Medicago truncatulaMolybdate Transporter type 1 (MtMOT1.3) is a plasma membrane molybdenum transporter required for nitrogenase activity in root nodules under molybdenum deficiency

SummaryMolybdenum, as a component of the iron-molybdenum cofactor of nitrogenase, is essential for symbiotic nitrogen fixation. This nutrient has to be provided by the host plant through molybdate transporters.Members of the molybdate transporters family MOT1 were identified in the model legumeMedicago truncatulaand their expression in nodules determined. Yeast toxicity assays, confocal microscopy, and phenotypical characterization of aTnt1insertional mutant line were carried out in the oneM. truncatulaMOT1 family member expressed specifically in nodules.Among the five MOT1 members present inM. truncatulagenome,MtMOT1.3is the only one uniquely expressed in nodules. MtMOT1.3 shows molybdate transport capabilities when expressed in yeast. Immunolocalization studies revealed that MtMOT1.3 is located in the plasma membrane of nodule cells. Amot1.3-1knockout mutant showed an impaired growth concomitant with a reduction in nitrogenase activity. This phenotype was rescued by increasing molybdate concentrations in the nutritive solution, or upon addition of an assimilable nitrogen source. Furthermore,mot1.3-1plants transformed with a functional copy ofMtMOT1.3showed a wild type-like phenotype.These data are consistent with a model in which MtMOT1.3 would be responsible for introducing molybdate into nodule cells, which will be later used to synthesize functional nitrogenase.

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Paraburkholderia phymatum Homocitrate Synthase NifV Plays a Key Role for Nitrogenase Activity during Symbiosis with Papilionoids and in Free-Living Growth Conditions

Cells ◽

10.3390/cells10040952 ◽

2021 ◽

Vol 10 (4) ◽

pp. 952

Author(s):

Paula Bellés-Sancho ◽

Martina Lardi ◽

Yilei Liu ◽

Sebastian Hug ◽

Marta Adriana Pinto-Carbó ◽

...

Keyword(s):

Nitrogenase Activity ◽

Root Nodules ◽

Growth Conditions ◽

Lysine Biosynthesis ◽

Metabolome Analysis ◽

Plant Host ◽

Free Living ◽

Bacterial Enzyme ◽

Homocitrate Synthase ◽

Iron Molybdenum Cofactor

Homocitrate is an essential component of the iron-molybdenum cofactor of nitrogenase, the bacterial enzyme that catalyzes the reduction of dinitrogen (N2) to ammonia. In nitrogen-fixing and nodulating alpha-rhizobia, homocitrate is usually provided to bacteroids in root nodules by their plant host. In contrast, non-nodulating free-living diazotrophs encode the homocitrate synthase (NifV) and reduce N2 in nitrogen-limiting free-living conditions. Paraburkholderia phymatum STM815 is a beta-rhizobial strain, which can enter symbiosis with a broad range of legumes, including papilionoids and mimosoids. In contrast to most alpha-rhizobia, which lack nifV, P. phymatum harbors a copy of nifV on its symbiotic plasmid. We show here that P. phymatum nifV is essential for nitrogenase activity both in root nodules of papilionoid plants and in free-living growth conditions. Notably, nifV was dispensable in nodules of Mimosa pudica despite the fact that the gene was highly expressed during symbiosis with all tested papilionoid and mimosoid plants. A metabolome analysis of papilionoid and mimosoid root nodules infected with the P. phymatum wild-type strain revealed that among the approximately 400 measured metabolites, homocitrate and other metabolites involved in lysine biosynthesis and degradation have accumulated in all plant nodules compared to uninfected roots, suggesting an important role of these metabolites during symbiosis.

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Diffusion Limitation of Oxygen Uptake and Nitrogenase Activity in the Root Nodules of Parasponia rigida Merr. and Perry

PLANT PHYSIOLOGY ◽

10.1104/pp.69.3.728 ◽

1982 ◽

Vol 69 (3) ◽

pp. 728-733 ◽

Cited By ~ 26

Author(s):

John D. Tjepkema ◽

Robert J. Cartica

Keyword(s):

Oxygen Uptake ◽

Nitrogenase Activity ◽

Root Nodules ◽

Diffusion Limitation

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Carbon Costs of Nitrogenase Activity in Legume Root Nodules determined using Acetylene and Oxygen

Journal of Experimental Botany ◽

10.1093/jxb/34.8.951 ◽

1983 ◽

Vol 34 (8) ◽

pp. 951-963 ◽

Cited By ~ 62

Author(s):

J. F. WITTY ◽

F. R. MINCHIN ◽

J. E. SHEEHY

Keyword(s):

