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. 2021:2192:211-226.
doi: 10.1007/978-1-0716-0834-0_16.

Sucrose Gradient Sedimentation Analysis of Mitochondrial Ribosomes

Austin Choi  1 , Antoni Barrientos  2   3
Affiliations

V体育平台登录 - Sucrose Gradient Sedimentation Analysis of Mitochondrial Ribosomes

Austin Choi et al. Methods Mol Biol. 2021.

Abstract

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 VSports手机版. 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. .

Keywords: Gradient fractionation; Gradient maker; Gradient master; Immunoblotting; Mitochondrial ribosome; Mitoribosome profile; Sucrose gradient. V体育安卓版.

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Figures

Figure 1.
Figure 1.. Sucrose gradient sedimentation analyses of the mtLSU assembly factor DDX28 and mitoribosomal proteins from wild-type (WT) HEK293T mitochondrial extracts.
(A) Sedimentation profile of mitoribosomes extracted in 5 mM EDTA or 10 mM MgCl2, (B) Sedimentation profile of mitoribosomes extracted in 5 mM EDTA and 150 or 300 mM KCl. Reproduced and modified from Tu and Barrientos, 2015 (11) with permission from Elsevier (license # 4470451296140).
Figure 2.
Figure 2.. Preparation of a continuous sucrose gradient using gradient makers.
(A) The automatized BioComp Instruments Gradient Master. This instrument is programmable, capable of producing 6 identical, linear gradients in less than 1 min. by utilizing tilted tube rotation technology. Inset depicts marker block described in section 3.2, step 3. (B) A classical manual gradient maker (C.B.S. Scientific). The device is linear with side outlets and has two chambers where the two sucrose solutions (of maximum and minimum concentrations) are placed. Gradient flow is controlled by a valve centered between the two chambers. The outflow channel is fitted with a male luer adapter, which can be used with any needle or luer valve to facilitate continuous and smooth flow into the ultracentrifugation tube. Only one gradient can be prepared at a time in some 10 min. The system includes a tubing adapter kit with a valve, tubing adapters, butterfly set, and silicone tubing. This kit can be used to adapt your gradient maker for either a gravity gradient or a pumping gradient.
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