Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB.
Periplasmic membrane fusion proteins (MFPs) are essential components of the type I protein secretion systems and drug efflux pumps in Gram-negative bacteria. Previous studies suggested that MFPs connect the inner and outer membrane components of the transport systems and by this means co-ordinate the transfer of substrates across the two membranes. In this study, we purified and reconstituted the macrolide transporter MacAB from Escherichia coli. Here, MacA is a periplasmic MFP and MacB is an ABC-type transporter. Similar to other MFP-dependent transporters from E. coli, the in vivo function of MacAB requires the outer membrane channel TolC. The purified MacB displayed a basal ATPase activity in detergent micelles. This activity conformed to Michaelis-Menten kinetics but was unresponsive to substrates or accessory proteins. Upon reconstitution into proteoliposomes, the ATPase activity of MacB was strictly dependent on MacA. The catalytic efficiency of MacAB ATPase was more than 45-fold higher than the activity of MacB alone. Both the N- and C-terminal regions of MacA were essential for this activity. MacA stimulated MacB ATPase only in phospholipid bilayers and did not need the presence of macrolides. Our results suggest that MacA is a functional subunit of the MacB transporter.
Tikhonova
Elena B
EB
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, OK 73019, USA.
Devroy
Vishakha K
VK
Lau
Sze Yi
SY
Zgurskaya
Helen I
HI
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis / Q000187:drug effects; D019897:Periplasm / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism*; D010743:Phospholipids / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D029968:Escherichia coli Proteins; C436312:MacA protein, E coli; C436313:MacB protein, E coli; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases