Mitochondrial ATP synthase

cDNA cloning, amino acid sequence, overexpression, and properties of the rat liver α subunit

James H. Lee, David N. Garboczi, Philip J. Thomas, Peter L Pedersen

Research output: Contribution to journalArticle

Abstract

The predicted amino acid sequence of the α subunit of the rat liver mitochondrial ATP synthase has been obtained by sequencing a cDNA for the α subunit. Analysis of the sequence shows that it contains the A and B consensus sequences found in many nucleotide-binding proteins. Twelve amino acids of the rat liver α subunit differ from the sequence of the bovine heart a subunit; four of these involve differences in charge. The rat liver α subunit, from arginine 15 to the C-terminal proline 510, has been overexpressed in Escherichia coli using the alkaline phosphatase promoter (phoA) and leader peptide to direct the export of the expressed protein to the bacterial periplasm. By treating the cells with lysozyme, osmotic shock, and alkaline pH washes, the α subunit can be extracted in high yield (>25 mg/liter) and in a high state of purity. The expressed α subunit remains soluble at pH 9.5 or greater and precipitates when treated with Mg2+ ions at low millimolar concentration. The bacterially expressed α subunit interacts with 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate (TNP-ATP), resulting in a marked fluorescence enhancement upon binding. An enhancement of fluorescence is also observed upon the interaction of the a subunit with TNP-ADP. Preincubating the α subunit with 1.5 mM ATP significantly reduces the fluorescence enhancement seen with TNP-ATP. The α subunit binds TNP-ATP with an apparent Kd in the low micromolar range (1-5 μM) and binds TNP-ADP with an affinity at least 10-fold lower. This work shows that the rat liver α subunit can be overexpressed in E. coli to yield a large amount of functional protein. With the acquisition of the overexpressed α subunit, it is now possible to test the reconstitution of ATPase activity from a mixture of recombinant and rat liver-derived subunits and to test the formation of complexes by the overexpressed α and β subunits of the rat liver F1-ATPase.

Original languageEnglish (US)
Pages (from-to)4664-4669
Number of pages6
JournalJournal of Biological Chemistry
Volume265
Issue number8
StatePublished - Mar 15 1990

Fingerprint

Mitochondrial Proton-Translocating ATPases
Cloning
Liver
Rats
Organism Cloning
Amino Acid Sequence
Complementary DNA
Amino Acids
Adenosine Triphosphate
Adenosine
Fluorescence
Escherichia coli
Periplasm
Proton-Translocating ATPases
Bacterial Proteins
Osmotic Pressure
Consensus Sequence
Muramidase
Protein Sorting Signals
Proline

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mitochondrial ATP synthase : cDNA cloning, amino acid sequence, overexpression, and properties of the rat liver α subunit. / Lee, James H.; Garboczi, David N.; Thomas, Philip J.; Pedersen, Peter L.

In: Journal of Biological Chemistry, Vol. 265, No. 8, 15.03.1990, p. 4664-4669.

Research output: Contribution to journalArticle

Lee, James H. ; Garboczi, David N. ; Thomas, Philip J. ; Pedersen, Peter L. / Mitochondrial ATP synthase : cDNA cloning, amino acid sequence, overexpression, and properties of the rat liver α subunit. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 8. pp. 4664-4669.
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