The extreme C terminus of the ABC protein DrrA contains unique motifs involved in function and assembly of the DrrAB complex.
ABSTRACT Two novel regulatory motifs, LDEVFL and C-terminal regulatory Glu (E)-rich motif (CREEM), are identified in the extreme C terminus of the ABC protein DrrA, which is involved in direct interaction with the N-terminal cytoplasmic tail of the membrane protein DrrB and in homodimerization of DrrA. Disulfide cross-linking analysis showed that the CREEM and the region immediately upstream of CREEM participate directly in forming an interaction interface with the N terminus of DrrB. A series of mutations created in the LDEVFL and CREEM motifs drastically affected overall function of the DrrAB transporter. Mutations in the LDEVFL motif also significantly impaired interaction between the C terminus of DrrA and the N terminus of DrrB as well as the ability of DrrA and DrrB to co-purify, therefore suggesting that the LDEVFL motif regulates CREEM-mediated interaction between DrrA and DrrB and plays a key role in biogenesis of the DrrAB complex. Modeling analysis indicated that the LDEVFL motif is critical for conformational integrity of the C-terminal domain of DrrA and confirmed that the C terminus of DrrA forms an independent domain. This is the first report which describes the presence of an assembly domain in an ABC protein and uncovers a novel mechanism whereby the ABC component facilitates the assembly of the membrane component. Homology sequence comparisons showed the presence of the LDEVFL and CREEM motifs in close prokaryotic and eukaryotic homologs of DrrA, suggesting that these motifs may play a similar role in other homologous drug and lipid export systems.
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ABSTRACT: ATP-binding cassette (ABC) systems are found in all three domains of life and in some giant viruses and form one of the largest protein superfamilies. Most family members are transport proteins that couple the free energy of ATP hydrolysis to the translocation of solutes across a biological membrane. The energizing module is also used to drive non-transport processes associated, e.g., with DNA repair and protein translation. Many ABC proteins are of considerable medical importance. In humans, dysfunction of at least eighteen out of 49 ABC transporters is associated with disease, such as cystic fibrosis, Tangier disease, adrenoleukodystrophy or Stargardt’s macular degeneration. In prokaryotes, ABC proteins confer resistance to antibiotics, secrete virulence factors and envelope components, or mediate the uptake of a large variety of nutrients. Canonical ABC transporters share a common structural organization comprising two transmembrane domains (TMDs) that form the translocation pore and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. In this Mini-Review, we summarize recent structural and biochemical data obtained from both prokaryotic and eukaryotic model systems.Central European Journal of Biology 10/2011; 6(5):785-801. DOI:10.2478/s11535-011-0054-4 · 0.63 Impact Factor
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ABSTRACT: Background The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production. Results In order to investigate OtrC’s function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively). Conclusions The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.BMC Biotechnology 08/2012; 12(1):52. DOI:10.1186/1472-6750-12-52 · 2.59 Impact Factor
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ABSTRACT: This study provides the first direct evidence for the dual role of the metalloprotease FtsH in membrane protein biogenesis. Using the physiological substrate DrrAB, it is shown that FtsH is not only responsible for proteolysis of unassembled DrrB protein but it also plays a much broader role in biogenesis of the DrrAB complex. Previous studies showed that the stable expression of DrrB in the membrane depends on simultaneous expression of DrrA. Here we show that DrrB is proteolyzed by FtsH when it is expressed alone. Moreover, DrrA and DrrB proteins expressed together in a temp-sensitive ftsH mutant strain of E. coli were found to be non-functional due to their incorrect assembly. Simultaneous expression of wild-type FtsH in trans resulted in normal doxorubicin efflux. Strikingly, doxorubicin efflux could be restored in mutant cells irrespective of whether FtsH was expressed simultaneously with DrrAB or expressed after these proteins had already accumulated in an inactive conformation, thus providing crucial evidence for the ability of FtsH to refold the misassembled proteins. Complementation experiments also showed that the catalytic AAA domain of FtsH contains a chaperone-like activity, however, unlike wild-type FtsH, it was unable to restore function. Our results therefore show for the first time that FtsH contains the protease as well as refolding functions, and both the AAA and the proteolytic domains of FtsH are required for each of these activities.Journal of Biological Chemistry 03/2013; 288. DOI:10.1074/jbc.M112.441915 · 4.57 Impact Factor