Integration of b subunits of unequal lengths into F1F0-ATP synthase.
In Escherichia coli the peripheral stalk of F1F0-ATP synthase consists of a parallel dimer of identical b subunits. However, the length of the two b subunits need not be fixed. This led us to ask whether it is possible for two b subunits of unequal length to dimerize in a functional enzyme complex. A two-plasmid expression system has been developed that directs production of b subunits of unequal lengths in the same cell. Two b subunits differing in length have been expressed with either a histidine or V5 epitope tag to facilitate nickel-affinity resin purification (Ni-resin) and Western blot analysis. The epitope tags did not materially affect enzyme function. The system allowed us to determine whether the different b subunits segregate to form homodimers or, conversely, whether a heterodimer consisting of both the shortened and lengthened b subunits can occur in an intact enzyme complex. Experiments expressing different b subunits lengthened and shortened by up to 7 amino acids were detected in the same enzyme complex. The V5-tagged b subunit shortened by 7 amino acids (b Delta 7-V5) was detected in Ni-resin-purified membrane preparations only when coexpressed with a histidine-tagged b subunit in the same cell. The results demonstrate that the enzyme complex can tolerate a size difference between the two b subunits of up to 14 amino acids. Moreover, the experiments demonstrated the feasibility of constructing enzyme complexes with non-identical b subunits that will be valuable for research requiring specific chemical modification of a single b subunit.
Grabar
Tammy Bohannon
TB
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610, USA.
Cain
Brian D
BD
D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D021122:Protein Subunits; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D017384:Sequence Deletion
D017931:DNA Primers; D000939:Epitopes; D021122:Protein Subunits; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases