A single negatively charged residue affects the orientation of a membrane protein in the inner membrane of Escherichia coli only when it is located adjacent to a transmembrane domain

The orientation of membrane proteins is determined by the asymmetric distri bution of charged residues in the sequences flanking the transmembrane doma ins. For the inner membrane of Escherichia coli, numerous studies have show n that an excess of positively charged residues defines a cytoplasmic domai n of a membrane protein ("positive inside" rule), The role of negatively ch arged residues in establishing membrane protein topology, however, is not c ompletely understood. To investigate the influence of negatively charged re sidues on this process in detail, we have constructed a single spanning chi meric receptor fragment comprising the N terminus and first transmembrane d omain of the heptahelical G protein-coupled vasopressin V-2 receptor and th e first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane off. coli with its N terminus facing the cytoplasm (N-in-C-out ori entation), although both membrane-flanking domains had rather similar topog enic determinants. The orientation of the receptor fragment was changed aft er the introduction of single glutamate residues into the N terminus. Orien tation inversion, however, was found to be dependent on the location of the glutamate substitutions, which had to lie within a narrow window up to 6 r esidues distant from the transmembrane domain. These results demonstrate th at a single negatively charged residue can play an active role as a topogen ic determinant of membrane proteins in the inner membrane of E. coli, but o nly if it is located adjacent to a transmembrane domain.