ATP Binding to the C Terminus of the Arabidopsis thaliana Nitrate/Proton Antiporter, AtCLCa, Regulates Nitrate Transport into Plant Vacuoles

Abstract

Nitrate is among the major nitrogen sources for plants in aerobic soils. It is taken up by root cells through plasma membrane transporters of nitrate-nitrite transporter and peptide transporter families. Once in the cytoplasm it can enter the amino acid biosynthesis pathway (1) or be accumulated in the vacuolar lumen via tonoplast transporters (2).The vacuolar nitrate transporter of the model plant Arabidopsis thaliana, AtCLCa, has been shown to work as an anion/ proton antiporter (3, 4), similarly to the bacterial CLCec-1 (5) and human hCLC-4 (6) as well as hCLC-5 (7). However, whereas bacterial and animal CLCs 2 transport chloride ions, the AtCLCa antiporter is more selective for nitrate, and therefore, it is able to mediate the accumulation of nitrate into the plant vacuole.Little is known on the modulation of CLC-proteins by nucleotides. The effects of ATP on the ion channel hCLC-1 are a matter of debate (8). Indeed, some reports have shown that ATP inhibits hCLC-1 currents, probably interacting with the C terminus of the protein (9 -11). Conversely, other reports indicate that ATP does not modify the properties of hCLC-1 current (12). This discrepancy has been attributed to the oxidation state of the channel, as ATP would be effective only in the presence of reducing agents (13).The C terminus domain of all eukaryotic CLC proteins has two cystathionine ␤-synthetase motifs (CBS (14, 15)), each one characterized by a ␤␣␤␤␣ topology (16,17). A structural and biochemical study of the hCLC-5 C-terminal part demonstrates that this region binds nucleotides (14). However, the effect of ATP binding on the transport activity of hCLC-5 is still unknown.The presence of analogous CBS domains in the C terminus of the AtCLCa antiporter suggested the hypothesis that ATP binds to this plant transporter and modulates its transport activity. Hence, we undertook a functional analysis of the effect of adenosine nucleotides on AtCLCa and found that ATP inhibits the AtCLCa-mediated transport. Based on a homology model of the C terminus of the channel, we identified two residues that would be putatively involved in the protein-nucleotide interaction.

The yeast CLC protein counteracts vesicular acidification during iron starvation

“…In this view, the overshoot in late Golgi and prevacuolar alkalinisation (Fig. 5B) of the presumably ATP-binding deficient Gef1 D732A would be consistent with the results presented by De Angeli and colleagues (De Angeli et al, 2009), who report a reversible inhibition of Arabidopsis CLC-a by ATP. In conclusion, our in vivo investigation of Gef1 antiporter activity under iron starvation suggests that the activity of CLC anion transport proteins is subject to metabolic regulation that results in an adaptation of compartmental function.…”

“…A recent structure of the CBS domains from ClC-5 revealed the presence of an ATP-binding site (Meyer et al, 2007), which is conserved in Gef1. The functional consequences of ATP binding have been studied for ClC-5 and Arabidopsis ClC-a (De Angeli et al, 2009;Zifarelli and Pusch, 2009). Although both reports suggest nucleotide regulation of CLC activity, the studies reach opposing mechanistic conclusions, with nucleotides stimulating ClC-5 function and inhibiting ClC-a.…”

ClC transporters: discoveries and challenges in defining the mechanisms underlying function and regulation of ClC-5

“…Higher coupling for nitrate is also observed for the plant ClC from Arabidopsis thaliana (AtClCa) which harbors a proline at the equivalent position and suggests this site, Site int , may exhibit a conserved property in eukaryotic ClC antiporters [22]. Unlike ClC-5 that retained its coupling ability in the face of the S168G mutation, mutation of the identical site in ClC-ec1 (S107G) completely uncoupled transport [59,133].…”

“…Conservation of residues in either identity or similarity which line the binding site for ClC-3 and ClC-4 indicate they likely bind nucleotides in a similar manner. Nucleotide binding to the CBS domains has been credited for the functional regulation that ATP exhibits on several ClC family members, such as ClC-2, ClC-4, ClC-5, and the plant ClC, AtClCa [22,83,91,116,134]. Conflicting results prevent the confident addition of ClC-1 to the list of nucleotide-regulated ClC proteins [5,6,112,131,132].…”

Nitrogen Nutrition, Its Regulation and Biotechnological Approaches to Improve Crop Productivity

“…This transport is dependent on proton coupling which is a unique feature in plants when compared to animal and bacterial CLC proteins where it is not dependent on proton coupling [180]. The nitrate selectivity of CLC proteins is based on the presence of conserved amino acids [204] [205]. A serine residue (Ser107) is con-served in human and bacterial CLC proteins while in plants a proline residue (Pro160) is conserved in the conserved motif which selects nitrate over chloride [201] [206].…”