Journal Article

Cell-free mitochondrial fusion assay detected by specific protease reaction revealed Ca2+ as regulator of mitofusin-dependent mitochondrial fusion

Naotada Ishihara, Maki Maeda, Tadato Ban and Katsuyoshi Mihara

in The Journal of Biochemistry

Volume 162, issue 4, pages 287-294
Published in print October 2017 | ISSN: 0021-924X
Published online April 2017 | e-ISSN: 1756-2651 | DOI: https://dx.doi.org/10.1093/jb/mvx029
Cell-free mitochondrial fusion assay detected by specific protease reaction revealed Ca2+ as regulator of mitofusin-dependent mitochondrial fusion

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Abstract

Mitochondrial dynamic by frequent fusion and fission have important roles in various cellular signalling processes and pathophysiology in vivo. However, the molecular mechanisms that regulate mitochondrial fusion, especially in mammalian cells, are not well understood. Accordingly, we developed a novel biochemical cell-free mitochondrial fusion assay system using isolated human mitochondria. We used a protease and its specific substrate that are essential for yeast autophagy; Atg4 protease is required for maturation and the de-conjugation of the ubiquitin-like modifier Atg8. Atg4-FLAG and Atg8-GFP were separately expressed in the mitochondrial matrix of HeLa cells. Isolated mitochondria were then mixed and packed in the presence of energy regeneration mix. Immunoblotting with an anti-GFP antibody revealed Atg8 processing, suggesting that the double membranes of isolated mitochondria were indeed fused. The mitochondrial fusion reaction required GTP hydrolysis, mitochondrial membrane potential and intact outer membrane proteins containing two mitofusin isoforms. Using this assay, we searched for stimulators of mitochondrial fusion and found that rabbit reticulocyte lysate and Ca2+ chelator EGTA stimulate mitochondrial fusion. This novel cell-free assay system using isolated human mitochondria is simple, sensitive and reproducible; thus, it is useful for screening proteins and molecules that modulate mitochondrial fusion.

Keywords: mitochondria; subcellular organelles; fusion; membrane; degradation; protein; autophagy/lysosome; proteins

Journal Article.  4478 words.  Illustrated.

Subjects: Cell Biology ; Biotechnology ; Biochemistry ; Molecular and Cell Biology

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