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Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae

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Dynamin Superfamily GTPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2159))

Abstract

Mitochondrial fission, an essential process for mitochondrial and cellular homeostasis, is accomplished by evolutionarily conserved members of the dynamin superfamily of large GTPases. These enzymes couple the hydrolysis of guanosine triphosphate to the mechanical work of membrane remodeling that ultimately leads to membrane scission. The importance of mitochondrial dynamins is exemplified by mutations in the human family member that causes neonatal lethality. In this chapter, we describe the subcloning, purification, and preliminary characterization of the budding yeast mitochondrial dynamin, DNM1, from Saccharomyces cerevisiae, which is the first mitochondrial dynamin isolated from native sources. The yeast-purified enzyme exhibits assembly-stimulated hydrolysis of GTP similar to other fission dynamins, but differs from the enzyme isolated from non-native sources.

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Acknowledgement

The reagents pEG(KT), SEY6210, and DDY1810 were kind gifts from B. Wendland. This work was supported by the National Institutes of Health grant R01GM067180.

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Author notes

  1. Nolan W. Kennedy and Lora K. Picton contributed equally to this work.

Authors and Affiliations

  1. Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, USA

    Nolan W. Kennedy, Lora K. Picton & R. Blake Hill

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  1. Nolan W. Kennedy
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  2. Lora K. Picton
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Correspondence to R. Blake Hill .

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Editors and Affiliations

  1. School of Medicine, Case Western Reserve University, Cleveland, OH, USA

    Rajesh Ramachandran

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Kennedy, N.W., Picton, L.K., Hill, R.B. (2020). Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae. In: Ramachandran, R. (eds) Dynamin Superfamily GTPases. Methods in Molecular Biology, vol 2159. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0676-6_1

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  • DOI: https://doi.org/10.1007/978-1-0716-0676-6_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0675-9

  • Online ISBN: 978-1-0716-0676-6

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