Two novel regulatory motifs LDEVFL and C-terminal rin a translationally coupled manner (8). role in function and assembly of the DrrAB complex. One motif present at the extreme C terminus of DrrA is usually rich in glutamic acid residues and is termed the C-terminal rbinds molybdenum and is involved in trans-inhibition CAL-130 of the ATPase activity which results in a decrease of the transport rate in response to an increase in concentration of the substrate in the cytoplasm (17). Similarly C-terminal extensions present in MetN of the MetNI system (18) and in Wzt of the Wzt/Wzm system of are able to bind their respective pump substrates (19 20 Crystal structure analysis suggests that the C-terminal domains of these proteins contain a comparable β-sheet fold although they contain diverse amino acid sequences and perform different functions in ABC MGC34923 transporters. In the studies described here we report that this CREEM and LDEVFL motifs present in the extreme C terminus of CAL-130 DrrA are critical for function of the DrrAB complex. We also show that this region of DrrA forms the point of contact with the N-terminal cytoplasmic tail of DrrB thus leading to the proposal that this major role of the CREEM and LDEVFL motifs may be in assembly of the DrrAB complex. Interestingly a 33-amino acid region in the C terminus of DrrA encompassing residues in the LDEVFL motif was also found to be involved in homodimerization of DrrA. The significance of these two interactions both localized to the C-terminal end of DrrA in protein assembly is discussed. EXPERIMENTAL PROCEDURES Bacterial Strains Plasmids and Antibodies The bacterial strains used in this study were TG1 N43 LE392Δin pSU2718) and pDx119 (in pET 16b). Various substitutions and deletions were created in the and genes in these plasmids. Rabbit polyclonal antibodies generated against DrrA and DrrB previously (4) were used for Western blot analysis. Anti-SecY antibody was provided by the laboratory of Dr. P. C. Tai. CAL-130 Media and Growth Conditions For doxorubicin efflux experiments cells were produced in TEA medium (50 mm triethanolamine HCl (pH 6.9) 15 mm KCl 10 mm (NH4)2SO4 1 mm MgSO4) supplemented with 0.5% (w/v) glycerol 2.5 μg/ml thiamine 0.5% (w/v) peptone and 0.15% (w/v) succinate at 37 °C (21). For site-directed mutagenesis XL1-Blue cells were produced at 37 °C in NYZ+ broth (pH 7.5; 1% (w/v) casein hydrolysate 0.5% (w/v) yeast extract 0.5% (w/v) NaCl) supplemented with 12.5 mm MgCl2 12.5 mm MgSO4 and 0.4% (w/v) CAL-130 glucose (Stratagene La Jolla CA). For all other experiments cells were produced in LB medium. Chloramphenicol was added to 20 μg/ml and ampicillin was added to 75 μg/ml where needed. Site-directed Mutagenesis of DrrA A QuikChange multisite-directed mutagenesis kit (Stratagene) was used to produce various mutations in the at position 23 (S23C) was used as the template (22). Single cysteine substitution mutants were created at amino acid position CAL-130 325 323 319 311 302 287 253 or 232 in DrrA in this clone. Deletion of the C Terminus of DrrA This was achieved by removing 27 bases (positions 961-987) from the 3′-end of while retaining the last 3 bases of the sequence in order to maintain translational stop/start overlap with N43 cells which are doxorubicin-sensitive. A single colony was incubated in 5 ml of LB made up of the desired antibiotic for 8 h. 1 μl of the above cells were streaked on M9 plates with a top layer made up of 0 4 6 8 or 10 μg/ml doxorubicin. Plates were covered with foil because doxorubicin is usually light-sensitive. Growth was recorded after incubation of plates at 37 °C for 24 h. Doxorubicin Efflux Assay The efflux assay was carried out according to the protocol previously developed in this laboratory.3 Briefly LE392Δcells (24) were transformed with the indicated plasmids; the cells were produced to mid-log phase (TG1 cells made up of the indicated plasmids were produced to mid-log phase and induced with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside. Growth was continued for an additional 3 h at 37 °C. The membrane fraction was prepared as follows. Cells were spun down and resuspended in 10 ml of buffer A (25 mm Tris-Cl (pH 7.5) 20 glycerol 2 mm EDTA (pH 8.0) 1 mm DTT) and passed through a French press cell at 16 0 p.s.i. followed by centrifugation at 10 0 × at 4 °C for 30 min to remove unbroken cells. The supernatant was centrifuged at 100 0 × at 4 °C for 1 h. The pellet was resuspended.