Heterologous expression of the Crassostrea gigas (Pacific oyster) alternative oxidase in the yeast Saccharomyces cerevisiae.
Alternative oxidase (AOX) is a terminal oxidase within the inner mitochondrial membrane (IMM) present in many organisms where it functions in the electron transport system (ETS). AOX directly accepts electrons from ubiquinol and is therefore capable of bypassing ETS Complexes III and IV. The human genome does not contain a gene coding for AOX, so AOX expression has been suggested as a gene therapy for a range of human mitochondrial diseases caused by genetic mutations that render Complex III and/or IV dysfunctional. An effective means of screening mutations amenable to AOX treatment remains to be devised. We have generated such a tool by heterologously expressing AOX from the Pacific oyster (Crassostrea gigas) in the yeast Saccharomyces cerevisiae under the control of a galactose promoter. Our results show that this animal AOX is monomeric and is correctly targeted to yeast mitochondria. Moreover, when expressed in yeast, Pacific oyster AOX is a functional quinol oxidase, conferring cyanide-resistant growth and myxothiazol-resistant oxygen consumption to yeast cells and isolated mitochondria. This system represents a high-throughput screening tool for determining which Complex III and IV genetic mutations in yeast will be amenable to AOX gene therapy. As many human genes are orthologous to those found in yeast, our invention represents an efficient and cost-effective way to evaluate viable research avenues. In addition, this system provides the opportunity to learn more about the localization, structure, and regulation of AOXs from animals that are not easily reared or manipulated in the lab.
Department of Biology, Wilfrid Laurier University, 75 University Ave. W, Waterloo, ON, N2L 3C5, Canada.
Aaron
A
Robertson
Department of Biology, Wilfrid Laurier University, 75 University Ave. W, Waterloo, ON, N2L 3C5, Canada.
Kyle
K
Schaltz
Department of Biology, Wilfrid Laurier University, 75 University Ave. W, Waterloo, ON, N2L 3C5, Canada.
Karina
K
Neimanis
Department of Biology, Western University, 1151 Richmond St, London, ON, N6A 5B7, Canada.
James F
JF
Staples
Department of Biology, Wilfrid Laurier University, 75 University Ave. W, Waterloo, ON, N2L 3C5, Canada. amcdonald@wlu.ca.
Allison E
AE
McDonald
D000818:Animals; D049896:Crassostrea / Q000201:enzymology* / Q000235:genetics; D004579:Electron Transport; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D028361:Mitochondrial Diseases / Q000628:therapy; D051336:Mitochondrial Membranes / Q000737:chemistry / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000235:genetics*; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D000099188:Alternative Oxidase
D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D000099188:Alternative Oxidase; C020094:duroquinol oxidase
Bioenergetics; Electron transport system; High-resolution respirometry; Mitochondria; Mitochondrial disease; Respiration