Assembly of Escherichia coli fumarate reductase holoenzyme

Abstract

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.

YidC Is Involved in the Biogenesis of Anaerobic Respiratory Complexes in the Inner Membrane of Escherichia coli

“…Fumarate reductase also has an ␣␤␥ structure with FrdA and FrdB forming a cytoplasmically located ␣␤ subcomplex that attaches to the membrane via the integral membrane proteins FrdC and -D. The decrease in the levels of FrdA and FrdB associated with the inner membrane is suggestive of a role for YidC in the insertion of FrdC and FrdD because FrdA and FrdB cannot assemble at the bacterial membrane without first having the correct assembly of integral membrane parts FrdC and FrdD (44). FrdC and FrD are both small (15 and 13 kDa, respectively) three TMS-containing proteins that have their N termini located in the cytoplasm and their C termini in the periplasm (45).…”

Assembly of the Cytochrome bo3 Complex

“…29 Plasmids were transformed into the E. coli strain BL21 (DE3). A colony was inoculated into a 2 ml tube containing 1 ml of LB medium with 50 μg/ml of kanamycin, and incubated with vigorous shaking at 37°C for 16 h. The culture was back-diluted, and incubated as before until the absorbance at 600 nm was between 0.3 and 0.5.…”

Multiple Roles for the Twin Arginine Leader Sequence of Dimethyl Sulfoxide Reductase of Escherichia coli

“…Rifampicin (at 250 g/ml of culture), an inhibitor of chromosomally encoded RNA polymerase, was added, and the incubation was continued at 42°C for 15 min. All subsequent incubations were at 37°C (20). Aliquots (1.0 ml) were drawn at various time points and spun in an Eppendorf centrifuge, and the resulting bacterial pellets were resuspended in 50 l of the SDS solubilization buffer.…”