Over-production, renaturation and reconstitution of delta and epsilon subunits from chloroplast and cyanobacterial F1.
We studied the functioning of chimeric F0F1-ATPases by replacing subunits delta and epsilon of spinach CF1 with their counterparts from Synechocystis sp. PCC 6803. The sequence identities between these subunits are 26 and 41%, respectively. For a systematic approach to such studies and later extension to genetically modified subunits recombinant proteins are required. The genes coding for spinach and Synechocystis delta and epsilon were cloned into pET3 expression vectors and expressed in Escherichia coli. Upon expression at 37 degrees C the recombinant subunits formed inclusion bodies within the host cells except for spinach delta, which was soluble. Synechocystis delta and epsilon could be obtained in soluble form upon expression at 20 degrees C. After purification (and refolding of spinach epsilon) both epsilon subunits inhibited the Ca(2+)-ATPase activity of soluble CF1(- epsilon). Subunits delta and epsilon from both species raised the rate of ATP synthesis in partially CF1-depleted spinach thylakoids when added together with CF1(- delta) or CF1(- delta, epsilon). This showed the functionality of recombinant Synechocystis and spinach delta and epsilon together with spinach alpha 3 beta 3 gamma. The molar excess of epsilon necessary for saturation was higher for Ca(2+)-ATPase inhibition than for reconstitution of photophosphorylation thus pointing to a direct interaction between epsilon and both CF1 and CF0.
Steinemann
D
D
Fachbereich Biologie/Chemie, Universität Osnabrück, Germany.
Lill
H
H
Junge
W
W
Engelbrecht
S
S
D000595:Amino Acid Sequence; D002736:Chloroplasts / Q000737:chemistry / Q000201:enzymology*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000737:chemistry / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D018724:Spinacia oleracea / Q000235:genetics
D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases