Five uncoupled mutant strains of Escherichia coli carrying mutations in the uncD gene have been studied. In each of these mutant strains the beta-subunit of the F1 portion of the membrane-bound adenosine triphosphatase is abnormal. In one of the mutant strains (carrying the uncD12 allele) in F1-ATPase aggregate was formed which was purified and found to have low ATPase activity. ATPase activity was absent in the other four strains and the abnormal beta-subunits were tightly bound to the membranes. However, membranes from these strains exhibited various proton permeabilities as indicated by NADH-dependent atebrin-fluorescence quenching and bound different amounts of normal F1-ATPase. The amounts of reconstitution of energy-linked reactions after the addition of normal F1-ATPase also varied depending on the mutant allele. It is apparent that considerable phenotypic variations can occur between strains carrying mutations in the same unc gene.

The effects of two protease inhibitors on the solubilization of the membranebound Mg2+-adenosine triphosphatase (Mg-ATPase) of Escherichia coli were investigated. p-Aminobenzamidine prevented the solubilization of the Mg-ATPase during treatment of membranes with low-ionic-strength buffers containing ethylenediaminetetraacetic acid. p-Aminobenzamidine did not prevent subsequent solubilization of the Mg-ATPase by treatment of the membranes with chloroform. This method of solubilization yielded a preparation of similar apparent molecular weight but with a 10-fold-increased specific activity as compared with the Mg-ATPase solubilized by washing with low-ionic-strength buffer. However, in contrast to the latter preparation, the chloroform-solubilized Mg-ATPase did not reconstitute ATP-dependent energization of stripped membranes, which were prepared by low-ionic-strength washing in the absence of p-aminobenzamidine. Another protease inhibitor, E-amino-n-caproic acid, did not affect the solubilization of the Mg-ATPase, but did inhibit the loss of activity occurring during concentration, by ultrafiltration, of the Mg-ATPase solubilized by the low-ionic-strength treatment.

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...

Membranes from a mutant strain of Escherichia coli K12 carrying the uncD409 allele were washed in low-ionic-strength buffers in the presence or absence of the proteinase inhibitor p-aminobenzamidine. Unlike membranes from a normal strain, those from strain AN463 (uncD409) did not become proton-permeable, as judged by NADH-induced atebrinfluorescence quenching, when the membranes were washed in the absence of p-aminobenzamide. Furthermore, ATP-dependent atebrin-fluorscence quenching in such washed membranes could not be reconstituted by the addition of solubilized Mg2+-stimulated adenosine triphosphatase preparations. The examination by two-dimensional polyacrylamide-gel electrophoresis of the polypeptide composition of the washed membranes from strain AN463 (uncD409) indicated the presence of a polypeptide of similar molecular weight to the normal beta-subunit of the Mg2+-stimulated adenosine triphosphatase, but with an altered isoelectric point. Both the normal and abnormal beta-subunits were identified in membranes prepared from a partial diploid strain carrying both the unc+ and uncD409 alleles. It is concluded that the uncD gene codes for the beta-subunit of the Mg2+-stimulated adenosine triphosphatase.

Four mutant strains of Escherichia coli which lack membrane-bound adenosine triphosphatase activity were shown by genetic-complementation tests to carry mutations in the uncA gene. A soluble inactive F1-ATPase aggregate was released from the membranes of three of the uncA mutant strains by low-ionic-strength washing, and purified by procedures developed for the purification of F1-ATPase from normal strains. Analysis of the subunit structure by two-dimensional gel electrophoresis indicated that the F1-ATPase in strains carrying the uncA401 or uncA453 alleles had a subunit structure indistinguishable from normal F1-ATPase. In contrast, the F1-ATPase from the strain carrying the uncA447 allele contained an alpha-subunit of normal molecular weight, but abnormal net charge. Membranes from strains carrying the uncA450 allele did not have F1-ATPase aggregates that could be solubilized by low-ionic-strength washing. However, a partial dipolid strain carrying both the uncA+ and uncA450 alleles formed an active F1-ATPase aggregate which could be solubilized by low-ionic-strength washing of the membranes and which contained two types of alpha-subunit, one of which was normal and the other had abnormal net charge. It is concluded that the uncA gene codes for the alpha-subunit of the adenosine triphosphatase.