Nitrogenase Activity ◽

Root Nodules ◽

Carbon Costs

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Seasonal pattern of energy use, respiration, and nitrogenase activity in root nodules of Myrica gale

Canadian Journal of Botany ◽

10.1139/b83-328 ◽

1983 ◽

Vol 61 (11) ◽

pp. 2937-2942 ◽

Cited By ~ 14

Author(s):

Christa R. Schwintzer ◽

John D. Tjepkema

Keyword(s):

Energy Cost ◽

Energy Use ◽

Nitrogenase Activity ◽

Root Nodules ◽

Seasonal Pattern ◽

Uptake Ratio ◽

Experimental Conditions ◽

Myrica Gale ◽

Net Production ◽

N Requirement

Annual CO2 evolution, H2 evolution, and C2H2 reduction were measured in root nodules from a vigorous Myrica gale stand in a Massachusetts peatland at 3-week intervals in 1980. Nodule activity was approximately the same under the experimental conditions (excised nodules reducing C2H2) as in nature (attached nodules reducing N2) and the CO2 evolution to O2 uptake ratio averaged 1.07. Nitrogenase activity was first detectable in late May, reached its maximum [Formula: see text] in mid-July, and disappeared in late October. The seasonal pattern of CO2 evolution was similar except that it continued at low rates when nitrogenase activity was absent. Hydrogen evolution was barely detectable. The energy cost of nitrogen fixation, expressed as the molar CO2:C2H4 ratio, was relatively low [Formula: see text] throughout the period of substantial nitrogenase activity and had a mean annual value of 4.9. Annual N2 fixation was estimated to be 2.8 g N m−2year−1, contributing about 33% of the annual N requirement measured in 1979. Annual C use by nodules was about 21.0 g C m−2 year−1. If this C were available for additional net production, it would increase it by about 5.5%.

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Role of the Gag Matrix Domain in Targeting Human Immunodeficiency Virus Type 1 Assembly

Journal of Virology ◽

10.1128/jvi.74.6.2855-2866.2000 ◽

2000 ◽

Vol 74 (6) ◽

pp. 2855-2866 ◽

Cited By ~ 185

Author(s):

Akira Ono ◽

Jan M. Orenstein ◽

Eric O. Freed

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Virus Particle ◽

Particle Formation ◽

Membrane Targeting ◽

Basic Domain ◽

Golgi Vesicles ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) particle formation and the subsequent initiation of protease-mediated maturation occur predominantly on the plasma membrane. However, the mechanism by which HIV-1 assembly is targeted specifically to the plasma membrane versus intracellular membranes is largely unknown. Previously, we observed that mutations between residues 84 and 88 of the matrix (MA) domain of HIV-1 Gag cause a retargeting of virus particle formation to an intracellular site. In this study, we demonstrate that the mutant virus assembly occurs in the Golgi or in post-Golgi vesicles. These particles undergo core condensation in a protease-dependent manner, indicating that virus maturation can occur not only on the plasma membrane but also in the Golgi or post-Golgi vesicles. The intracellular assembly of mutant particles is dependent on Gag myristylation but is not influenced by p6Gag or envelope glycoprotein expression. Previous characterization of viral revertants suggested a functional relationship between the highly basic domain of MA (amino acids 17 to 31) and residues 84 to 88. We now demonstrate that mutations in the highly basic domain also retarget virus particle formation to the Golgi or post-Golgi vesicles. Although the basic domain has been implicated in Gag membrane binding, no correlation was observed between the impact of mutations on membrane binding and Gag targeting, indicating that these two functions of MA are genetically separable. Plasma membrane targeting of Gag proteins with mutations in either the basic domain or between residues 84 and 88 was rescued by coexpression with wild-type Gag; however, the two groups of MA mutants could not rescue each other. We propose that the highly basic domain of MA contains a major determinant of HIV-1 Gag plasma membrane targeting and that mutations between residues 84 and 88 disrupt plasma membrane targeting through an effect on the basic domain.

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Requirement for an Intact T-Cell Actin and Tubulin Cytoskeleton for Efficient Assembly and Spread of Human Immunodeficiency Virus Type 1

Journal of Virology ◽

10.1128/jvi.01469-06 ◽

2007 ◽

Vol 81 (11) ◽

pp. 5547-5560 ◽

Cited By ~ 134

Author(s):

Clare Jolly ◽

Ivonne Mitar ◽

Quentin J. Sattentau

Keyword(s):

