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Abstract: Some intracellular organelles found in eukaryotes such as plants have arisen through the endocytotic engulfment of prokaryotic cells. This accounts for the presence of plant membrane intrinsic proteins that have homologs in prokaryotic cells. Other organelles, such as those of the endomembrane system, are thought to have evolved through infolding of the plasma membrane. Acquisition of intracellular components (organelles) in the cells supplied additional functions for survival in various natural environments. The organelles are surrounded by biological membranes, which contain membrane-embedded K+ transport systems allowing K+ to move across the membrane. K+ transport systems in plant organelles act coordinately with the plasma membrane intrinsic K+ transport systems to maintain cytosolic K+ concentrations. Since it is sometimes difficult to perform direct studies of organellar membrane proteins in plant cells, heterologous expression in yeast and Escherichia coli has been used to elucidate the function of plant vacuole K+ channels and other membrane transporters. The vacuole is the largest organelle in plant cells; it has an important task in the K+ homeostasis of the cytoplasm. The initial electrophysiological measurements of K+ transport have categorized three classes of plant vacuolar cation channels, and since then molecular cloning approaches have led to the isolation of genes for a number of K+ transport systems. Plants contain chloroplasts, derived from photoautotrophic cyanobacteria. A novel K+ transport system has been isolated from cyanobacteria, which may add to our understanding of K+ flux across the thylakoid membrane and the inner membrane of the chloroplast. This chapter will provide an overview of recent findings regarding plant organellar K+ transport proteins. [Copyright &y& Elsevier]

Plant cells are sensitive to salinity stress and do not require sodium as an essential element for their growth and development. Saline soils reduce crop yields and limit available land. Research shows that HKT transporters provide a potent mechanism for mediating salt tolerance in plants. Knowledge of the molecular ion transport and regulation mechanisms and the control of HKT gene expression are crucial for understanding the mechanisms by which HKT transporters enhance crop performance under salinity stress. This review focuses on HKT transporters in monocot plants and in Arabidopsis as a dicot plant, as a guide to efforts toward improving salt tolerance of plants for increasing the production of crops and bioenergy feedstocks. [ABSTRACT FROM AUTHOR]

The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers (CHX), represented by 28 genes in A rabidopsis thaliana, are associated with diverse endomembrane compartments and tissues in plants, although their roles are poorly understood. We expressed a phylogenetically related cluster of CHX genes, encoded by CHXJ5-CHX2O, in yeast and bacterial cells engineered to lack multiple cation- handling mechanisms, Of these, CHX16-CHX2O were impli- cated in pH homeostasis because their expression rescued the alkaline pH-sensitive growth phenotype of the host yeast strain. A smaller subset, CHX17-CHX19, also conferred tolerance to hygromycin B. Further differences were observed in K+- and low pH-dependent growth phenotypes. Although CHX17 did not alter cytoplasmic or vacuolar pH in yeast, CHX2O elicited acidification and alkalization of the cytosol and vacuole, respectively. Using heterologous expression in Escherichia coli strains lacking K+ uptake systems, we provide evidence for K+ (86Rb) transport mediated by CHX17 and CHX2O. Finally, we showthat CHX17 and CHX2O affected protein sorting as measured by carboxypeptidase Y secretion in yeast mutants grown at alkaline pH. In plant cells, CHX2O-RFP co-localized with an endoplasmic reticulum marker, whereas RFP-tagged CHX17-CHX19 co-localized with prevacuolar compartment and endosome markers. Together, these results suggest that in response to environmental cues, multiple CHX transporters differentially modulate K+ and pH homeostasis of distinct intracellular compartments, which alter membrane trafficking events likely to be critical for adaptation and survival. [ABSTRACT FROM AUTHOR]

