Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from leaf, root, and seed tissues by optimized sucrose density gradient centrifugation o...

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Bibliographic Details
Published in:Plants (Basel) 2020-07, Vol.9 (7), p.892
Main Authors: Firmino, Alexandre Augusto Pereira, Gorka, Michal, Graf, Alexander, Skirycz, Aleksandra, Martinez-Seidel, Federico, Zander, Kerstin, Kopka, Joachim, Beine-Golovchuk, Olga
Format: Article
Language:English
Subjects:
Biosynthesis
Centrifugation
Chloroplasts
Composition
Crosslinking
Cytoplasm
Flowers & plants
Fractionation
in vivo protein complex stabilization
Leaves
Mammals
Microfluidics
Plant extracts
plant proteomics
Plant tissues
Polyribosomes
Protected plants
Protein composition
Proteins
Proteomes
Proteomics
ribo-proteome
ribosome complexes
Ribosomes
rRNA
Sedimentation
Seeds
Sucrose
Sugar
tissue specific separation
Yeast
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Summary:Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.
ISSN:2223-7747
2223-7747
DOI:10.3390/plants9070892