200 to 2,060 nmol/mg (dry weight) when the salinity of the medium was raised from 1 to 2 M. Indeed, the imported compatible solutes play an actual role in regulating the intracellular Na(+) content and confer a much higher salt tolerance to T. halophila.  相似文献   

10.
The food-borne pathogen Listeria monocytogenes grows actively under high-salt conditions by accumulating compatible solutes such as glycine betaine and carnitine from the medium. We report here that the dominant transport system for glycine betaine uptake, the Gbu porter, may act as a secondary uptake system for carnitine, with a K(m) of 4 mM for carnitine uptake and measurable uptake at carnitine concentrations as low as 10 microM. This porter has a K(m) for glycine betaine uptake of about 6 micro M. The dedicated carnitine porter, OpuC, has a K(m) for carnitine uptake of 1 to 3 microM and a V(max) of approximately 15 nmol/min/mg of protein. Mutants lacking either opuC or gbu were used to study the effects of four carnitine analogs on growth and uptake of osmolytes. In strain DP-L1044, which had OpuC and the two glycine betaine porters Gbu and BetL, triethylglycine was most effective in inhibiting growth in the presence of glycine betaine, but trigonelline was best at inhibiting growth in the presence of carnitine. Carnitine uptake through OpuC was inhibited by gamma-butyrobetaine. Dimethylglycine inhibited both glycine betaine and carnitine uptake through the Gbu porter. Carnitine uptake through the Gbu porter was inhibited by triethylglycine. Glycine betaine uptake through the BetL porter was strongly inhibited by trigonelline and triethylglycine. These results suggest that it is possible to reduce the growth of L. monocytogenes under osmotically stressful conditions by inhibiting glycine betaine and carnitine uptake but that to do so, multiple uptake systems must be affected.  相似文献   

11.
R M Kappes  B Kempf    E Bremer 《Journal of bacteriology》1996,178(17):5071-5079
The accumulation of the osmoprotectant glycine betaine from exogenous sources provides a high degree of osmotic tolerance to Bacillus subtilis. We have identified, through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, a new glycine betaine transport system from B. subtilis. The DNA sequence of a 2,310-bp segment of the cloned region revealed a single gene (opuD) whose product (OpuD) was essential for glycine betaine uptake and osmoprotection in E. coli. The opuD gene encodes a hydrophobic 56.13-kDa protein (512 amino acid residues). OpuD shows a significant degree of sequence identity to the choline transporter BetT and the carnitine transporter CaiT from E. coli and a BetT-like protein from Haemophilus influenzae. These membrane proteins form a family of transporters involved in the uptake of trimethylammonium compounds. The OpuD-mediated glycine betaine transport activity in B. subtilis is controlled by the environmental osmolarity. High osmolarity stimulates de novo synthesis of OpuD and activates preexisting OpuD proteins to achieve maximal glycine betaine uptake activity. An opuD mutant was constructed by marker replacement, and the OpuD-mediated glycine betaine uptake activity was compared with that of the previously identified multicomponent OpuA and OpuC (ProU) glycine betaine uptake systems. In addition, a set of mutants was constructed, each of which synthesized only one of the three glycine betaine uptake systems. These mutants were used to determine the kinetic parameters for glycine betaine transport through OpuA, OpuC, and OpuD. Each of these uptake systems shows high substrate affinity, with Km values in the low micromolar range, which should allow B. subtilis to efficiently acquire the osmoprotectant from the environment. The systems differed in their contribution to the overall glycine betaine accumulation and osmoprotection. A triple opuA, opuC, and opuD mutant strain was isolated, and it showed no glycine betaine uptake activity, demonstrating that three transport systems for this osmoprotectant operate in B. subtilis.  相似文献   

12.
Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.  相似文献   

13.
Cellular turgor is of fundamental importance to bacterial growth and survival. Changes in external osmolarity as a consequence of fluctuating environmental conditions and colonization of diverse environments can significantly impact cytoplasmic water content, resulting in cellular lysis or plasmolysis. To ensure maintenance of appropriate cellular turgor, bacteria import ions and small organic osmolytes, deemed compatible solutes, to equilibrate cytoplasmic osmolarity with the extracellular environment. Here, we show that elevated levels of c‐di‐AMP, a ubiquitous second messenger among bacteria, result in significant susceptibility to elevated osmotic stress in the bacterial pathogen Listeria monocytogenes. We found that levels of import of the compatible solute carnitine show an inverse correlation with intracellular c‐di‐AMP content and that c‐di‐AMP directly binds to the CBS domain of the ATPase subunit of the carnitine importer OpuC. Biochemical and structural studies identify conserved residues required for this interaction and transport activity in bacterial cells. Overall, these studies reveal a role for c‐di‐AMP mediated regulation of compatible solute import and provide new insight into the molecular mechanisms by which this essential second messenger impacts bacterial physiology and adaptation to changing environmental conditions.  相似文献   

