Expression of a Plastidic ATP/ADP Transporter Gene inEscherichia coli Leads to a Functional Adenine Nucleotide Transport System in the Bacterial Cytoplasmic Membrane

Tjaden, Joachim; Schwöppe, Christian; Möhlmann, Torsten; Quick, Paul; Neuhaus, H. Ekkehard

doi:10.1074/jbc.273.16.9630

Cited by 78 publications

(90 citation statements)

References 32 publications

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“…Several plant membrane proteins, including the A. thaliana Shaker-type K ϩ channels KAT1 and AKT2 (25,38), the A. thaliana K ϩ translocating protein AtKUP1-2 homologs of the E. coli K ϩ transporter Kup (39), and the A. thaliana plastidic ADP͞ATP translocator (40) have been found to be functionally expressed in E. coli cells. The same appears to be true for AtHKT1 (6).…”

Section: Discussionmentioning

confidence: 99%

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na ⁺ /K ⁺ translocating AtHKT1 protein, a member of the superfamily of K ⁺ transporters

Kato

Sakaguchi

Mori

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

127

107

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The Arabidopsis thaliana AtHKT1 protein, a Na ؉ ͞K ؉ transporter, is capable of mediating inward Na ؉ currents in Xenopus laevis oocytes and K ؉ uptake in Escherichia coli. HKT1 proteins are members of a superfamily of K ؉ transporters. These proteins have been proposed to contain eight transmembrane segments and four pore-forming regions arranged in a mode similar to that of a K ؉ channel tetramer. However, computer analysis of the AtHKT1 sequence identified eleven potential transmembrane segments. We have investigated the membrane topology of AtHKT1 with three different techniques. First, a gene fusion alkaline phosphatase study in E. coli clearly defined the topology of the N-terminal and middle region of AtHKT1, but the model for membrane folding of the C-terminal region had to be refined. Second, with a reticulocyte-lysate supplemented with dog-pancreas microsomes, we demonstrated that N-glycosylation occurs at position 429 of AtHKT1. An engineered unglycosylated protein variant, N429Q, mediated Na ؉ currents in X. laevis oocytes with the same characteristics as the wild-type protein, indicating that N-glycosylation is not essential for the functional expression and membrane targeting of AtHKT1. Five potential glycosylation sites were introduced into the N429Q. Their pattern of glycosylation supported the model based on the E. coli-alkaline phosphatase data. Third, immunocytochemical experiments with FLAG-tagged AtHKT1 in HEK293 cells revealed that the N and C termini of AtHKT1, and the regions containing residues 135-142 and 377-384, face the cytosol, whereas the region of residues 55-62 is exposed to the outside. Taken together, our results show that AtHKT1 contains eight transmembrane-spanning segments.

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Section: Discussionmentioning

confidence: 99%

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na ⁺ /K ⁺ translocating AtHKT1 protein, a member of the superfamily of K ⁺ transporters

Kato

Sakaguchi

Mori

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

127

107

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“…This enables the determination of the transport activity by measuring the import or export of radiolabeled substances into or out of intact E. coli cells. Several plastid transporters have been analyzed using this approach including the NTTs and BT1 from maize and Arabidopsis (Tjaden et al, 1998;Kirchberger et al, 2007). As the production of membrane proteins in E. coli is in some cases difficult to achieve, yeast cells have been widely used for this purpose as an alternative.…”

Section: The Meaning Of Plastid Transportersmentioning

confidence: 99%

The Import and Export Business in Plastids: Transport Processes across the Inner Envelope Membrane

Fischer

2011

Plant Physiology

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“…To check for correct modifications, all constructs were sequenced by the dideoxy chain-termination method [22] using an ABI Prism 377 TM and Big Dye TM Terminator CycleSequencer (PE Applied Systems, Weiterstadt, Germany). The oligonucleotides used to modify the sequence of the plastidic ATP/ADP transporter, AATP1(At) (cloned in plasmid pET16b, [23]) are shown inTable 1.…”

Section: Cloning Strategiesmentioning

confidence: 99%

Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from Arabidopsis are important for transport efficiency, substrate specificity, and counter exchange properties

