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

Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO₃⁻/H⁺ exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine β-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of nitrate influx into the vacuole at cytosolic physiological nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.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 CLCs² 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 β-synthetase motifs (CBS (14, 15)), each one characterized by a βαββα 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.     

NEDD4-2 associates with γc and regulates its degradation rate

Interleukin-2 (IL-2) is a cytokine that regulates proliferation, differentiation and survival of various lymphoid cell subsets. Its actions are mediated through its binding to the IL-2 receptor which is composed of three subunits (IL-2Rα, IL-2Rβ and γ_c). Only β and γ_c have been shown to transduce intra cellular signals. The γ_c chain is shared by the interleukin-2, 4, 7, 9, 15 and 21 receptors, and is essential for lymphocyte functions. The regulation of γ_c expression level is therefore critical for the ability of cells to respond to these cytokines. In the present work, we show that the IL-2R constitutively associates with the ubiquitin ligase NEDD4-2, and to a lesser extent NEDD4-1. We identified the specific binding site on γ_c. And we show that the loss of NEDD4 association on γ_c is accompanied by a dramatic increase of the half-life of the receptor subunit.     

Mobilization of Neural Stem Cells and Generation of New Neurons in 6-OHDA�Clesioned Rats by Intracerebroventricular Infusion of Liver Growth Factor

Neural stem cells with self-renewal and multilineage potential persist in the subventricular zone of the adult mammalian forebrain. These cells remain relatively quiescent but, under certain conditions, can be stimulated, giving rise to new neurons. Liver growth factor (LGF) is a mitogen for liver cells that shows biological activity in extrahepatic sites and is useful for neuroregenerative therapies. The aim of this study was to investigate the potential neurogenic activity of LGF in the 6-hydroxydopamine rat model of Parkinson''s disease. Proliferation was significantly increased in the subventricular zone and denervated striatum of rats receiving ICV LGF infusions, and 25% of the proliferating cells were doublecortin-positive neurons. Doublecortin-positive cells with the morphology of migrating neuroblasts were also observed in the dorsal and ventral regions of the striatum of LGF-infused animals. Moreover, some newly generated cells were neuronal nuclei-positive mature neurons. LGF also stimulated microglia and induced astrogliosis, both phenomena associated with generation and migration of new neurons in the adult brain. In summary, our study shows that LGF stimulates neurogenesis when applied intraventricularly in 6-hydroxydopamine–lesioned rats. Considering that this factor also promotes neuronal migration into damaged tissue, we propose LGF as a novel factor useful for neuronal replacement in neurodegenerative diseases. (J Histochem Cytochem 57:491–502, 2009)