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17 results on '"Kuhnert, Franziska"'

6. Physiological functions of malate shuttles in plants and algae

Author

Dao, Ousmane, Kuhnert, Franziska, Weber, Andreas P. M., Peltier, Gilles, Li-Beisson, Yonghua, Dao, Ousmane, Kuhnert, Franziska, Weber, Andreas P. M., Peltier, Gilles, and Li-Beisson, Yonghua

Abstract

Subcellular compartmentalization confers evolutionary advantage to eukaryotic cells but entails the need for efficient interorganelle communication. Malate functions as redox carrier and metabolic intermediate. It can be shuttled across membranes through translocators. The interconversion of malate and oxaloacetate mediated by malate dehydrogenases requires oxidation/reduction of NAD (P)H/NAD(P)(+); therefore, malate trafficking serves to transport reducing equivalents and this is termed the 'malate shuttle'. Although the term 'malate shuttle' was coined more than 50 years ago, novel functions are still emerging. This review highlights recent findings on the functions of malate shuttles in photorespiration, fatty acid beta-oxidation, interorganelle signaling and its putative role in CO2-concentrating mechanisms. We compare and contrast knowledge in plants and algae, thereby providing an evolutionary perspective on redox trafficking in photosynthetic eukaryotes.

Published
2022

7. Transport Proteins Enabling Plant Photorespiratory Metabolism

Author

Kuhnert, Franziska, Schlueter, Urte, Linka, Nicole, Eisenhut, Marion, Kuhnert, Franziska, Schlueter, Urte, Linka, Nicole, and Eisenhut, Marion

Abstract

Photorespiration (PR) is a metabolic repair pathway that acts in oxygenic photosynthetic organisms to degrade a toxic product of oxygen fixation generated by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase. Within the metabolic pathway, energy is consumed and carbon dioxide released. Consequently, PR is seen as a wasteful process making it a promising target for engineering to enhance plant productivity. Transport and channel proteins connect the organelles accomplishing the PR pathway-chloroplast, peroxisome, and mitochondrion-and thus enable efficient flux of PR metabolites. Although the pathway and the enzymes catalyzing the biochemical reactions have been the focus of research for the last several decades, the knowledge about transport proteins involved in PR is still limited. This review presents a timely state of knowledge with regard to metabolite channeling in PR and the participating proteins. The significance of transporters for implementation of synthetic bypasses to PR is highlighted. As an excursion, the physiological contribution of transport proteins that are involved in C-4 metabolism is discussed.

Published
2021

10. Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria

Author

Kuhnert, Franziska, Stefanski, Anja, Overbeck, Nina, Drews, Leonie, Reichert, Andreas S., Stuehler, Kai, Weber, Andreas P. M., Kuhnert, Franziska, Stefanski, Anja, Overbeck, Nina, Drews, Leonie, Reichert, Andreas S., Stuehler, Kai, and Weber, Andreas P. M.

Abstract

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis (Arabidopsis thaliana) with a triple hemagglutinin tag for rapid purification via a single affinity-purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial Glu transporter BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.

Published
2020

12. The Photorespiratory BOU Gene Mutation Alters Sulfur Assimilation and Its Crosstalk With Carbon and Nitrogen Metabolism in Arabidopsis thaliana

Author

Samuilov, Sladjana, Brilhaus, Dominik, Rademacher, Nadine, Flachbart, Samantha, Arab, Leila, Alfarraj, Saleh, Kuhnert, Franziska, Kopriva, Stanislav, Weber, Andreas P. M., Mettler-Altmann, Tabea, and Rennenberg, Heinz

Subjects
photorespiration, food and beverages, nitrogen assimilation, Plant Science, glutathione, glycolysis, cysteine synthesis, serine production, TCA cycle, Original Research, glycine
Abstract

