The delta- and epsilon-subunits of bovine F1-ATPase interact to form a heterodimeric subcomplex.
The delta-subunit of bovine F1-ATPase was expressed from a bacterial vector at fairly high level in Escherichia coli, but the yield of bovine epsilon-subunit was rather low under similar conditions. However, co-expression of the proteins from a dicistronic operon delta-epsilon in the same expression vector, produced both of them in good yield in a soluble form in the bacterial cytoplasm, and by chromatography it was found that the delta- and epsilon-subunits were associated in a stable complex. The amino groups in the complex were labelled exhaustively by chemical reaction under denaturing conditions with ethyl-[1-14C]acetimidate. The alpha-amino groups of the proteins were unmodified, but complete reaction of all epsilon-amino groups in both proteins was demonstrated by determination of the molecular masses of the modified proteins by electrospray MS. The modified subunits were separated by denaturing gel electrophoresis, and from measurements of the ratio of incorporated radioactivities and the lysine contents of the proteins, it was calculated that the subcomplex contains equimolar amounts of the two proteins. As the apparent molecular mass of the complex determined by gel filtration was 29 kDa, it appears that the complex contains one copy of each protein. It is likely that the delta- and epsilon subunits are associated in a similar manner in the bovine F1-ATPase complex, and that, like a bacterial homologue of the delta-subunit, they interact with the gamma- and beta-subunits.
Orriss
G L
GL
The Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.
Runswick
M J
MJ
Collinson
I R
IR
Miroux
B
B
Fearnley
I M
IM
Skehel
J M
JM
Walker
J E
JE
D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D002417:Cattle; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001704:Biopolymers; D017931:DNA Primers; D006180:Proton-Translocating ATPases