Functional characterization of a plasma membrane Na+/H+ antiporter from alkali grass (Puccinellia tenuiflora)

Radish is a kind of moderately salt-sensitive vegetable. Salt stress seriously decreases the yield and quality of radish. The plasma membrane Na+/H+ antiporter protein Salt Overly Sensitive 1 (SOS1) plays a crucial role in protecting plant cells against salt stress, but the biological function of the RsSOS1 gene in radish remains to be elucidated. In this study, the RsSOS1 gene was isolated from radish genotype ‘NAU-TR17’, and contains an open reading frame of 3414 bp encoding 1137 amino acids. Phylogenetic analysis showed that RsSOS1 had a high homology with BnSOS1, and clustered together with Arabidopsis plasma membrane Na+/H+ antiporter (AtNHX7). The result of subcellular localization indicated that the RsSOS1 was localized in the plasma membrane. Furthermore, RsSOS1 was strongly induced in roots of radish under 150 mmol/L NaCl treatment, and its expression level in salt-tolerant genotypes was significantly higher than that in salt-sensitive ones. In addition, overexpression of RsSOS1 in Arabidopsis could significantly improve the salt tolerance of transgenic plants. Meanwhile, the transformation of RsSOS1△999 could rescue Na+ efflux function of AXT3 yeast. In summary, the plasma membrane Na+/H+ antiporter RsSOS1 plays a vital role in regulating salt-tolerance of radish by controlling Na+ homeostasis. These results provided useful information for further functional characterization of RsSOS1 and facilitate clarifying the molecular mechanism underlying salt stress response in radish.

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Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

Journal of Biological Chemistry ◽

10.1074/jbc.m110.179853 ◽

2011 ◽

Vol 286 (12) ◽

pp. 10419-10428 ◽

Cited By ~ 56

Author(s):

Rasmus J. N. Frandsen ◽

Claes Schütt ◽

Birgitte W. Lund ◽

Dan Staerk ◽

John Nielsen ◽

...

Keyword(s):

Plasma Membrane ◽

Fusarium Graminearum ◽

Polyketide Synthase ◽

Functional Characterization ◽

Extracellular Enzyme ◽

Gene Replacement ◽

Orphan Genes ◽

Pigment Biosynthesis ◽

Targeted Gene Replacement

Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurO genes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis.

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Functional characterization of a wheat plasma membrane Na+/H+ antiporter in yeast

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2008.02.018 ◽

2008 ◽

Vol 473 (1) ◽

pp. 8-15 ◽

Cited By ~ 69

Author(s):

Haixia Xu ◽

Xingyu Jiang ◽

Kehui Zhan ◽

Xiyong Cheng ◽

Xinjian Chen ◽

...

Keyword(s):

Plasma Membrane ◽

Functional Characterization

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Functional characterization of NIPA2, a selective Mg2+ transporter

AJP Cell Physiology ◽

10.1152/ajpcell.00091.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. C944-C953 ◽

Cited By ~ 49

Author(s):

Angela Goytain ◽

Rochelle M. Hines ◽

Gary A. Quamme

Keyword(s):

Plasma Membrane ◽

Functional Characterization ◽

Affinity Constant ◽

Fluorescence Studies ◽

Low Magnesium ◽

Early Endosomes ◽

Electrode Voltage ◽

Voltage Dependent ◽

Transmembrane Regions

We used microarray analysis to identify renal cell transcripts that were upregulated with low magnesium. One transcript, identified as NIPA2 (nonimprinted in Prader-Willi/Angelman syndrome) subtype 2, was increased over twofold relative to cells cultured in normal magnesium. The deduced sequence comprises 129 amino acids with 8 predicted transmembrane regions. As the secondary structure of NIPA2 conformed to a membrane transport protein, we expressed it in Xenopus oocytes and determined that it mediated Mg2+ uptake with two-electrode voltage-clamp and fluorescence studies. Mg2+ transport was electrogenic, voltage dependent, and saturable, demonstrating a Michaelis affinity constant of 0.31 mM. Unlike other reported Mg2+ transporters, NIPA2 was very selective for the Mg2+ cation. NIPA2 mRNA is found in many tissues but particularly abundant in renal cells. With the use of immunofluorescence, it was shown that NIPA2 protein was normally localized to the early endosomes and plasma membrane and was recruited to the plasma membrane in response to low extracellular magnesium. We conclude that NIPA2 plays a role in magnesium metabolism and regulation of renal magnesium conservation.

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HvPAA1 Encodes a P-Type ATPase, a Novel Gene for Cadmium Accumulation and Tolerance in Barley (Hordeum vulgare L.)