This study was aimed at elucidating the significance of photorespiratory serine (Ser) production for cysteine (Cys) biosynthesis. For this purpose, sulfur (S) metabolism and its crosstalk with nitrogen (N) and carbon (C) metabolism were analyzed in wildtype Arabidopsis and its photorespiratory bou-2 mutant with impaired glycine decarboxylase (GDC) activity. Foliar glycine and Ser contents were enhanced in the mutant at day and night. The high Ser levels in the mutant cannot be explained by transcript abundances of genes of the photorespiratory pathway or two alternative pathways of Ser biosynthesis. Despite enhanced foliar Ser, reduced GDC activity mediated a decline in sulfur flux into major sulfur pools in the mutant, as a result of deregulation of genes of sulfur reduction and assimilation. Still, foliar Cys and glutathione contents in the mutant were enhanced. The use of Cys for methionine and glucosinolates synthesis was reduced in the mutant. Reduced GDC activity in the mutant downregulated Calvin Cycle and nitrogen assimilation genes, upregulated key enzymes of glycolysis and the tricarboxylic acid (TCA) pathway and modified accumulation of sugars and TCA intermediates. Thus, photorespiratory Ser production can be replaced by other metabolic Ser sources, but this replacement deregulates the cross-talk between S, N, and C metabolism.

Published
2018
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15. Isolation of Plant Mitochondria Using Affinity Purification.

Author

Kuhnert F and Weber APM

Subjects
Arabidopsis genetics, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Mitochondrial Membranes, Organelles metabolism, Plants, Mitochondria
Abstract

Mitochondria are highly dynamic organelles that are vital for eukaryotic cellular metabolism. Mitochondria account only for a small fraction of the total cell volume. As such, intact organelle isolation is a basic requirement to study mitochondrial biochemistry and physiology. However, established isolation procedures are tedious, and require a substantial amount of tissue material to start with. We addressed this matter and developed a single step purification for Arabidopsis thaliana (Arabidopsis) mitochondria. Ubiquitous expression of an 3xHA-sGFP-TOM5 fusion protein in the outer mitochondrial membrane allows affinity purification by immunocapture in a single step. We here provide a detailed step-by-step protocol for rapid immunocapture of intact and physiologically active mitochondria from Arabidopsis plants., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Published
2022
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16. Rapid Single-Step Affinity Purification of HA-Tagged Plant Mitochondria.

Author

Kuhnert F, Stefanski A, Overbeck N, Drews L, Reichert AS, Stühler K, and Weber APM

Subjects
Arabidopsis genetics, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Glutamine metabolism, Hemagglutinin Glycoproteins, Influenza Virus genetics, Membrane Transport Proteins genetics, Microscopy, Confocal, Mitochondrial Membrane Transport Proteins genetics, Mitochondrial Membrane Transport Proteins metabolism, Peptide Fragments genetics, Plants, Genetically Modified, Proteome genetics, Proteome metabolism, Proteomics, Transcription Factors genetics, Transcription Factors metabolism, Arabidopsis metabolism, Chromatography, Affinity methods, Mitochondria enzymology, Mitochondria metabolism, Seedlings metabolism
Abstract

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolating mitochondria from plant cells for physiological and biochemical analyses is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis ( Arabidopsis thaliana ) with a triple hemagglutinin tag for rapid purification via a single affinity-purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial Glu transporter À BOUT DE SOUFFLE (BOU) and identified 27 differentially expressed mitochondrial proteins compared with tagged Col-0 controls. Our work sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Published
2020
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17. In Vitro Analysis of Metabolite Transport Proteins.

Author

Roell MS, Kuhnert F, Zamani-Nour S, and Weber APM

Subjects
Arabidopsis chemistry, Arabidopsis genetics, Cell Membrane chemistry, Chloroplasts chemistry, Chloroplasts genetics, Chromatography, Gel methods, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genetic Vectors chemistry, Genetic Vectors metabolism, Membrane Transport Proteins chemistry, Membrane Transport Proteins genetics, Membrane Transport Proteins metabolism, Oryza chemistry, Oryza genetics, Oryza metabolism, Oxygen Consumption physiology, Pisum sativum chemistry, Pisum sativum genetics, Pisum sativum metabolism, Phosphatidylcholines chemistry, Photosynthesis physiology, Plant Leaves chemistry, Plant Leaves genetics, Plant Leaves metabolism, Plant Proteins chemistry, Plant Proteins genetics, Plant Proteins metabolism, Proteolipids chemistry, Proteolipids metabolism, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Triticum chemistry, Triticum genetics, Triticum metabolism, Arabidopsis metabolism, Biological Assay, Chloroplasts metabolism, Gene Expression Regulation, Plant, Membrane Transport Proteins isolation & purification, Plant Proteins isolation & purification
Abstract

The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges.Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.

Published
2017
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