14.
The food-borne pathogen Listeria monocytogenes is notable for its ability to grow under osmotic stress and at low temperatures. It is known to accumulate the compatible solutes glycine betaine and carnitine from the medium in response to osmotic or chill stress, and this accumulation confers tolerance to these stresses. Two permeases that transport glycine betaine have been identified, both of which are activated by hyperosmotic stress and one of which is activated by low temperature. An osmotically activated transporter for carnitine, OpuC, has also been identified. We have isolated a Tn917-LTV3 insertional mutant that could not be rescued from hyperosmotic stress by exogenous carnitine. The mutant, LTS4a, grew indistinguishably from a control strain (DP-L1044) in the absence of stress or in the absence of carnitine, but DP-L1044 grew substantially faster under osmotic or chill stress in the presence of carnitine. LTS4a was found to be strongly impaired in KCl-activated as well as chill-activated carnitine transport. 13C nuclear magnetic resonance spectroscopy of perchloric acid extracts showed that accumulation of carnitine by LTS4a was negligible under all conditions tested. Direct sequencing of LTS4a genomic DNA with a primer based on Tn917-LTV3 yielded a 487-bp sequence, which allowed us to determine that the opuC operon had been interrupted by the transposon. It can be concluded that opuC encodes a carnitine transporter that can be activated by either hyperosmotic stress or chill and that the transport system plays a significant role in the tolerance of L. monocytogenes to both forms of environmental stress.  相似文献   

15.
The plant pathogen Pseudomonas syringae derives better osmoprotection from choline than from glycine betaine, unlike most bacteria that have been characterized. In this report, we identified a betaine/carnitine/choline family transporter (BCCT) in P. syringae pv. tomato strain DC3000 that mediates the transport of choline and acetylcholine. This transporter has a particularly low affinity (K(m) of 876 microM) and high capacity (V(max) of 80 nmol/min/mg of protein) for choline transport relative to other known BCCTs. Although BetT activity increased in response to hyperosmolarity, BetT mediated significant uptake under low-osmolarity conditions, suggesting a role in transport for both osmoprotection and catabolism. Growth studies with mutants deficient in BetT and other choline transporters demonstrated that BetT was responsible for the superior osmoprotection conferred to P. syringae by choline over glycine betaine when these compounds were provided at high concentrations (>100 microM). These results suggest that P. syringae has evolved to survive in relatively choline-rich habitats, a prediction that is supported by the common association of P. syringae with plants and the widespread production of choline, but genus- and species-specific production of glycine betaine, by plants. Among the three putative BCCT family transporters in Pseudomonas aeruginosa and six in Pseudomonas putida, different transporters were predicted to function based on similarity to Escherichia coli BetT than to P. syringae BetT. Functional P. putida and P. aeruginosa transporters were identified, and their possession of a long C-terminal tail suggested an osmoregulatory function for this tail; this function was confirmed for P. syringae BetT using deletion derivatives.  相似文献   

16.
Transport of L-[3H]carnitine and acetyl-L-[3H]carnitine at the blood-brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-L-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-L-carnitine and in the absence of sodium ions. Similar transport properties for L-[3H]carnitine and/or acetyl-L-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of L-[3H]carnitine and acetyl-L-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-L-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of L-[3H]carnitine and acetyl-L-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of L-carnitine and acetyl-L-carnitine from the circulating blood to the brain across the BBB.  相似文献   

17.
Carnitine (L-3-hydroxy-4-N-trimethylaminobutyric acid) forms esters with a wide range of acyl groups and functions to transport and excrete these groups. It is found in most cells at millimolar levels after uptake via the sodium-dependent carrier, OCTN2. The acylation state of the mobile carnitine pool is linked to that of the limited and compartmentalised coenzyme A pools by the action of the family of carnitine acyltransferases and the mitochondrial membrane transporter, CACT. The genes and sequences of the carriers and the acyltransferases are reviewed along with mutations that affect activity. After summarising the accepted enzymatic background, recent molecular studies on the carnitine acyltransferases are described to provide a picture of the role and function of these freely reversible enzymes. The kinetic and chemical mechanisms are also discussed in relation to the different inhibitors under study for their potential to control diseases of lipid metabolism.  相似文献   

