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Ammonium is a primary source of N for plants, so knowing how it is transported, stored, and assimilated in plant cells is important for rational approaches to optimise N-use in agriculture. Electrophysiological studies of Arabidopsis AtAMT1;1 expressed in oocytes revealed passive, Delta psi-driven transport of NH4+ through this protein. Expression of AtAMT1;1 in a novel yeast mutant defective in endogenous ammonium transport and vacuolar acidification supported the above mechanism for AtAMT1;1 and revealed a central role for acid vacuoles in storage and retention of ammonia in cells. These results highlight the mechanistic differences between plant AMT proteins and related transporters in bacteria and animal cells, and suggest novel strategies to enhance nitrogen use efficiency in agriculture. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved
The group of voltage-independent K+ channels in Arabidopsis thaliana consists of six members, five tandem-pore channels (TPK1-TPK5) and a single K-ir-like channel (KCO3). All TPK/KCO channels are located at the vacuolar membrane except for TPK4, which was shown to be a plasma membrane channel in pollen. The vacuolar channels interact with 14-3-3 proteins (also called General Regulating Factors, GRFs), indicating regulation at the level of protein-protein interactions. Here we review current knowledge about these ion channels and their genes, and highlight open questions that need to be urgently addressed in future studies to fully appreciate the physiological functions of these ion channels.
The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.
The permeation pore of K+ channels is formed by four copies of the pore domain. AtKCO3 is the only putative voltage-independent K+ channel subunit of Arabidopsis thaliana with a single pore domain. KCO3-like proteins recently emerged in evolution and, to date, have been found only in the genus Arabidopsis (A. thaliana and A. lyrata). We show that the absence of KCO3 does not cause marked changes in growth under various conditions. Only under osmotic stress we observed reduced root growth of the kco3-1 null-allele line. This phenotype was complemented by expressing a KCO3 mutant with an inactive pore, indicating that the function of KCO3 under osmotic stress does not depend on its direct ability to transport ions. Constitutively overexpressed AtKCO3 or AtKCO3::G FP are efficiently sorted to the tonoplast indicating that the protein is approved by the endoplasmic reticulum quality control. However, vacuoles isolated from transgenic plants do not have significant alterations in current density. Consistently, both AtKCO3 and AtKCO3::GFP are detected as homodimers upon velocity gradient centrifugation, an assembly state that would not allow for activity. We conclude that if AtKCO3 ever functions as a K+ channel, active tetramers are held by particularly weak interactions, are formed only in unknown specific conditions and may require partner proteins.
Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories-hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.
Expressed sequence tags (ESTs) represent a huge resource for the discovery of previously unknown genetic information and functional genome assignment. In this study we screened a collection of 178 292 ESTs from Arabidopsis thaliana by testing them against previously annotated genes of the Arabidopsis genome. We identified several hundreds of new transcripts that match the Arabidopsis genome at so far unassigned loci. The transcriptional activity of these loci was independently confirmed by comparison with the Salk Whole Genome Array Data. To a large extent, the newly identified transcriptionally active genomic regions do not encode 'classic' proteins, but instead generate non-coding RNAs and/or small peptide-coding RNAs of presently unknown biological function. More than 560 transcripts identified in this study are not represented by the Affymetrix GeneChip arrays currently widely used for expression profiling in A. thaliana. Our data strongly support the hypothesis that numerous previously unknown genes exist in the Arabidopsis genome
Members of the Shaker-like plant K+ channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K-in) channels, or leak-like (K-weak) channels, or depolarization-activated outward-rectifying (K-out) channels. Here we created 256 chimeras between the K-in channel KAT1 and the K-out channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed Lis to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N- terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K-out Channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-1314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range. (C) 2005 Elsevier Inc. All rights reserved
Voltage-gated potassium channels are formed by the assembly of four identical (homotetramer) or different (heterotetramer) subunits. Tetramerization of plant potassium channels involves the C-terminus of the protein. We investigated the role of the C-terminus of KDC1, a Shaker-like inward-rectifying K+ channel that does not form functional homomeric channels, but participates in the formation of heteromeric complexes with other potassium alpha- subunits when expressed in Xenopus oocytes. The interaction of KDC1 with KAT1 was investigated using the yeast two- hybrid system, fluorescence and electrophysiological studies. We found that the KDC1-EGFP fusion protein is not targeted to the plasma membrane of Xenopus oocytes unless it is coexpressed with KAT1. Deletion mutants revealed that the KDC1 C- terminus is involved in heteromerization. Two domains of the C-terminus, the region downstream the putative cyclic nucleotide binding domain and the distal part of the C-terminus called K-HA domain, contributed to a different extent to channel assembly. Whereas the first interacting region of the C-terminus was necessary for channel heteromerization, the removal of the distal KHA domain decreased but did not abolish the formation of heteromeric complexes. Similar results were obtained when coexpressing KDC1 with the KAT1-homolog KDC2 from carrots, thus indicating the physiological significance of the KAT1/KDC1 characterization. Electrophysiological experiments showed furthermore that the heteromerization capacity of KDC1 was negatively influenced by the presence of the enhanced green fluorescence protein fusion.
Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (K-weak channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of + 100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique K-weak gating. Instead, a lysine residue in S4, highly conserved among all K-weak channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the "open-lock" characteristic and converted AKT2 into an inward- rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the K-in channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is similar to 6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it