ATP-DEPENDENT FERRIC HYDROXAMATE TRANSPORT-SYSTEM IN ESCHERICHIA-COLI- PERIPLASMIC FHUD INTERACTS WITH A PERIPLASMIC AND WITH A TRANSMEMBRANE CYTOPLASMIC REGION OF THE INTEGRAL MEMBRANE-PROTEIN FHUB, AS REVEALED BY COMPETITIVE PEPTIDE-MAPPING

The Escherichia coli iron transport system via ferrichrome belongs to the group of ATP-dependent transporters that are widely distributed in prokaryotes and eukaryotes. Transport across the cytoplasmic membrane is mediated by three proteins: FhuD in the periplasm, FhuB in the cyt oplasmic membrane and FhuC (ATPase) associated with the inside of the cytoplasmic membrane. Interaction of FhuD with FhuB was studied in vit ro with biotinylated synthetic 10 residue and 20-24 residue peptides o f FhuB by determining the activity of beta-galactosidase linked to the peptides via streptavidin. Peptides identical in sequence to only one of the four periplasmic loops (loop 2), predicted by a transmembrane model of FhuB, and peptides representing a transmembrane segment and p art of the adjacent cytoplasmic loop 7 of FhuB bound to FhuD, Decapept ides were transferred into the periplasm of cells through a FhuA delet ion derivative that forms permanently open channels three times as lar ge as the porins in the outer membrane. FhuB peptides that bound to Fh uD inhibited ferrichrome transport, while peptides that did not bind t o FhuD did not affect transport. These data led us to propose that the periplasmic FhuD interacts with a transmembrane region and the cytopl asmic segment 7 of FhuB, The transmembrane region may be part of a por e through which a portion of FhuD inserts into the cytoplasmic membran e during transport, The cytoplasmic segment 7 of FhuB contains the con served amino acid sequence EAA...G (in FhuB DTA...G) found in ABC tran sporters, which is predicted to interact with the cytoplasmic FhuC ATP ase, Triggering of ATP hydrolysis by substrate-loaded FhuD may occur b y physical interaction between FhuD and FhuC, which bind close to each other on loop 7, Although FhuB consists of two homologous halves, Fhu B(N) and FhuB(C), the sites identified for FhuD-mediated ferrichrome t ransport are asymmetrically arranged.