Rapid and specific isolation of intact mitochondria from Arabidopsis leaves

Lilan Luo^{1, 1},
Yajun He^{2, 3, 1},
Qian Xu^{4, 5, 1},
Wenhui Lyu⁶,
Jijun Yan⁷,
Peiyong Xin⁸,
Dahan Zhang^{9, 10},
Jinfang Chu¹¹,
Jiayang Li^{12, 13},
Hong Yu^{14
, ,}

1.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
2.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
3.
University of the Chinese Academy of Sciences, Beijing, 100049, China
4.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
5.
University of the Chinese Academy of Sciences, Beijing, 100049, China
6.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
7.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
8.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
9.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
10.
University of the Chinese Academy of Sciences, Beijing, 100049, China
11.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
12.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China
13.
University of the Chinese Academy of Sciences, Beijing, 100049, China
14.
State Key Laboratory of Plant Genomics, and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, The Innovative Academy of Seed Design, Chinese Academy of Sciences, Beijing, 100101, China

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Corresponding author: E-mail address: hyu@genetics.ac.cn (Hong Yu)

These authors contributed equally to this work.

摘要

These authors contributed equally to this work.

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Fig. 1. Rapid and specific isolation of intact mitochondria from Arabidopsis seedlings by affinity purification method. A: The MIMIS protein specifically localizes to mitochondria. Schematic structure of MIMIS protein is shown. Green fluorescent signal from eGFP indicates the localization of MIMIS protein in Arabidopsis seedling leaves. Mitochondria stained with MitoTrack Red are shown in red. Scale bar = 5 μm. B: Workflow for rapid isolation of mitochondria and determination of matrix metabolites. C: The affinity-purified mitochondria from Arabidopsis leaves are intact. Micrographs of beads with isolated mitochondria (GFP) and MitoTracker Red signal are shown in the merged picture. Right: magnifications of several beads with mitochondria. Scale bar = 5 μm. D: The affinity-purified mitochondria are highly pure compared to those isolated by the traditional density gradient centrifugation (DC) method. Immunoblot analysis of whole-cell lysates (whole-cell) and lysates of mitochondria captured with anti-HA beads (anti-HA AP) or DC are shown. The eGFP-OMP25 without HA tag was used as the affinity-purification control. The organelle protein markers used for immunoblot are present in the left, and their corresponding organelles are indicated in the right. ER, endoplastic reticulum. E: Absolute quantification of matrix pyridine nucleotides and ATP is highly consistent between experiments. Matrix contents of metabolites from two biological replicates each with three technical replicates were compared. F: The ATP contents in mitochondria isolated from mock (mock) or oligomycin treated (OM) Arabidopsis seedlings by DC method (left) or AP method (right). Values are means ± SD (n = 3). ** represents significant differences (P < 0.01) between mock and treated mitochondria determined by two-tailed Student'st-test. NS, no significant difference.

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