Triton-stimulated nucleoside diphosphatase: Characterization

Nagahashi, Jerry; Nagahashi, Susan L.

doi:10.1007/bf01284091

Cited by 34 publications

(28 citation statements)

References 20 publications

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“…13 shows that the GFP distribution profile very closely resembled that of latent inosine diphosphatase activity. Because latent inosine diphosphatase is exclusively associated with Golgi membranes (36), this result further supports the Golgi localization of MTP11.…”

Section: Arabidopsis and Poplar Mtp11 Proteins Are Targeted To A Golgsupporting

confidence: 67%

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

Peiter

Montanini

Gobert

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

253

262

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Manganese toxicity is a major problem for plant growth in acidic soils, but cellular mechanisms that facilitate growth in such conditions have not been clearly delineated. Established mechanisms that counter metal toxicity in plants involve chelation and cytoplasmic export of the metal across the plasma or vacuolar membranes out of the cell or sequestered into a large organelle, respectively. We report here that expression of the Arabidopsis and poplar MTP11 cation diffusion facilitators in a manganesehypersensitive yeast mutant restores manganese tolerance to wild-type levels. Microsomes from yeast expressing AtMTP11 exhibit enhanced manganese uptake. In accord with a presumed function of MTP11 in manganese tolerance, Arabidopsis mtp11 mutants are hypersensitive to elevated levels of manganese, whereas plants overexpressing MTP11 are hypertolerant. In contrast, sensitivity to manganese deficiency is slightly decreased in mutants and increased in overexpressing lines. Promoter-GUS studies showed that AtMTP11 is most highly expressed in root tips, shoot margins, and hydathodes, but not in epidermal cells and trichomes, which are generally associated with manganese accumulation. Surprisingly, imaging of MTP11-EYFP fusions demonstrated that MTP11 localizes neither to the plasma membrane nor to the vacuole, but to a punctate endomembrane compartment that largely coincides with the distribution of the trans-Golgi marker sialyl transferase. Golgi-based manganese accumulation might therefore result in manganese tolerance through vesicular trafficking and exocytosis. In accord with this proposal, Arabidopsis mtp11 mutants exhibit enhanced manganese concentrations in shoots and roots. We propose that Golgi-mediated exocytosis comprises a conserved mechanism for heavy metal tolerance in plants.Golgi ͉ heavy metal transport ͉ metal tolerance protein ͉ metal trafficking ͉ manganese transporter T ransition metals are required by living systems where they perform a wide variety of functions as cofactors for enzymes and transcription factors. Transition metals are also present in many environments at potentially toxic concentrations, and this has led to the evolution of mechanisms that counter toxicity. In plants exposed to high concentrations of transition metals in the soil, binding of the metals to phytochelatins in the cytosol lowers metal activity (1). Additionally, metals can be removed from the cytosol through the action of metal transporters. Transporters involved in metal tolerance localize to the plasma membrane, thereby removing metals from the cell, or to the vacuolar membrane, where the metal can be sequestered into a large and metabolically relatively inert intracellular compartment (2).Manganese is the second most prevalent transition metal, after iron, in the Earth's crust and an essential micronutrient for all organisms, including humans and plants (3). In addition to being a cofactor for a variety of enzymes (including various decarboxylases of the tricarboxylic acid cycle, RNA polymerases, and numerous glyco...

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Section: Arabidopsis and Poplar Mtp11 Proteins Are Targeted To A Golgsupporting

confidence: 67%

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

Peiter

Montanini

Gobert

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

253

262

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“…Thylakoid membranes were identified by spectrophotometric analysis of the chlorophyll content (Arnon, 1949). The identification of Golgi membranes was based on the measurement of Triton X-100-stimulated latent UDPase activity (Nagahashi and Nagahashi, 1982).…”

Section: Membrane Markersmentioning

confidence: 99%

The Cytokinin Receptors of Arabidopsis Are Located Mainly to the Endoplasmic Reticulum

et al. 2011

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The plant hormone cytokinin is perceived by membrane-located sensor histidine kinases. Arabidopsis (Arabidopsis thaliana) possesses three cytokinin receptors: ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and CYTOKININ RESPONSE1/ AHK4. The current model predicts perception of the cytokinin signal at the plasma membrane. However, cytokinin-binding studies with membrane fractions separated by two-phase partitioning showed that in the wild type, as well as in mutants retaining only single cytokinin receptors, the major part of specific cytokinin binding was associated with endomembranes. Leaf epidermal cells of tobacco (Nicotiana benthamiana) expressing receptor-green fluorescent protein fusion proteins and bimolecular fluorescence complementation analysis showed strong fluorescence of the endoplasmic reticulum (ER) network for all three receptors. Furthermore, separation of the microsomal fraction of Arabidopsis plants expressing Myc-tagged AHK2 and AHK3 receptors by sucrose gradient centrifugation followed by immunoblotting displayed the Mg 2+ -dependent density shift typical of ER membrane proteins. Cytokinin-binding assays, fluorescent fusion proteins, and biochemical fractionation all showed that the large majority of cytokinin receptors are localized to the ER, suggesting a central role of this compartment in cytokinin signaling. A modified model for cytokinin signaling is proposed.

