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Article

Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

1
Max Planck Institute of Molecular Plant Physiology, 14476 Potsdam-Golm, Germany
2
School of BioSciences, University of Melbourne, Melbourne, VIC 3010, Australia
3
Heidelberg University, Biochemie-Zentrum, Nuclear Pore Complex and Ribosome Assembly, 69120 Heidelberg, Germany
*
Author to whom correspondence should be addressed.
Plants 2020, 9(7), 892; https://doi.org/10.3390/plants9070892
Received: 8 May 2020 / Revised: 24 June 2020 / Accepted: 9 July 2020 / Published: 14 July 2020
(This article belongs to the Special Issue Plant Ribosome Biogenesis)
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 Arabidopsis thaliana 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. View Full-Text
Keywords: in vivo protein complex stabilization; plant proteomics; ribo-proteome; ribosome complexes; tissue specific separation in vivo protein complex stabilization; plant proteomics; ribo-proteome; ribosome complexes; tissue specific separation
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MDPI and ACS Style

Firmino, A.A.P.; Gorka, M.; Graf, A.; Skirycz, A.; Martinez-Seidel, F.; Zander, K.; Kopka, J.; Beine-Golovchuk, O. Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes. Plants 2020, 9, 892. https://doi.org/10.3390/plants9070892

AMA Style

Firmino AAP, Gorka M, Graf A, Skirycz A, Martinez-Seidel F, Zander K, Kopka J, Beine-Golovchuk O. Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes. Plants. 2020; 9(7):892. https://doi.org/10.3390/plants9070892

Chicago/Turabian Style

Firmino, Alexandre A.P.; Gorka, Michal; Graf, Alexander; Skirycz, Aleksandra; Martinez-Seidel, Federico; Zander, Kerstin; Kopka, Joachim; Beine-Golovchuk, Olga. 2020. "Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes" Plants 9, no. 7: 892. https://doi.org/10.3390/plants9070892

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