SUBCELLULAR-LOCALIZATION AND TOPOLOGY OF THE K88 USHER FAED IN ESCHERICHIA-COLI

The subcellular localization of the K88 usher FaeD was studied in Esch erichia coli whole cells by using isopycnic sucrose density gradient c entrifugation of isolated membranes, the detergents Triton X-100 and s odium lauryl sarcosinate and immunoblotting with a specific FaeD antis erum. Cells containing the complete K88 operon, as well as cells conta ining the subcloned faeD gene in various expression vectors, were used . Most of the FaeD was present in the outer membranes in a detergent-r esistant form. Agglutination experiments with E. coli cells expressing FaeD confirmed an outer membrane localization and indicated the prese nce of FaeD at the cell surface. Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amin o acid residues. Twelve different FaeD-PhoA fusion proteins were prepa red and characterized by nucleotide sequencing and immuno-blotting. Mo st of these fusion sites were located in the amino-terminal and carbox yl-terminal regions of FaeD. Six amino-terminal fusion proteins were s oluble proteins in the periplasm, whereas the other fusion proteins we re associated with the outer membrane. The protease accessibility of F aeD and of the six outer membrane-bound FaeD-PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, a nd isolated membranes. Collagenase H, kallikrein, trypsin and proteina se K were used. Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane-s panning segments and two relatively large periplasmic regions, at the amino-terminal and carboxyl-terminal end of the protein, respectively.