A High Affinity Fungal Nitrate Carrier with Two Transport Mechanisms

Zhou, Jing‐Jiang; Trueman, Laurence; Boorer, Kathryn J.; Theodoulou, F. L.; Forde, Brian G.; Miller, Anthony J.

doi:10.1074/jbc.m004610200

Cited by 57 publications

(44 citation statements)

References 28 publications

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“…Glutamate transporters from the human brain have been shown previously to form dimers and trimers (17). There are now several examples of transporters that have dual affinity for their substrate and/or two mechanisms (13,19,21,35,55). Further investigation is needed to determine if this applies to PitB.…”

Section: Discussionmentioning

confidence: 99%

Characterization of PitA and PitB from Escherichia coli

Harris

Webb

Howitt³

et al. 2001

J Bacteriol

123

115

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Escherichia coli contains two major systems for transporting inorganic phosphate (P i ). The low-affinity P i transporter (pitA) is expressed constitutively and is dependent on the proton motive force, while the highaffinity Pst system (pstSCAB) is induced at low external P i concentrations by the pho regulon and is an ABC transporter. We isolated a third putative P i transport gene, pitB, from E. coli K-12 and present evidence that pitB encodes a functional P i transporter that may be repressed at low P i levels by the pho regulon. While a pitB ؉ cosmid clone allowed growth on medium containing 500 M P i , E. coli with wild-type genomic pitB (pitA ⌬pstC345 double mutant) was unable to grow under these conditions, making it indistinguishable from a pitA pitB ⌬pstC345 triple mutant. The mutation ⌬pstC345 constitutively activates the pho regulon, which is normally induced by phosphate starvation. Removal of pho regulation by deleting the phoB-phoR operon allowed the pitB ؉ pitA ⌬pstC345 strain to utilize P i , with P i uptake rates significantly higher than background levels. In addition, the apparent K m of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional pitA gene and lacks Pit activity when the Pst system is mutated. The pitA mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.Escherichia coli contains at least two major systems for transporting inorganic phosphate (P i ). The low-affinity inorganic phosphate transporter (Pit) is dependent on the proton motive force for energy and is constitutively expressed (30,31,49). When P i is plentiful, this is the major uptake system for phosphate, with a reported apparent K m (K m app ) of 25 M (30) to 38 M (50) in whole cells and 11.9 M in membrane vesicles (43). If the external P i concentration is below the millimolar range, the high-affinity phosphate-specific transport (Pst) system is induced. This has a K m app of around 0.2 M (30, 50). The Pst system is a complex of four proteins, including a periplasmic binding protein, which is energized by ATP and belongs to the ABC transporter family (7,15,47). The pst operon contains five genes under pho regulon control (1,40,41), which induces a range of genes when the phosphate supply is limited. Both the Pit and Pst systems are highly specific for P i (30). Another two transporters accept P i as a low-affinity analogue for either glycerol-3-phosphate (glpT) (18) or glucose-6-phosphate (uhpT) (29, 53), but in the absence of Pit and Pst activity, these latter two systems cannot support cell growth when supplied with P i (38).Divalent cations, such as Mg 2ϩ or Ca 2ϩ

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Section: Discussionmentioning

confidence: 99%

Characterization of PitA and PitB from Escherichia coli

Harris

Webb

Howitt³

et al. 2001

J Bacteriol

123

115

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“…These include the ability of TbNrt2 to support high-affinity transport of nitrate and nitrite, but not chlorate ions. With the sole exception of A. nidulans NrtA assayed in Xenopus oocytes [5], the nitrite transport capacity of other fungal NTs has not, so far, been determined directly. However, the observation that growth on nitrite is not impaired in either an A. nidulans nrtA/ntrB double mutant [6], or in an N. crassa nit-10 mutant [9], has been taken as an indication that none of the above NTs is capable of nitrite transport.…”

Section: The Tbnrt2 Transportermentioning

confidence: 99%

“…A large group of NTs (nitrate transporters), from both prokaryotes and eukaryotes, belong to the MFS (major facilitator superfamily) and, specifically, to the NRT2 family of NT-encoding genes [1][2][3]. The most well characterized fungal members of this family are NrtA (formerly known as CrnA) and NrtB from Aspergillus nidulans [4][5][6] and Ynt1 from Hansenula polymorpha [7,8]. They share a similar membrane topology, in which two sets of six TM (transmembrane) helices, with an N IN C IN (N-terminus intracellular/C-terminus intracellular) orientation, are connected by a large cytosolic loop.…”

