SUBUNIT INTERACTIONS INVOLVED IN THE ASSEMBLY OF PYRIDINE-NUCLEOTIDE TRANSHYDROGENASE IN THE MEMBRANES OF ESCHERICHIA-COLI

The pyridine nucleotide transhydrogenase (PNT) of Escherichia coli con sists of two different subunits (alpha and beta) and assembles as a te tramer (alpha(2)beta(2)) in the inner membrane. The pnt genes from E. coli have been cloned on a multicopy plasmid resulting in high level e xpression of the enzyme activity. We have studied the influence of the different segments of the polypeptide chains of the alpha and beta su bunits on the assembly and function of the enzyme by constructing a se ries of deletion mutants for both of the subunits. Our results show th at the assembly of the beta subunit is contingent upon the insertion o f the alpha subunit into the membrane, while the alpha subunit can ass emble independently of the beta subunit. All deletions constructed for the cytosolic portion of the alpha subunit gave no incorporation of t he alpha subunit and, as a consequence, of the beta subunit, also. Of the four membrane-spanning regions of the alpha subunit, the last two were indispensable, while the deletion of the first two still allowed the association of alpha as well as of the beta subunit with the membr ane. However, the enzyme was not functional. The two subunits were als o loosely associated as mild detergent treatment released them from th e membrane in contrast with the wild-type enzyme. Deletions within the beta subunit had little effect on the assembly of the alpha subunit, although less was incorporated. All deletions involving the cytosolic portion of the beta subunit resulted in loss of incorporation into the membrane. Of the eight membrane-spanning regions of the beta subunit, the deletion of regions 2-3, 2-4, 2-6, and 2-7 yielded significant as sociation of both the subunits with the membrane. However, none of the se mutants assembled a functional enzyme, and again the two subunits w ere loosely associated with the membrane. Based on the stringent requi rement of the cytosolic portions of alpha and beta subunits for assemb ly, a model is proposed that suggests interactions between these two r egions must occur prior to assembly.