Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits

Abstract

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-Fo adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the Fo portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional Fo might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight Fo subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight Fo subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the a-and ,f-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight Fo subunit and to the formation of a functional Fo. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the FI-Fo ATPase complex to be proposed.The membrane-bound, energy-coupling ATPase complex has been purified from several species of bacteria, including the thermophilic bacterium PS3 (31), Escherichia coli (13,14), and Mycobacterium phlei (4, 21). The enzyme complexes from these three sources probably consist of eight nonidentical subunits and are readily separated into two portions: Fl, containing five subunits Fo containing three subunits. There is some disagreement about the molecular weights of the Fo subunits of E. coli. However, those proposed by Foster and Fillingame (13) of 24,000, 19,000, and 8,400 agree well with the results of experiments with the cloned unc operon of E. coli in an in vitro transcription-translation system (7). Similar molecular weights have also been obtained for Fo components of M. phlei (4). t Present address: Fo portions from strain PS3 and E. coli have been incorporated into phospholipid vesicles and shown to function as a proton pore (24, 28). F1 ATPase is water soluble, has ATPase activity, and has been separated into its component subunits. These can be reassembled and combined with Fl-depleted membranes to give a fully functional F1-Fo ATPase complex (11,35). The similarity of the ATPase structures found in such diverse bacteria as strain PS3 and E. coli is emphasized by the experiments in which the ysubunits from the F1 ATPase from each species have been shown to be partly interchangeable (15). The Fo portion of the ATPase complex has not yet been dissociated into individual subunits and reassembled into a functional complex.Information concerning the assembly of the ATPase complex in strain PS3 has been obtained from experiments involving purified Fo incorporated into phospholipid vesicles an...

Use of lambda unc transducing bacteriophages in genetic and biochemical characterization of H+-ATPase mutants of Escherichia coli

“…Loo and Bragg (45) noted that a peptide, tentatively identified as the psi subunit, was missing from strain N144 membranes. The absence of psi would be consistent with the assembly model of Cox et al (7), which requires the presence of a and l for integration of this subunit. A better understanding of these and other complex mutants will require a more detailed analysis.…”

An additional acidic residue in the membrane portion of the b-subunit of the energy-transducing adenosine triphosphatase of Escherichia coli affects both assembly and function

“…However, further experimentation indicated that both the aand P-subunits of the F -ATPase were required for the assembly in vivo of the b-subunit into the Fo portion of the ATPase (Klionsky et al, 1983). Friedl et al (1983) also studied the integration of the b-subunit into the membrane by using various mutant strains and plasmid-bearing strains and, although they had some problems with proteolysis, reported data consistent with the assembly pathway previously reported by Cox et al (1981). However, Friedl et al (1983) also obtained some evidence for the membrane association of the bsubunit in the absence of other subunits.…”

Mutations in the uncE gene affecting assembly of the c-subunit of the adenosine triphosphatase of Escherichia coli

“…The growth yields of all four strains were also similar (Table 2) and indicated that the partial-diploid strains had regained some capacity for oxidative phosphorylation as compared with the haploid controls. The ATPase activities (Table 2) of membrane preparations from these four strains also indicated that some assembly of the complete F1FO-ATPase had occurred compared with the interrupted assembly (Cox et al, 1981a) that occurs in the haploid strains.…”