Abstract
F1‐ATPase (F1) is a multisubunit water‐soluble domain of FoF1‐ ATP synthase and is a rotary enzyme by itself. Earlier genetic studies using yeast suggested that two factors, Atp11p and Atp12p, contribute to F1 assembly. Here, we show that their mammalian counterparts, AF1 and AF2, are essential and sufficient for efficient production of recombinant bovine mitochondrial F1 in Escherichia coli cells. Intactness of the function and conformation of the E. coli‐expressed bovine F1 was verified by rotation analysis and crystallization. This expression system opens a way for the previously unattempted mutation study of mammalian mitochondrial F1.
Highlights
FoF1-ATP synthase (FoF1) is ubiquitously found in membranes of bacteria, chloroplasts, and mitochondria, and synthesizes ATP from ADP and inorganic phosphate (Pi) driven by downhill proton flow across the membranes [1,2,3]
F1 isolated from bovine heart appeared as two split bands in native-PAGE for an unknown reason and bovine F1 produced in E. coli gives two bands
These results show that expression of the ATP11 and ATP12 is essential for efficient production of bovine F1
Summary
FoF1-ATP synthase (FoF1) is ubiquitously found in membranes of bacteria, chloroplasts, and mitochondria, and synthesizes ATP from ADP and inorganic phosphate (Pi) driven by downhill proton flow across the membranes [1,2,3]. We recently succeeded in expressing human mitochondrial F1 in Escherichia coli cells and reported its nine-step rotation in one revolution [5]. Here, we report the expression of bovine mitochondrial F1 in E. coli. We expressed the five subunits of bovine mitochondrial F1 in E. coli cells with or without coexpression of AF1 and AF2.
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