The genome of Synechocystis PCC 6803 contains a single gene encoding an aquaporin, aqpZ. The AqpZ protein functioned as a water-permeable channel in the plasma membrane. However, the physiological importance of AqpZ in Synechocystis remains unclear. We found that growth in glucose-containing medium inhibited proper division of ΔaqpZ cells and led to cell death. Deletion of a gene encoding a glucose transporter in the ΔaqpZ background alleviated the glucose-mediated growth inhibition of the ΔaqpZ cells. The ΔaqpZ cells swelled more than the wild type after the addition of glucose, suggesting an increase in cytosolic osmolarity. This was accompanied by a down-regulation of the pentose phosphate pathway and concurrent glycogen accumulation. Metabolite profiling by GC/TOF-MS of wild-type and ΔaqpZ cells revealed a relative decrease of intermediates of the tricarboxylic acid cycle and certain amino acids in the mutant. The changed levels of metabolites may have been the cause for the observed decrease in growth rate of the ΔaqpZ cells along with decreased PSII activity at pH values ranging from 7.5 to 8.5. A mutant in sll1961, encoding a putative transcription factor, and a Δhik31 mutant, lacking a putative glucose-sensing kinase, both exhibited higher glucose sensitivity than the ΔaqpZ cells. Examination of protein expression indicated that sll1961 functioned as a positive regulator of aqpZ gene expression but not as the only regulator. Overall, the ΔaqpZ cells showed defects in macronutrient metabolism, pH homeostasis, and cell division under photomixotrophic conditions, consistent with an essential role of AqpZ in glucose metabolism. [ABSTRACT FROM AUTHOR]

Iron deficiency is one of the major agricultural problems, as 30% of the arable land of the world is too alkaline for optimal crop production, rendering plants short of available iron despite its abundance. To take up apoplasmic precipitated iron, plants secrete phenolics such as protocatechuic acid (PCA) and caffeic acid. The molecular pathways and genes of iron uptake strategies are already characterized, whereas the molecular mechanisms of phenolics synthesis and secretion have not been clarified, and no phenolics efflux transporters have been identified in plants yet. Here we describe the identification of a phenolics efflux transporter in rice. We identified a cadmium-accumulating rice mutant in which the amount of PCA and caffeic acid in the xylem sap was dramatically reduced and hence named it ...henolics ...fflux ...ero 1 (pez1). PEZ1 localized to the plasma membrane and transported PCA when expressed in Xenopus laevis oocytes. PEZ1 localized mainly in the stele of roots. In the roots of pez1, precipitated apoplasmic iron increased. The growth of PEZ1 overexpression lines was severely restricted, and these lines accumulated more iron as a result of the high solubilization of precipitated apoplasmic iron in the stele. We show that PEZ1 is responsible for an increase of PCA concentration in the xylem sap and is essential for the utilization of apoplasmic precipitated iron in the stele. [ABSTRACT FROM AUTHOR]

Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released 14C-labeled deoxymugineic acid but not 14C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants. [ABSTRACT FROM AUTHOR]

Na[sup+]/H[sup+] antiporters influence proton or sodium motive force across the membrane. Synechocystis sp. PCC 6803 has six genes encoding Na[sup+]/H[sup+] antiporters, nhaS1-5 and s110556. In this study, the function of NhaS3 was examined. NhaS3 was essential for growth of Synechocystis, and loss of nhaS3 was not complemented by expression of the Escherichia coli Na[sup+]/H[sup+] antiporter NhaA. Membrane fractionation followed by immunoblotting as well as immunogold labeling revealed that NhaS3 was localized in the thylakoid membrane of Synechocystis. NhaS3 was shown to be functional over a pH range from pH 6.5 to 9.0 when expressed in F. coli. A reduction in the copy number of nhaS3 in the Synechocystis genome rendered the cells more sensitive to high Na[sup+] concentrations. NhaS3 had no K[sup+]/H[sup+] exchange activity itself but enhanced ICuptake from the medium when expressed in an F. coli potassium uptake mutant. Expression of nhaS3 increased after shifting from low CO[sub2] to high CO[sub2] conditions. Expression of nhaS3 was also found to be controlled by the circadian rhythm. Gene expression peaked at the beginning of subjective night. This coincided with the time of the lowest rate of CO[sub2] consumption caused by the ceasing of O[sub2]-evolving photosynthesis. This is the first report of a Na[sup+]/H[sup+] antiporter localized in thylakoid membrane. Our results suggested a role of NhaS3 in the maintenance of ion homeostasis of H[sup+], Na[sup+] and K[sup+] in supporting the conversion of photosynthetic products and in the supply of energy in the dark. [ABSTRACT FROM AUTHOR]

The subunit KtrB of bacterial Na+-dependent K+-translocating KtrAB systems belongs to a superfamily of K+ transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly70, Gly185, Gly290, and Gly402 from p-loops PA to PD of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrBA70, KtrBA185, and KtrBA290 maintained a substantial activity in KtrAB-mediated K+ uptake in Escherichia coil This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on Vmax. Minor activities were also observed for three other variants: KtrBA402, KtrBS70, and KtrBD185. With all of these variants, the property of Na+ dependence of K+ transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K+/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex. [ABSTRACT FROM AUTHOR]