Trentmann¹,

Decker²,

Winkler³

et al. 2000

Eur J Biochem

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Structure±function relationships of the plastidic ATP/ADP transporter from Arabidopsis thaliana have been determined using site-directed mutants at positions K155, E245, E385, and K527. These charged residues are found within highly conserved domains of homologous transport proteins from plants and bacteria and are located in predicted transmembrane regions. Mutants of K155 to K155E, K155R, or K155Q reduced ATP transport to values between 4 and 16% of wild-type uptake, whereas ADP transport was always less then 3% of the wild-type value. Site-directed mutations in which glutamate at positions 245 or 385 was replaced with lysine, abolished transport. However, conservative (E245D, E385D) or neutral (E245Q, E385Q) replacement at these two positions allowed transport. The fourth reciprocal exchange, K527E, also abolished uptake of both adenylates. K527R and K527Q were unable to transport ATP, but ADP transport remained at 35 and 27%, respectively, of the wild-type activity. There was a 70-fold decreased apparent affinity of K527R for ATP, but only a twofold decrease for ADP. The efflux of ATP, but not ADP, was also greatly reduced in K527R. These observations show strikingly that K527 plays a role in substrate specificity that is manifest in both the influx and efflux components of this antiporter.Keywords: ATP/ADP transporter; structure/function relationship; site-specific mutations; plastid; Arabidopsis thaliana.Transport of solutes across cellular membranes, whether catalyzed by channels or carriers, is necessary for complex metabolism. Although adenine nucleotides, the universal cellular energy currency, fulfil an irreplaceable function, they are viewed as part of the intracellular compartment and often are not transported across biological membranes. However, there are three exceptions to this generalization in which transport of ATP and ADP appropriately occurs: in obligate intracellular bacteria, in mitochondria, and in plastids.In heterotrophic eukaryotic cells, most ATP is synthesized during oxidative phosphorylation and accumulates in the mitochondrial matrix, making it necessary to export this intermediate into the cytosol to fuel anabolic reactions. This transport is mediated by the mitochondrial ADP/ATP carrier (AAC), one of the best-characterized membrane carrier proteins [1±7]. AAC is a member of a large group of transporters comprising the mitochondrial carrier family [3]. These carriers share the same evolutionary ancestor, exhibit an internal sequence triplication of about 100 amino acids, and in the functional state probably form a homodimer from the monomer that has six transmembrane domains [8±10].A second type of adenylate transporter resides in the inner envelope membrane of plant plastids [11±14]. This transporter displays a high structural similarity to the third type of ATP/ADP transporter, that from rickettsiae [15] and chlamydiae [16], obligate intracellular bacteria that grow within eukaryotic cells. Neither plastidic nor bacterial types show homology to the mitochondrial ADP/ATP carrier...

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Expression of a Plastidic ATP/ADP Transporter Gene inEscherichia coli Leads to a Functional Adenine Nucleotide Transport System in the Bacterial Cytoplasmic Membrane

Cited by 78 publications

References 32 publications

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na ⁺ /K ⁺ translocating AtHKT1 protein, a member of the superfamily of K ⁺ transporters

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na ⁺ /K ⁺ translocating AtHKT1 protein, a member of the superfamily of K ⁺ transporters

The Import and Export Business in Plastids: Transport Processes across the Inner Envelope Membrane

Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from Arabidopsis are important for transport efficiency, substrate specificity, and counter exchange properties

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Expression of a Plastidic ATP/ADP Transporter Gene inEscherichia coli Leads to a Functional Adenine Nucleotide Transport System in the Bacterial Cytoplasmic Membrane

Cited by 78 publications

References 32 publications

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na + /K + translocating AtHKT1 protein, a member of the superfamily of K + transporters

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na + /K + translocating AtHKT1 protein, a member of the superfamily of K + transporters

The Import and Export Business in Plastids: Transport Processes across the Inner Envelope Membrane

Charged amino-acid residues in transmembrane domains of the plastidic ATP/ADP transporter from Arabidopsis are important for transport efficiency, substrate specificity, and counter exchange properties

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Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na ⁺ /K ⁺ translocating AtHKT1 protein, a member of the superfamily of K ⁺ transporters

Evidence in support of a four transmembrane-pore-transmembrane topology model for the Arabidopsis thaliana Na ⁺ /K ⁺ translocating AtHKT1 protein, a member of the superfamily of K ⁺ transporters