Section: Introductionmentioning

confidence: 99%

Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

Montanini

Viscomi

Bolchi

et al. 2006

Biochemical Journal

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Nitrogen assimilation by plant symbiotic fungi plays a central role in the mutualistic interaction established by these organisms, as well as in nitrogen flux in a variety of soils. In the present study, we report on the functional properties, structural organization and distinctive mode of regulation of TbNrt2 (Tuber borchii NRT2 family transporter), the nitrate transporter of the mycorrhizal ascomycete T. borchii. As revealed by experiments conducted in a nitrate-uptake-defective mutant of the yeast Hansenula polymorpha, TbNrt2 is a high-affinity transporter (K m = 4.7 µM nitrate) that is bispecific for nitrate and nitrite. It is expressed in free-living mycelia and in mycorrhizae, where it preferentially accumulates in the plasma membrane of root-contacting hyphae. The TbNrt2 mRNA, which is transcribed from a single-copy gene clustered with the nitrate reductase gene in the T. borchii genome, was specifically up-regulated following transfer of mycelia to nitrate-(or nitrite)-containing medium. However, at variance with the strict nitrate-dependent induction commonly observed in other organisms, TbNrt2 was also up-regulated (at both the mRNA and the protein level) following transfer to a nitrogen-free medium. This unusual mode of regulation differs from that of the adjacent nitrate reductase gene, which was expressed at basal levels under nitrogen deprivation conditions and required nitrate for induction. The functional and expression properties, described in the present study, delineate TbNrt2 as a versatile transporter that may be especially suited to cope with the fluctuating (and often low) mineral nitrogen concentrations found in most natural, especially forest, soils.

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“…Early studies on nitrate uptake in plants have identified nitrate transporters as mediators of the influx of chlorate, giving support to the use of 36 CIO { 3 as a radiotracer transport analogue for nitrate (Deane-Drummond & Glass, 1982;Ruiz-Cristin & Briskin, 1991). This view has been subsequently modified in the light of results obtained by studying the kinetics of chlorate absorption, and interactions between chlorate and nitrate in intact plants (Kosola & Bloom, 1996), and results from electrophysiological experiments with Xenopus oocytes expressing individual nitrate transporter genes (Zhou et al, 1998(Zhou et al, , 2000. Currently, it appears that the ability to transport chlorate or chlorite is restricted to some members of the proton-dependent oligopeptide transport (POT) family (Galván & Fernández, 2001).…”

Section: Introductionmentioning

confidence: 99%

Interference of chlorate and chlorite with nitrate reduction in resting cells of Paracoccus denitrificans

Kučera

2006

Microbiology

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When grown anaerobically on a succinate+nitrate (SN) medium, Paracoccus denitrificans forms the membrane-bound, cytoplasmically oriented, chlorate-reducing nitrate reductase Nar, while the periplasmic enzyme Nap is expressed during aerobic growth on butyrate+oxygen (BO) medium. Preincubation of SN cells with chlorate produced a concentration-dependent decrease in nitrate utilization, which could be ascribed to Nar inactivation. Toluenization rendered Nar less sensitive to chlorate, but more sensitive to chlorite, suggesting that the latter compound may be the true inactivator. The Nap enzyme of BO cells was inactivated by both chlorate and chlorite at concentrations that were at least two orders of magnitude lower than those shown to affect Nar. Partial purification of Nap resulted in insensitivity to chlorate and diminished sensitivity to chlorite. Azide was specific for SN cells in protecting nitrate reductase against chlorate attack, the protective effect of nitrate being more pronounced in BO cells. The results are discussed in terms of different metabolic activation of chlorine oxoanions in both types of cells, and limited permeation of chlorite across the cell membrane.

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A High Affinity Fungal Nitrate Carrier with Two Transport Mechanisms

Cited by 57 publications

References 28 publications

Characterization of PitA and PitB from Escherichia coli

Characterization of PitA and PitB from Escherichia coli

Functional properties and differential mode of regulation of the nitrate transporter from a plant symbiotic ascomycete

Interference of chlorate and chlorite with nitrate reduction in resting cells of Paracoccus denitrificans

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