Transmembrane ion transport processes play a key role in the adaptation of cells to hyperosmotic conditions. Previous work has shown that the disruption of a ktrB/ntpJ-like putative Na+/K+ transporter gene in the cyanobacterium Synechocystis sp. PCC 6803 confers increased Na+ sensitivity, and inhibits HCO3- uptake. Here, we report on the mechanistic basis of this effect. Heterologous expression experiments in Escherichia coli show that three Synechocystis genes are required for K+ transport activity. They encode an NAD+-binding peripheral membrane protein (ktrA; s110493), an integral membrane protein, belonging to a superfamily of K+ transporters (ktrB; formerly ntpJ; sir1509), and a novel type of ktr gene product, not previously found in Ktr systems (ktrE; slr1508). In E. coli, Synechocystis KtrABE- mediated K+ uptake occurred with a moderately high affinity (Km of about 60 μM), and depended on both Na+ and a high membrane potential, but not on ATP. KtrABE neither mediated Na+ uptake nor Na+ efflux. In Synechocystis sp. PCC 6803, KtrB-mediated K+ uptake required Na+ and was inhibited by protonophore. A ΔktrB strain was sensitive to long term hyperosmotic stress elicited by either NaC1 or sorbitol. Hyperosmotic shock led initially to loss of net K+ from the cells. The ΔktrB cells shocked with sorbitol failed to reaccumulate K+ up to its original level. These data indicate that in strain PCC 6803 K+ uptake via KtrABE plays a crucial role in the early phase of cell turgor regulation after hyperosmotic shock. [ABSTRACT FROM AUTHOR]

Gprotein-activated inwardly rectifying K+ channel (GIRK or Kir3) currents are inhibited by mechanical stretch of the cell membrane, but the underlying mechanisms are not understood. In Xenopus oocytes heterologously expressing GIRK channels, membrane stretch induced by 50% reduction of osmotic pressure caused a prompt reduction of GIRK¼, GIRK1, and GIRK4 currents by 16.6-42.6%. Comparable GIRK current reduction was produced by protein kinase C (PKC) activation (phorbol 12-myristate 13-acetate). The mechanosensitivity of the GIRK4 current was abolished by pretreatment with PKC inhibitors (staurosporine or calphostin C). Neither hypo-osmotic challenge nor PKC activation affected IRK1 currents. GIRK4 chimera (GIRK4-IRK1(Lys207-Leu245)) and single point mutant (GIRK4(I229L)), in which the phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain or residue was replaced by the corresponding region of IRK1 to strengthen the channel-PIP2 interaction, showed no mechanosensitivity and minimal PKC sensitivity. IRK1 gained mechanosensitivity and PKC sensitivity by reverse double point mutation of the PIP2 binding domain (L222I/R213Q). Overexpression of Gβγ, which is known to strengthen the channel-PIP2 interaction, attenuated the mechanosensitivity of GIRK4 channels. In oocytes expressing a pleckstrin homology domain of PLC-δ tagged with green fluorescent protein, hypo-osmotic challenge or PKC activation caused a translocation of the fluorescence signal from the cell membrane to the cytosol, reflecting PIP2 hydrolysis. The translocation was prevented by pretreatment with PKC inhibitors. Involvement of PKC activation in the mechanosensitivity of muscarinic K+ channels was confirmed in native rabbit atrial myocytes. These results suggest that the mechanosensitivity of GIRK channels is mediated primarily by channel-PIP2 interaction, with PKC playing an important role in modulating the interaction probably through PIP2 hydrolysis. [ABSTRACT FROM AUTHOR]

Potassium channels, which control the passage of K[sup +] across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K[sup +] channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K[sup +] channel, KAT1, the MI segment in KcsA, was found to have a strong type H signal-anchor function, which favors the N[sub cyt]/C[sub exo] topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of MI in Kit 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signal-anchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1. [ABSTRACT FROM AUTHOR]

Discusses the molecular dissection of the contribution of negatively and positively charged residues in S2, S3 and S4 to the final membrane topology of the voltage sensor in the K[sup +] channel, KAT1. Use of in vitro translation and translocation experiments; Indication that Asp[sup 95] assists in the membrane insertion S3-S4.

We tested a one batch reaction method for the transcription and the insect cell-free translation from undigested plasmids without any centrifugation steps. The efficiency of protein synthesis reached 74–112% of that achieved using the conventional procedure. This simplified method will help expedite the high throughput insect cell-free protein production. [ABSTRACT FROM AUTHOR]