Biochemical coupling between the DrrA and DrrB proteins of the doxorubicin efflux pump of Streptomyces peucetius.
The drrAB operon of Streptomyces peucetius encodes for resistance to the antibiotics doxorubicin and daunorubicin. Subcloning of the drrAB genes in Escherichia coli has previously been shown to result in expression of DrrA and DrrB proteins and resistance to doxorubicin in a sensitive strain of E. coli. DrrA, a peripheral membrane protein, binds ATP in a UV-catalyzed reaction in a doxorubicin-dependent manner; DrrB, a hydrophobic protein, is localized to the inner membrane of E. coli (Kaur, P. (1997) J. Bacteriol. 179, 569-575). The present study provides evidence that DrrB, the membrane component of the complex, is stably maintained in the cell only if DrrA is present. Furthermore, it was found that the catalytic component DrrA is in an active conformation only when it is in a complex with DrrB. In a subclone containing the drrB gene by itself, no DrrB protein could be detected, although a translational fusion of the first 15 amino acids of DrrB to beta-galactosidase indicated that DrrB is translated in the absence of DrrA. Upon co-transformation with a plasmid containing the drrA gene in trans, DrrB could again be detected in these cells. UV cross-linking studies with [alpha-32P]ATP showed that only the membrane-bound form of DrrA in cells containing both DrrA and DrrB was in a conformation competent to bind ATP. Chemical cross-linking studies also provided direct evidence for interaction between the two proteins. Based on these analyses, a model for interaction between DrrA and DrrB proteins is presented.
Kaur
P
P
Department of Biology, Georgia State University, Atlanta, Georgia 30303, USA. bioppk@panther.gsu.edu
Russell
J
J
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D017931:DNA Primers; D004317:Doxorubicin / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011485:Protein Binding; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013302:Streptomyces / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D017931:DNA Primers; D004317:Doxorubicin; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; C104037:DrrA protein, Streptomyces peucetius; C104038:DrrB protein, Streptomyces peucetius