The expression of fumarate reductase in Escherichia coli has been amplified over 30-fold by utilizing a recombinant plasmid, pFR63, carrying the fumarate reductase operon. More than 50% of the innermembrane protein could be accounted for by the enzyme, whereas the total amount of protein associated with the inembrane fraction doubled. The membrane accommodated this excess fumarate reductase without reducing the levels of other membrane-associated enzymes. At the same time, the amount of membrane lipid increased such that the lipid/protein ratio remained constant, indicating that the total amount of membrane had doubled. Small alterations in fatty acid composition as well as a large increase in cardiolipin were detected in the fumarate reductase-enriched memnbranes. The excess membrane was localized in novel tubular structures which were observed in thin-section and negatively stained electron-microscopic preparations. The tubules only appeared after the cytoplasmic membrane became highly enriched in fumarate reductase. They branched from the cytoplasmic membrane and were comnposed of an aggregate of fumarate reductase and lipid. * Corresponding author. amplification of fumarate reductase does not reduce the levels of other membrane-bound activities. MATERIALS AND METHODS Strains and plasmids. E. coli HB101 is F_ hsdR hsdM pro leu gal lac thi recA rpsL. Plasmids pBR322 and pFRD63 have previously been described (15). Preparation of everted envelopes, inner membranes, and

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.