International Journal of Molecular Sciences ◽

10.3390/ijms20071732 ◽

2019 ◽

Vol 20 (7) ◽

pp. 1732 ◽

Cited By ~ 2

Author(s):

Xin-Ke Wang ◽

Xue Gong ◽

Fangbin Cao ◽

Yizhou Wang ◽

Guoping Zhang ◽

...

Keyword(s):

Plasma Membrane ◽

Functional Characterization ◽

Cadmium Accumulation ◽

Hordeum Vulgare L ◽

Dh Population ◽

Cd Accumulation ◽

Novel Gene ◽

Cd Tolerance ◽

P Type

The identification of gene(s) that are involved in Cd accumulation/tolerance is vital in developing crop cultivars with low Cd accumulation. We developed a doubled haploid (DH) population that was derived from a cross of Suyinmai 2 (Cd-sensitive) × Weisuobuzhi (Cd-tolerant) to conduct quantitative trait loci (QTL) mapping studies. We assessed chlorophyll content, traits that are associated with development, metal concentration, and antioxidative enzyme activity in DH population lines and parents under control and Cd stress conditions. A single QTL, designated as qShCd7H, was identified on chromosome 7H that was linked to shoot Cd concentration; qShCd7H explained 17% of the phenotypic variation. Comparative genomics, map-based cloning, and gene silencing were used in isolation, cloning, and functional characterization of the candidate gene. A novel gene HvPAA1, being related to shoot Cd concentration, was identified from qShCd7H. Sequence comparison indicated that HvPAA1 carried seven domains with an N-glycosylation motif. HvPAA1 is predominantly expressed in shoots. Subcellular localization verified that HvPAA1 is located in plasma membrane. The silencing of HvPAA1 resulted in growth inhibition, greater Cd accumulation, and a significant decrease in Cd tolerance. We conclude HvPAA1 is a novel plasma membrane-localized ATPase that contributes to Cd tolerance and accumulation in barley. The results provide us with new insights that may aid in the screening and development of Cd-tolerant and low-Cd-accumulation crops.

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Functional characterization of Rat Plasma Membrane Monoamine Transporter in the Blood–Brain and Blood–Cerebrospinal Fluid Barriers

Journal of Pharmaceutical Sciences ◽

10.1002/jps.22594 ◽

2011 ◽

Vol 100 (9) ◽

pp. 3924-3938 ◽

Cited By ~ 33

Author(s):

Takashi Okura ◽

Sayaka Kato ◽

Yusuke Takano ◽

Takenori Sato ◽

Atsushi Yamashita ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Plasma Membrane ◽

Functional Characterization ◽

Rat Plasma ◽

Monoamine Transporter ◽

Blood Brain

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Functional identification of SLC43A3 as an equilibrative nucleobase transporter involved in purine salvage in mammals

Scientific Reports ◽

10.1038/srep15057 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 22

Author(s):

Junji Furukawa ◽

Katsuhisa Inoue ◽

Junya Maeda ◽

Tomoya Yasujima ◽

Kinya Ohta ◽

...

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Cellular Uptake ◽

Functional Characterization ◽

Salvage Pathway ◽

Functional Identification ◽

Purine Salvage ◽

Transporter Family ◽

Mdckii Cells

Abstract The purine salvage pathway plays a major role in the nucleotide production, relying on the supply of nucleobases and nucleosides from extracellular sources. Although specific transporters have been suggested to be involved in facilitating their transport across the plasma membrane in mammals, those which are specifically responsible for utilization of extracellular nucleobases remain unknown. Here we present the molecular and functional characterization of SLC43A3, an orphan transporter belonging to an amino acid transporter family, as a purine-selective nucleobase transporter. SLC43A3 was highly expressed in the liver, where it was localized to the sinusoidal membrane of hepatocytes and the lung. In addition, SLC43A3 expressed in MDCKII cells mediated the uptake of purine nucleobases such as adenine, guanine and hypoxanthine without requiring typical driving ions such as Na+ and H+, but it did not mediate the uptake of nucleosides. When SLC43A3 was expressed in APRT/HPRT1-deficient A9 cells, adenine uptake was found to be low. However, it was markedly enhanced by the introduction of SLC43A3 with APRT. In HeLa cells, knock-down of SLC43A3 markedly decreased adenine uptake. These data suggest that SLC43A3 is a facilitative and purine-selective nucleobase transporter that mediates the cellular uptake of extracellular purine nucleobases in cooperation with salvage enzymes.

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