Human Immunodeficiency Virus ◽

T Cells ◽

Plasma Membrane ◽

T Cell ◽

Immunodeficiency Virus ◽

Fixed Cell ◽

Cell Spread ◽

Hiv 1 ◽

Cell Cell

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection of CD4+ T cells leads to the production of new virions that assemble at the plasma membrane. Gag and Env accumulate in the context of lipid rafts at the inner and outer leaflets of the plasma membrane, respectively, forming polarized domains from which HIV-1 buds. HIV-1 budding can result in either release of cell-free virions or direct cell-cell spread via a virological synapse (VS). The recruitment of Gag and Env to these plasma membrane caps in T cells is poorly understood but may require elements of the T-cell secretory apparatus coordinated by the cytoskeleton. Using fixed-cell immunofluorescence labeling and confocal microscopy, we observed a high percentage of HIV-1-infected T cells with polarized Env and Gag in capped, lipid raft-like assembly domains. Treatment of infected T cells with inhibitors of actin or tubulin remodeling disrupted Gag and Env compartmentalization within the polarized raft-like domains. Depolymerization of the actin cytoskeleton reduced Gag release and viral infectivity, and actin and tubulin inhibitors reduced Env incorporation into virions. Live- and fixed-cell confocal imaging and assay of de novo DNA synthesis by real-time PCR allowed quantification of HIV-1 cell-cell transfer. Inhibition of actin and tubulin remodeling in infected cells interfered with cell-cell spread across a VS and reduced new viral DNA synthesis. Based on these data, we propose that HIV-1 requires both actin and tubulin components of the T-cell cytoskeleton to direct its assembly and budding and to elaborate a functional VS.

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Opposing Effects of Human Immunodeficiency Virus Type 1 Matrix Mutations Support a Myristyl Switch Model of Gag Membrane Targeting

Journal of Virology ◽

10.1128/jvi.73.4.2604-2612.1999 ◽

1999 ◽

Vol 73 (4) ◽

pp. 2604-2612 ◽

Cited By ~ 117

Author(s):

Jean-Christophe Paillart ◽

Heinrich G. Göttlinger

Keyword(s):

Human Immunodeficiency Virus ◽

Plasma Membrane ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Virus Assembly ◽

Membrane Binding ◽

Single Amino Acid ◽

Immunodeficiency Virus ◽

N Terminus

ABSTRACT Targeting of the human immunodeficiency virus type 1 (HIV-1) Gag precursor Pr55 gag to the plasma membrane, the site of virus assembly, is primarily mediated by the N-terminal matrix (MA) domain. N-myristylation of MA is essential for the stable association of Pr55 gag with membranes and for virus assembly. We now show that single amino acid substitutions near the N terminus of MA can dramatically impair assembly without compromising myristylation. Subcellular fractionation demonstrated that Gag membrane binding was compromised to a similar extent as in the absence of the myristyl acceptor site, indicating that the myristyl group was not available for membrane insertion. Remarkably, the effects of the N-terminal modifications could be completely suppressed by second-site mutations in the globular core of MA. The compensatory mutations enhanced Gag membrane binding and increased viral particle yields above wild-type levels, consistent with an increase in the exposure of the myristyl group. Our results support a model in which the compact globular core of MA sequesters the myristyl group to prevent aberrant binding to intracellular membranes, while the N terminus is critical to allow the controlled exposure of the myristyl group for insertion into the plasma membrane.

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Internalisation of the type I angiotensin II receptor (ATI) and angiotensin II function in the rat adrenal zona glomerulosa cell.

Journal of Endocrinology ◽

10.1677/joe.0.141r005 ◽

1994 ◽

Vol 141 (2) ◽

pp. R5-R9 ◽

Cited By ~ 10

Author(s):

G. P. Vinson ◽

M. M. Ho. ◽

J.R. Puddefoot ◽

R. Teja ◽

S. Barker

Keyword(s):

Monoclonal Antibody ◽

Plasma Membrane ◽

Angiotensin Ii ◽

Zona Glomerulosa ◽

Type I ◽

Specific Monoclonal Antibody ◽

Angiotensin Ii Receptor ◽

Cellular Localisation ◽

Glomerulosa Cells

ABSTRACT Little is known about the cellular localisation of the angiotensin II (AII) type 1 receptor (ATI) in the rat adrenal glomerulosa cell, but some studies have suggested that receptor internalisation and recycling may occur. Using a specific monoclonal antibody (6313/G2) to the first extracellular domain, we show here that most of the receptor is internalised in the unstimulated cell. When viable glomerulosa cells are incubated with 6313/G2, the receptor is transiently concentrated on the cell surface, and aldosterone output is stimulated. This stimulated output is enhanced by neither threshold nor maximal stimulatory concentrations of All amide, although the antibody does not inhibit All binding to the receptor. Conversely, the stimulatory actions of the antibody and those of ACTH are additive. The data suggest that recycling to the plasma membrane is constitutive, or regulated by unknown factors. Retention of the ATI receptor in the membrane is alone enough to allow sufficient G protein interaction to generate maximal stimulatory events.

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