ELSEVIER Biochimica et Biophysica Acta 1186 (1994) 243-246 liB, Biochi~ic~a et Biophysica Acta Rapid Report Complementation of Escherichia coli uncD mutant strains by a chimeric F1- fl subunit constructed from E. coli and spinach chloroplast Fl-fl Andreas Burkovski a,1, Holger Lill b, Siegfried Engelbrecht b,, a Mikrobiologie, Fachbereich Biologie/ Chemie, Universitiit Osnabriick, D-49069 Osnabriick, Germany b Biophysik, Fachbereich Biologie/Chemie, Universitiit Osnabriick, Barbarastr. 11, D-49069 Osnabriick, Germany Received 29 March 1994 Abstract ATP-synthesizing FoF1-ATPases are complex enzymes consisting of at least eight different subunits. These subunits are conserved during evolution to a very variable degree ranging in pairwise comparison between, for example, Escherichia coli and spinach chloroplast from 20% to 66% identical residues. It was surprising to find that some of the less well conserved subunits like 8 and e could replace their E. coli counterparts, whereas the highly conserved/3 subunit, which carries the active site, in the E. coli enzyme could not be substituted by spinach chloroplast/3 (Lill et al. (1993) Biochim. Biophys. Acta 1144, 278-284). We constructed a chimeric F1-/3 subunit consisting of spinach /3 in which the 96 N-terminal amino acids were replaced by the respective residue sequence from E. coli/3. Whereas spinach/3 did not complement E. coli uncD mutant strains, the chimeric/3 subunit restored growth under conditions of oxidative phosphorylation. Key words: ATPase, FoF1-; ATPase, F1-; Complementation; Chimeric protein FoF1-ATPases synthesize ATP at the expense of protonmotive force [1-6]. They are found in bacteria, mitochondria and chloroplasts. The enzyme consists of two distinct subcomplexes, the membrane-embedded proton channel F0, and the extrinsic, water-soluble part, F1, which carries the catalytic sites. F 1 of Es- cherichia coli and chloroplasts consists of five different subunits, a (56 kDa),/3 (54 kDa), y (31-36 kDa), 8 (21 kDa), and E (15 kDa) witha stoichiometry of 3 : 3 : 1 : 1 : 1 and nucleotide binding sites on or between a and/3. While the general architecture of FoF1-ATPases from different sources is very similar, their subunits are conserved to very variable degrees. The /3 subunits contain the catalytic nucleotide binding sites and it is not surprising that F1-/3 is the best conserved subunit: 66% of the residues are identical in pairwise compari- son between spinach and E. coli /3. Subunits /5 and ~, Abbreviations: F0F 1, FoF:-ATPase; Fo, proton channel (mem- brane-embedded); F1, ATPase (soluble part). * Corresponding author. Fax: + 49 541 9692870. 1 Present address: Forschungszentrum Jiilich, Institut ffir Biotech- nologie I, 52425 Jiilich, Germany. 0005-2728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0005-2728(94)00052-7 on the other hand, share only about 25% of identical residues in pairwise comparison. This degree of conservation does not, however, cor- relate with the 'exchangeability' of the subunits: Table 1 summarizes the data which have been published so far. It was surprising to find that the highly conserved subunit /3 from spinach did not complement the re- spective E. coli unc mutant strain, whereas cyano- bacterial /3 from Synechocystis sp. PCC6803 did [7]. The percentages of identical residues are 71% (E. coli ,~ Synechocystis), 66% (E. coli ¢~ spinach) and 80% (Synechocystis ,~, spinach). The degree of identity is thus very similar, with Synechocystis /3 resembling E. coli /3 just a little more than spinach /3 resembles E. coli/3. We tried to take advantage of this small differ- ence and constructed a chimeric /3 subunit consisting of ~ 20% of the E. coli sequence (at the N-terminus) and the remaining ~ 80% of the spinach /3 sequence. The chimeric protein allowed for significant growth of /3-defective E. coli mutant strains under conditions of oxidative phosphorylation, thus indicating that the N- terminal part of subunit /3 at least in E. coli F0F 1 has to fulfill some rather restricted structural require- ments.