Root-to-shoot iron partitioning in Arabidopsis requires IRON-REGULATED TRANSPORTER1 (IRT1)

ABSTRACTIRON-REGULATED TRANSPORTER1 (IRT1) is the root high-affinity ferrous iron uptake system and indispensable for the completion of the life cycle of Arabidopsis thaliana without vigorous iron (Fe) supplementation. Here we provide evidence supporting a second role of IRT1 in root-to-shoot mobilization of Fe. We show that the irt1-2 (pam42) mutant over-accumulates Fe in roots, most prominently in the cortex of the differentiation zone, when compared to the wild type. Shoots of irt1-2 are severely Fe-deficient according to Fe content and marker transcripts, as expected. We generated irt1-2 lines producing IRT1 mutant variants carrying single amino-acid substitutions of key residues in transmembrane helices IV and V, Ser206 and His232, which are required for transport activity in yeast. In the transgenic Arabidopsis lines, short-term root Fe uptake rates and secondary substrate Mn accumulation resemble those of irt1-2, suggesting that these plants remain incapable of IRT1-mediated root Fe uptake. Yet, IRT1S206A partially complements rosette dwarfing and leaf chlorosis, as well as root-to-shoot Fe partitioning and gene expression defects of irt1-2, all of which are fully complemented by wild-type IRT1. Taken together, these results suggest a function for IRT1 in root-to-shoot Fe partitioning that does not require Fe transport activity of IRT1. Among the genes of which transcript levels are partially dependent on IRT1, we identify MYB DOMAIN PROTEIN10, MYB DOMAIN PROTEIN72 and NICOTIANAMINE SYNTHASE4 as candidates for effecting IRT1-dependent Fe mobilization in roots. Understanding the biological functions of IRT1 will help to improve iron nutrition and the nutritional quality of agricultural crops.

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Single amino acid substitution of rat MRP2 results in acquired transport activity for taurocholate

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.2001.281.4.g1034 ◽

2001 ◽

Vol 281 (4) ◽

pp. G1034-G1043 ◽

Cited By ~ 31

Author(s):

Kousei Ito ◽

Hiroshi Suzuki ◽

Yuichi Sugiyama

Keyword(s):

Amino Acids ◽

Multidrug Resistance ◽

Amino Acid ◽

Membrane Vesicles ◽

Single Amino Acid ◽

Sf9 Cells ◽

Transport Activity ◽

Wild Type ◽

Cationic Amino Acids ◽

The Difference

Multidrug resistance-associated protein 3 (MRP3), unlike other MRPs, transports taurocholate (TC). The difference in TC transport activity between rat MRP2 and MRP3 was studied, focusing on the cationic amino acids in the transmembrane domains. For analysis, transport into membrane vesicles from Sf9 cells expressing wild-type and mutated MRP2 was examined. Substitution of Arg at position 586 with Leu and Ile and substitution of Arg at position 1096 with Lys, Leu, and Met resulted in the acquisition of TC transport activity, while retaining transport activity for glutathione and glucuronide conjugates. Substitution of Leu at position 1084 of rat MRP3 (which corresponds to Arg-1096 in rat MRP2) with Lys, but not with Val or Met, resulted in the loss of transport activity for TC and glucuronide conjugates. These results suggest that the presence of the cationic charge at Arg-586 and Arg-1096 in rat MRP2 prevents the transport of TC, whereas the presence of neutral amino acids at the corresponding position of rat MRP3 is required for the transport of substrates.

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Sensitivity of human immunodeficiency virus to bicyclam derivatives is influenced by the three-dimensional structure of gp120.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.12.2616 ◽

1997 ◽

Vol 41 (12) ◽

pp. 2616-2620 ◽

Cited By ~ 14

Author(s):

K De Vreese ◽

I Van Nerum ◽

K Vermeire ◽

J Anné ◽

E De Clercq

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Single Amino Acid ◽

Resistance Pattern ◽

V3 Loop ◽

Resistant Virus ◽

Wild Type ◽

Immunodeficiency Virus ◽

Resistant Strains ◽

Anti Hiv

The bicyclams are a new class of anti-human immunodeficiency virus (anti-HIV) compounds targeted at viral entry. From marker rescue experiments, it appears that the envelope gp120 glycoprotein plays an important role in the anti-HIV activity of the bicyclams. Bicyclam-resistant strains contain a number of amino acid changes scattered over the V2 to V5 region of gp120. Experiments aimed at estimating the relative importance of particular amino acid changes with regard to the overall resistance pattern are described. The sequences of some partially bicyclam-resistant virus strains, obtained during the resistance development process, were analyzed, and the corresponding 50% effective concentrations were determined. Selected mutations observed in bicyclam-resistant strains were introduced in the wild-type background by site-directed mutagenesis. In addition, some amino acids were back-mutated to their wild-type counterparts in an otherwise JM3100-resistant strain. The sensitivities of these mutant viruses to bicyclams were determined. Construction of chimeric viruses, carrying the V3 loop of JM3100-resistant virus in a wild-type HIV type 1 HXB2 background, enabled us to investigate the importance of the mutations in the V3 loop of JM3100-resistant virus. From the results described in the report, it can be concluded that single amino acid substitutions do not influence the observed resistance to JM3100. Also, the mutations in the V3 loop are not sufficient to engender even a partially resistant phenotype. We postulate that the overall conformation of gp120 determines the degree of sensitivity or resistance of HIV strains to bicyclams.

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Loss of wild-type carrier-mediatedL-carnitine transport activity in hepatocytes of juvenile visceral steatosis mice

Hepatology ◽

10.1002/hep.510300423 ◽

1999 ◽

Vol 30 (4) ◽

pp. 997-1001 ◽

Cited By ~ 30

Author(s):

Koichi Yokogawa ◽

Masayuki Yonekawa ◽

Ikumi Tamai ◽

Rikiya Ohashi ◽

Yasuaki Tatsumi ◽

...

Keyword(s):

Transport Activity ◽

Wild Type ◽

Carnitine Transport

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Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

Applied and Environmental Microbiology ◽

10.1128/aem.71.7.3468-3474.2005 ◽

2005 ◽

Vol 71 (7) ◽

pp. 3468-3474 ◽

Cited By ~ 19

Author(s):

Gyeong Tae Eom ◽

Jae Kwang Song ◽

Jung Hoon Ahn ◽

Yeon Soo Seo ◽

Joon Shick Rhee

Keyword(s):

Escherichia Coli ◽

Directed Evolution ◽

Abc Transporter ◽

Microtiter Plate ◽

Single Amino Acid ◽

Wild Type ◽

E Coli ◽

Single Amino Acid Change ◽

Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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Replacement of both tryptophan residues at 388 and 412 completely abolished cytochalasin B photolabelling of the GLUT1 glucose transporter

Biochemical Journal ◽

10.1042/bj3020355 ◽

1994 ◽

Vol 302 (2) ◽

pp. 355-361 ◽

Cited By ~ 31

Author(s):

K Inukai ◽

T Asano ◽

H Katagiri ◽

M Anai ◽

M Funaki ◽

...

Keyword(s):

Glucose Transporter ◽

Cytochalasin B ◽

Transmembrane Domain ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Site Directed Mutagenesis ◽

Transport Activity ◽

Wild Type ◽

In The Wild ◽

Glut1 Glucose Transporter

A mutated GLUT1 glucose transporter, a Trp-388, 412 mutant whose tryptophans 388 and 412 were both replaced by leucines, was constructed by site-directed mutagenesis and expressed in Chinese hamster ovary cells. Glucose transport activity was decreased to approx. 30% in the Trp-388, 412 mutant compared with that in the wild type, a similar decrease in transport activity had been observed previously in the Trp-388 mutant and the Trp-412 mutant which had leucine at 388 and 412 respectively. Cytochalasin B labelling of the Trp-388 mutant was only decreased rather than abolished, a result similar to that obtained previously for the Trp-412 mutant. Cytochalasin B labelling was finally abolished completely in the Trp-388, 412 mutant, while cytochalasin B binding to this mutant was decreased to approx. 30% of that of the wild-type GLUT1 at the concentration used for photolabelling. This level of binding is thought to be adequate to detect labelling, assuming that the labelling efficiency of these transporters is similar. These findings suggest that cytochalasin B binds to the transmembrane domain of the glucose transporter in the vicinity of helix 10-11, and is inserted covalently by photoactivation at either the 388 or the 412 site.

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Mutation of a single amino acid converts the human water channel aquaporin 5 into an anion channel

AJP Cell Physiology ◽

10.1152/ajpcell.00129.2013 ◽

2013 ◽

Vol 305 (6) ◽

pp. C663-C672 ◽

Cited By ~ 12

Author(s):

Xue Qin ◽

Walter F. Boron

Keyword(s):

Water Channel ◽

Anion Channel ◽

Single Amino Acid ◽

Side Chain ◽

Wild Type ◽

Aquaporin 5 ◽

Anion Conductance ◽

Electrode Voltage ◽

Microelectrode Measurements ◽

Positively Charged

Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H2O permeability ( Pf), microelectrode measurements of surface pH (pHS) to assess CO2 permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H2O or CO2 permeability of the wild-type protein but instead has a novel p-chloromercuribenzene sulfonate (pCMBS)-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I− > NO3− ≅ NO2− > Br− > Cl− > HCO3− > gluconate. Of the other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H2O and CO2 permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H2O or CO2 permeability. L51K (positively charged) has an intermediate H2O and CO2 permeability and anion conductance. L51H is unusual in having a relatively low CO2 permeability and anion conductance, but a moderate Pf. Thus, positively charged mutations of L51 can convert AQP5 from a H2O/CO2 channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO2, in part, takes a pathway different from H2O through AQP5.

