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 distribution of charged residues in the sequences flanking the transmembrane domains. For the inner membrane of Escherichia coli, numerous studies have shown that an excess of positively charged residues defines a cytoplasmic domain of a membrane protein ("positive inside" rule). The role of negatively charged residues in establishing membrane protein topology, however, is not completely understood. To investigate the influence of negatively charged residues on this process in detail, we have constructed a single spanning chimeric receptor fragment comprising the N terminus and first transmembrane domain of the heptahelical G protein-coupled vasopressin V(2) receptor and the first cytoplasmic loop of the beta(2)-adrenergic receptor. When fused to alkaline phosphatase (PhoA), the receptor fragment inserted into the inner membrane of E. coli with its N terminus facing the cytoplasm (N(in)-C(out) orientation), although both membrane-flanking domains had rather similar topogenic determinants. The orientation of the receptor fragment was changed after the introduction of single glutamate residues into the N terminus. Orientation 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 residues distant from the transmembrane domain. These results demonstrate that a single negatively charged residue can play an active role as a topogenic determinant of membrane proteins in the inner membrane of E. coli, but only if it is located adjacent to a transmembrane domain.
Rutz
C
C
Forschungsinstitut für Molekulare Pharmakologie, Alfred-Kowalke-Strasse 4, D-10315 Berlin, Germany.
Rosenthal
W
W
Schülein
R
R
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase