Sample Preparation for Analysis of the Plant Mitochondrial Membrane Proteome

Abstract

Containing plastids and vacuoles in addition to those organelles also found in other (heterotrophic) cells, the plant cell displays an extraordinary level of compartmentalization, largely obtained by the utilization of membranes. These membranes not only confine reaction spaces but must also facilitate cross-talk between organelles and other cell compartments. They also host important components of the plant energy metabolism, i.e., the electron transport chains of mitochondria and chloroplasts. Characterization of the proteomes of these membranes requires isolation of pure and intact organelles from plant tissues followed by subsequent purification of their respective membranes. Membrane fractions are then amenable for further analyses using gel electrophoresis procedures or gel-free proteomic approaches. Here, we describe the preparation of intact mitochondria from Arabidopsis thaliana cell-culture, the isolation of outer and inner mitochondrial membranes and downstream proteomic applications for analyzing their membrane protein content.

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

“…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.…”

Protein Extraction Methods Shape Much of the Extracted Proteomes

“…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.…”

The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum

“…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.…”

“…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.…”