Published: Vol 7, Iss 9, May 5, 2017 DOI: 10.21769/BioProtoc.2266 Views: 10051
Reviewed by: Nicoletta CordaniJianwei SunDaniel Kraus
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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: MitochondriaBackground
Interactions of proteins and membrane lipids are critical for the remodeling of biological membranes in cells such as organelle division. In mitochondrial division, Drp1 constricts the mitochondrial membranes and drives this membrane remodeling process. We have recently shown that a signaling phospholipid, phosphatidic acid, interacts with Drp1 and creates the priming step by restraining assembled division machinery on mitochondria (Adachi et al., 2016). Drp1 recognizes both the head group and acyl chains of phosphatidic acid. To analyze Drp1-phosphatidic acid binding, we set up several protein-lipid interaction assays, including a lipid dot blot assay, a competition assay and a liposome flotation assay. These assays allowed us to determine the function of the head group and acyl chain composition in protein-lipid interactions. In addition, we analyzed lipids under bilayer conditions in the liposome assay and lipids under non-bilayer conditions in dot blot and competition assays, which made it possible to examine the contribution of the membrane curvature and lipid packing to protein-lipid interactions. Below, we describe a liposome flotation assay that can be applied to many peripheral membrane proteins.
Materials and Reagents
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Data analysis
Notes
When loading the sample on SDS-page, please warm the sample with a heat block at 37 °C. Because the sample contains sucrose, the fluidity of the sample changes depending on the temperature.
Recipes
Acknowledgments
We thank past and present members of the Iijima and Sesaki labs for helpful discussions and technical assistance. This work was supported by NIH grants to M.I. (GM084015) and H.S. (GM089853).
References
Article Information
Copyright
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Adachi, Y., Itoh, K., Iijima, M. and Sesaki, H. (2017). Assay to Measure Interactions between Purified Drp1 and Synthetic Liposomes. Bio-protocol 7(9): e2266. DOI: 10.21769/BioProtoc.2266.
Category
Biochemistry > Lipid > Lipid-protein interaction
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