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. 2017 May 5;7(9):e2266.
doi: 10.21769/BioProtoc.2266.

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Yoshihiro Adachi  1 , Kie Itoh  1 , Miho Iijima  1 , Hiromi Sesaki  1
Affiliations

Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes

Yoshihiro Adachi et al. Bio Protoc. .

Abstract

A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.

Keywords: Dynamin superfamily; Mitochondria; Organelle division; Phospholipids.

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Figures

Figure 1.
Figure 1.. Analysis of purified Drp1 by SDS-PAGE and Coomassie Brilliant Blue staining
Figure 2.
Figure 2.. Confocal fluorescence microscopy of liposomes
Figure 3.
Figure 3.. Drp1 and liposomes are incubated and analyzed by sucrose gradient centrifugation.
Amounts of Drp1 that co-migrate with liposomes into fractions 1 and 2 are quantified.
See this image and copyright information in PMC

References

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