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“…Nucleoside diphosphatase was used as a marker for golgi (18). The specific activity of the nucleoside diphosphatase was highest in the second 10,000g pellet, but was also high in the 10,000 to 80,000g microsomal pellet and in the dextran interface fraction.…”

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confidence: 99%

“…Antimycin A insensitive NADH Cyt c reductase and Cyt c oxidase were assayed according to the method of Hodges and Leonard (10) except that 0.1% Triton X-100 was included during the Cyt c oxidase assay, and appeared to be much more effective than digitonin in stimulating activity. Nucleoside diphosphatase was assayed in the same manner as the ATPase activity, using 1.5 mm UDP or GDP as substrate plus 1.5 mM MnCl2 in 25 mm Bis-Tris-propane/Mes (pH 6.75) at 26°C (18). RESULTS Balance Sheet.…”

mentioning

confidence: 99%

Localization of a Proton-Translocating ATPase on Sucrose Gradients

DuPont

Bennett²,

Spanswick³

1982

Plant Physiol.

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lonophore-stimulated ATPase activity and ATP-dependent quinacrine quench were enriched in parallel when microsomal vesicles were prepared from corn (Crow Single Cross Hybrid WF9-Mo17) roots and collected on a cushion of 10% dextran. Activities were highest in the apical 1.5 centimeters of the roots. Vesicles collected on the dextran cushion also contained NADH cytochrome c reductase (enriched in the apical 0.5 cm of the root) and nucleoside diphosphatase (distributed throughout the first four cm). On continuous sucrose gradients, ATP-dependent proton transport and ionophore-stimulated ATPase In a previous paper (5) we described a proton-translocating ATPase in membranes from corn roots. It was stimulated by C1-and by ionophores, did not appear to be of mitochondrial origin, as it was not inhibited by oligomycin, and differed from the previously described plasma membrane ATPase of corn roots in that it was unaffected by monovalent cations, and was not inhibited by vanadate. Sze (23,24) suggested that similar vesicles obtained from tobacco callus originated from the plasma membrane and that the ionophore-stimulated ATPase, ATP-dependent methylamine uptake, and ATP-dependent SCN-uptake are the function of a proton translocating ATPase located on the plasma membrane. In this paper we describe preliminary attempts to determine the origin of the vesicles. We present evidence that the proton-translocating ATPase of corn root microsomal membranes is not of plasma membrane origin, and present evidence that the enzyme is likely to be of tonoplast origin. Similar findings have recently been reported for corn coleoptiles (8,9,15,17 Preparation of Vesicles. Sealed microsomal vesicles were prepared by a modification of the method of Sze (23). Corn seeds were germinated between damp paper towels in plastic trays. Root tips (approximately 15 mm in length) of primary and secondary roots of 4-to 5-day-old seedlings were excised into aerated 0.1 mM CaCl2 at room temperature. Roots were ground with a chilled mortar and pestle. The homogenization buffer consisted of 0.25 M sorbitol, 2 mm EGTA, 0.1% f8-mercaptoethanol, 0.1% BSA, and 25 mm Bis-Tris-propane adjusted to pH 7.5 with solid Mes. Approximately 10 ml of buffer/g fresh weight of root was used. The homogenate was centrifuged at 10,000g for 10 min, the mitochondrial pellet discarded, the supernatant centrifuged again at 10,000g for 10 min, the pellet discarded, and the supernatant centrifuged at 80,000g for 30 min to prepare the microsomal pellet. The microsomal pellet was suspended in suspension buffer consisting of 0.25 M sorbitol and 2.5 mm Bis-Tris-propane/Mes (pH 7.2), underlain with 8 ml of 10%o dextran T-70 in the same buffer, and centrifuged at 80,000g for 1 h. The cloudy interface (approximately 50%o of the protein applied to the gradient) was collected from the dextran cushion, and vesicles were stored on ice for 1 to 6 h before use. Vesicles showed little loss of activity for up to 10 h, and activity was preserved for several days if vesicles were frozen i...

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Triton-stimulated nucleoside diphosphatase: Characterization

Cited by 34 publications

References 20 publications

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

A secretory pathway-localized cation diffusion facilitator confers plant manganese tolerance

The Cytokinin Receptors of Arabidopsis Are Located Mainly to the Endoplasmic Reticulum

Localization of a Proton-Translocating ATPase on Sucrose Gradients

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