Membrane integration and function of the three F0 subunits of the ATP synthase of Escherichia coli K12.

Abstract

Integration into the cytoplasmic membrane and function of the three Fo subunits, a, b and c, of the membrane-bound ATP synthase of Escherichia coli K12 were analysed in situations where synthesis of only one or two types of subunits was possible. This was achieved by combined use of atp mutations and plasmids carrying and expressing one or two of the atp genes coding for ATP synthase subunits. An three Fo subunits were found to be required for the establishment of efficient H + conduction. Subunits a and b individually as well as together were found to bind F1 ATPase to the membrane while subunit c did not. The ATPase activity bound to either of these single subunits, or in pairwise combinations, was not inhibited by N,N '-dicyclohexylcarbodiimide. Also ATPdependent H + translocation was not catalysed unless all three Fo subunits were present in the membrane. The integration into the membrane of the subunits a and b was independent of the presence of other ATP synthase subunits.

The proton conducting F0-part of bacterial ATP synthases

“…Most likely, the contaet is formed by the large polar domain. This large polar domain binds F 1 as demonstrated in mutant membrane where the other Fa subunits a and c are deleted [102]. This is not the only contact side between Fa and F 1 , since -as already mentioned -subunit a also is involved in the binding of F 1 [102].…”

“…The binding affinity of F} has not yet been quantitatively analysed. This may explain discrepancies reported for the rebinding of F 1 to F o from certain mutants by different groups [102,106]. Despite these uncertainties, the most surprising result is that in mutant strains with apparently assembled F o , the binding of F} is altered [109].…”

“…The membranes of such overproducing strains offer advantages for structural studies. Furthermore, plasmids are now available containing only segments of the atp operon [101,102]. Thus, a subset of subunits or in certain cases even a single subunit can be expressed at an increased level in the cell (Fig.…”

“…10, allow the study of membranes in wh ich selected Fa subunits are missing. Strain CM 2080, characterized in some detail, has adeletion of about 500 basepairs at the start of the subunit a gene [102]. Membranes of this deletion mutant contain Fa subunits band e, but only if isolated from exponentially growing cells.…”

“…The strains obtained contain in the membrane either only Fa subunit a or only subunit b. The membranes of all these strains, in which single or multiple Fa subunits are genetically deleted, have been analysed for their capability to TABLEIV 13 rebind F] and to conduct H ' [102] (Table III). Also included in these studies was a mutant membrane which is devoid of subunit e due to a point mutation in the alp E (une E) gene (see below).…”