TY - CHAP
T1 - Sucrose Gradient Sedimentation Analysis of Mitochondrial Ribosomes
AU - Choi, Austin
AU - Barrientos, Antoni
N1 - Funding Information:
We thank Dr. Priyanka Maiti for critical reading of the manuscript. This research was supported by NIH R35 Grant GM118141 (to A. B.) and MDA Grant MDA-381828 (to A.B.).
PY - 2021
Y1 - 2021
N2 - Mitochondria contain ribosomes (mitoribosomes) specialized in the synthesis of a handful of proteins essential for oxidative phosphorylation. Therefore, mitoribosome integrity and function are essential for the life of eukaryotic cells and lesions that affect them result in devastating human disorders. To broadly analyze the integrity and assembly state of mitoribosomes it is useful to start by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts. During centrifugation, mitoribosome subunits, monosomes and polysomes, and potentially accumulated assembly intermediates will sediment through the gradient at different rates. Sedimentation will depend on the centrifugal force applied and the density and viscosity of the gradient. Importantly, it will also depend on the size, shape, and density of the mitoribosome particles present in the samples under study. Variations of this technique, often coupled with additional downstream approaches, have been used to analyze the process of mitoribosome biogenesis, the composition of assembly intermediates, or to monitor the interaction of extraribosomal proteins with individual mitoribosome subunits or monosomes.
AB - Mitochondria contain ribosomes (mitoribosomes) specialized in the synthesis of a handful of proteins essential for oxidative phosphorylation. Therefore, mitoribosome integrity and function are essential for the life of eukaryotic cells and lesions that affect them result in devastating human disorders. To broadly analyze the integrity and assembly state of mitoribosomes it is useful to start by determining the sedimentation profile of these structures by sucrose gradient centrifugation of mitochondrial extracts. During centrifugation, mitoribosome subunits, monosomes and polysomes, and potentially accumulated assembly intermediates will sediment through the gradient at different rates. Sedimentation will depend on the centrifugal force applied and the density and viscosity of the gradient. Importantly, it will also depend on the size, shape, and density of the mitoribosome particles present in the samples under study. Variations of this technique, often coupled with additional downstream approaches, have been used to analyze the process of mitoribosome biogenesis, the composition of assembly intermediates, or to monitor the interaction of extraribosomal proteins with individual mitoribosome subunits or monosomes.
KW - Gradient fractionation
KW - Gradient maker
KW - Gradient master
KW - Immunoblotting
KW - Mitochondrial ribosome
KW - Mitoribosome profile
KW - Sucrose gradient
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U2 - 10.1007/978-1-0716-0834-0_16
DO - 10.1007/978-1-0716-0834-0_16
M3 - Chapter
C2 - 33230776
AN - SCOPUS:85096726045
T3 - Methods in Molecular Biology
SP - 211
EP - 226
BT - Methods in Molecular Biology
PB - Humana Press Inc.
ER -