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Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbaa037 ◽

2021 ◽

Vol 85 (2) ◽

pp. 297-306

Author(s):

Zui Fujimoto ◽

Le Thi Thu Hong ◽

Naomi Kishine ◽

Nobuhiro Suzuki ◽

Keitarou Kimura

Keyword(s):

Bacillus Subtilis ◽

Quaternary Structure ◽

Single Amino Acid ◽

Wild Type ◽

Amino Acid Mutation ◽

X Ray Crystallography ◽

Ligand Binding Sites ◽

Potential Ligand ◽

Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

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The calcium-dependent ATP-Mg/Pi mitochondrial carrier is a target of glucose-induced calcium signalling in Saccharomyces cerevisiae

Biochemical Journal ◽

10.1042/bj20050806 ◽

2005 ◽

Vol 392 (3) ◽

pp. 537-544 ◽

Cited By ~ 37

Author(s):

Santiago Cavero ◽

Javier Traba ◽

Araceli Del Arco ◽

Jorgina Satrústegui

Keyword(s):

Calcium Binding ◽

Mitochondrial Protein ◽

Calcium Signalling ◽

Cytosolic Calcium ◽

Calcium Signal ◽

Transport Activity ◽

Wild Type ◽

Binding Motifs ◽

Mitochondrial Carrier ◽

Calcium Dependent

Sal1p is a mitochondrial protein that belongs to the SCaMC (short calcium-binding mitochondrial carrier) subfamily of mitochondrial carriers. The presence of calcium-binding motifs facing the extramitochondrial space allows the regulation of the transport activity of these carriers by cytosolic calcium and provides a new mechanism to transduce calcium signals in mitochondria without the requirement of calcium entry in the organelle. We have studied its transport activity, finding that it is a carboxyatractyloside-resistant ATP-Mg carrier. Mitochondria from a disruption mutant of SAL1 have a 50% reduction in the uptake of ATP. We have also found a clear stimulation of ATP-transport activity by calcium, with an S0.5 of approx. 30 μM. Our results also suggest that Sal1p is a target of the glucose-induced calcium signal which is non-essential in wild-type cells, but becomes essential for transport of ATP into mitochondria in yeast lacking ADP/ATP translocases.

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Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

Molecular and Cellular Biology ◽

10.1128/mcb.12.5.2372-2382.1992 ◽

1992 ◽

Vol 12 (5) ◽

pp. 2372-2382

Author(s):

K M Arndt ◽

S L Ricupero ◽

D M Eisenmann ◽

F Winston

Keyword(s):

Amino Acid ◽

Dna Binding ◽

Direct Repeat ◽

Single Amino Acid ◽

Wild Type ◽

Dna Binding Specificity ◽

Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

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Sinefungin shares AdoMet-uptake system to enter Leishmania donovani promastigotes

Biochemical Journal ◽

10.1042/bj3050133 ◽

1995 ◽

Vol 305 (1) ◽

pp. 133-137 ◽

Cited By ~ 21

Author(s):

M A Phelouzat ◽

M Basselin ◽

F Lawrence ◽

M Robert-Gero

Keyword(s):

Cell Membrane ◽

Concentration Gradient ◽

Leishmania Donovani ◽

Uptake System ◽

Wild Type ◽

Saturation Kinetics ◽

Carrier Systems ◽

Resistant Cells

The involvement of a carrier for sinefungin (SF) uptake in Leishmania donovani promastigotes is indicated by saturation kinetics, competition studies and SF accumulation against a 270-fold concentration gradient across the cell membrane. Whether SF uptake occurs via nucleoside- or AdoMet-carrier systems was investigated by competition experiments and comparison of the uptake of various molecules in wild-type and SF-resistant cells. Results show that SF did not inhibit purine or pyrimidine uptake whereas it competitively inhibited AdoMet uptake. Furthermore, the uptake of nucleosides in SF-resistant cells is similar to that in wild-type cells, whereas uptake of SF and AdoMet is lower.

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