18.
Since carnitine plays an important role in fat oxidation, influx of carnitine could be crucial for muscle metabolism. OCTN2 (SLC22A5), a sodium-dependent solute carrier, is assumed to transport carnitine into skeletal muscle cells. Acute regulation of OCTN2 activity in rat hindlimb muscles was investigated in response to electrically induced contractile activity. The tissue uptake clearance (CL(uptake)) of l-[(3)H]carnitine during muscle contraction was examined in vivo using integration plot analysis. The CL(uptake) of [(14)C]iodoantipyrine (IAP) was also determined as an index of tissue blood flow. To test the hypothesis that increased carnitine uptake involves the translocation of OCTN2, contraction-induced alteration in the subcellular localization of OCTN2 was examined. The CL(uptake) of l-[(3)H]carnitine in the contracting muscles increased 1.4-1.7-fold as compared to that in the contralateral resting muscles (p<0.05). The CL(uptake) of [(14)C]IAP was much higher than that of l-[(3)H]carnitine, but no association between the increase in carnitine uptake and blood flow was obtained. Co-immunostaining of OCTN2 and dystrophin (a muscle plasma membrane marker) showed an increase in OCTN2 signal in the plasma membrane after muscle contraction. Western blotting showed that the level of sarcolemmal OCTN2 was greater in contracting muscles than in resting muscles (p<0.05). The present study showed that muscle contraction facilitated carnitine uptake in skeletal muscles, possibly via the contraction-induced translocation of its specific transporter OCTN2 to the plasma membrane.  相似文献   

19.
Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. We report novel localization of the organic cation/carnitine transporter, OCTN1, to mitochondria. We made GFP- and RFP-human OCTN1 cDNA constructs and showed expression of hOCTN1 in several transfected mammalian cell lines. Immunostaining of GFP-hOCTN1 transfected cells with different intracellular markers and confocal fluorescent microscopy demonstrated mitochondrial expression of OCTN1. There was striking co-localization of an RFP-hOCTN1 fusion protein and a mitochondrial-GFP marker construct in transfected MEF-3T3 and no co-localization of GFP-hOCTN1 in transfected human skin fibroblasts with other intracellular markers. L-[(3)H]Carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a K(m) of 422 microM and Western blot with an anti-GFP antibody identified the expected GFP-hOCTN1 fusion protein (90 kDa). We showed endogenous expression of native OCTN1 in HepG2 mitochondria with anti-GST-hOCTN1 antibody. Further, we definitively confirmed intact L-[(3)H]carnitine uptake (K(m) 1324 microM), solely attributable to OCTN1, in isolated mitochondria of mutant human skin fibroblasts having <1% of carnitine acylcarnitine translocase activity (alternate mitochondrial carnitine transporter). This mitochondrial localization was confirmed by TEM of murine heart incubated with highly specific rabbit anti-GST-hOCTN1 antibody and immunogold labeled goat anti-rabbit antibody. This suggests an important yet different role for OCTN1 from other OCTN family members in intracellular carnitine homeostasis.  相似文献   

20.
Through functional complementation of an Escherichia coli mutant defective in glycine betaine uptake, we identified a single-component glycine betaine transporter from Tetragenococcus halophila, a moderate halophilic lactic acid bacterium. DNA sequence analysis characterized the ButA protein as a member of the betaine choline carnitine transporter (BCCT) family, that includes a variety of previously characterized compatible solute transporters such as OpuD from Bacillus subtilis, EctP and BetP from Corynebacterium glutamicum, and BetL from Listeria monocytogenes. When expressed in the heterologous host E. coli, the permease is specific for glycine betaine and does not transport the other osmoprotectants previously described for T. halophila (i.e. carnitine, choline, dimethylsulfonioacetate, dimethylsulfoniopropionate, and ectoine). In E. coli, statement of ButA is mainly constitutive and maximal uptake activity may result from a weak osmotic induction. This is the first study demonstrating a role for a permease in osmoregulation, and GB uptake, of a lactic acid bacterium.  相似文献   

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