Sample Preparation for Analysis of the Plant Mitochondrial Membrane Proteome

Schikowsky, Christine; Thal, Beate; Braun, Hans‐Peter; Eubel, Holger

doi:10.1007/978-1-4939-7411-5_11

Cited by 6 publications

(6 citation statements)

References 16 publications

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“…The mitochondria isolation procedure was described previously ( Schikowsky et al, 2018 ). About 200 g of 4-week-old Arabidopsis rosettes were collected (three independent isolations for combined lipidomics and proteomics analysis and three more independent isolations for lipid quantification by TLC–GC/FID analysis) and homogenized at 4°C with a waring blender in 1 L disruption buffer (0.3 m sucrose, 60 m m TES, 25 m m tetrasodium pyrophosphate, 10 m m potassium dihydrogen phosphate, 2 m m EDTA, 1 m m glycine, 1% PVP40, 1% BSA, 50 m m sodium ascorbate, 20 m m cysteine; pH 8.0) by three times for 10 s with 30 s intervals.…”

Section: Methodsmentioning

confidence: 99%

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and biosynthesis capacity

Liu¹,

Senkler

Herrfurth

et al. 2023

Plant Physiology

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Mitochondria are often considered the power stations of the cell, playing critical roles in various biological processes such as cellular respiration, photosynthesis, stress responses and programmed cell death. To maintain the structural and functional integrities of mitochondria, it is crucial to achieve a defined membrane lipid composition between different lipid classes wherein specific proportions of individual lipid species are present. Although mitochondria are capable of self-synthesizing a few lipid classes, many phospholipids are synthesized in the endoplasmic reticulum and transferred to mitochondria via membrane contact sites, as mitochondria are excluded from the vesicular transportation pathway. However, knowledge on the capability of lipid biosynthesis in mitochondria and the precise mechanism of maintaining the homeostasis of mitochondrial lipids is still scarce. Here we describe the lipidome of mitochondria isolated from Arabidopsis (Arabidopsis thaliana) leaves, including the molecular species of glycerolipids, sphingolipids and sterols, to depict the lipid landscape of mitochondrial membranes. In addition, we define proteins involved in lipid metabolism by proteomic analysis and compare our data with mitochondria from cell cultures since they still serve as model systems. Proteins putatively localized to the membrane contact sites are proposed based on the proteomic results and online databases. Collectively, our results suggest that leaf mitochondria are capable - with the assistance of membrane contact site-localized proteins - of generating several lipid classes including phosphatidylethanolamines, cardiolipins, diacylgalactosylglycerols and free sterols. We anticipate our work to be a foundation to further investigate the functional roles of lipids and their involvement in biochemical reactions in plant mitochondria.

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Section: Methodsmentioning

confidence: 99%

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and biosynthesis capacity

Liu¹,

Senkler

Herrfurth

et al. 2023

Plant Physiology

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“…For the organelles with complex structures, the separate extraction of proteins from each suborganelle fraction enables producing more detailed subproteome profiles. For example, subproteomic analysis involving the isolation of Arabidopsis chloroplasts as stroma, thylakoid membrane, and lumen fractions (Hall et al, 2011 ) and the separate isolation of inner and outer mitochondrial membrane fractions (Duncan et al, 2011 ; Schikowsky et al, 2018 ) have also provided information about the specific localization of proteins within the organelles.…”

Section: Protein Extraction For Organelle Proteomicsmentioning

confidence: 99%

Protein Extraction Methods Shape Much of the Extracted Proteomes

et al. 2018

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“…2A ). Second, enrichment of nuclei then involved ultracentrifugation with a three-layer Percoll-sucrose gradient as previously described for organelle enrichment in Arabidopsis thaliana ( 69 ). The nuclei-containing fraction was then further purified by another round of ultracentrifugation using a specifically designed five-layer Percoll-sucrose gradient ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…For cell disruption, the samples were sonicated for 1 minute (Branson Ultrasonics Sonifier 250 CE; ThermoFisher Scientific) with an output of 30% and a duty cycle of 25% on ice. Then, the sample was subjected to a first fractionation by a three-layer discontinuous Percoll-sucrose gradient (70,000 g, 4°C, 90 minutes; Beckmann Avanti J 25.5; Beckmann Coulter, Krefeld, Germany) according to Schikowsky et al ( 69 ). Further enrichment and purification of nuclei was achieved by a five-layer discontinuous Percoll-sucrose gradient.…”

Section: Methodsmentioning

confidence: 99%

The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum

Kalvelage,

Wöhlbrand,

Schoon

et al. 2023

mSphere

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The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum ) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum . High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4–6.7 µm 3 ), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

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Sample Preparation for Analysis of the Plant Mitochondrial Membrane Proteome

Cited by 6 publications

References 16 publications

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and biosynthesis capacity

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and biosynthesis capacity

Protein Extraction Methods Shape Much of the Extracted Proteomes

The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum

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