D. Kiefer et al., NEGATIVELY CHARGED AMINO-ACID-RESIDUES PLAY AN ACTIVE-ROLE IN ORIENTING THE SEC-INDEPENDENT PF3 COAT PROTEIN IN THE ESCHERICHIA-COLI INNER MEMBRANE, EMBO journal, 16(9), 1997, pp. 2197-2204
The coat protein of Pseudomonas aeruginosa phage Pf3 is transiently in
serted into the bacterial inner membrane with a single transmembrane a
nchor sequence in the NoutCin orientation. The N-terminal sequence imm
ediately flanking the membrane anchor contains one negatively charged
residue, whereas the C-terminal hydrophilic segment has two positively
charged residues, To investigate how the orientation of this protein
is achieved, the three flanking charged amino acid residues were alter
ed, Membrane insertion was analyzed in vivo using the accessibility to
externally added protease and in vitro by testing the insertion into
inverted Escherichia coil membrane vesicles, In both systems, the orie
ntation of the protein was completely reversed for the oppositely char
ged mutant coat protein (RD mutant). In addition, we show in vivo that
the electrochemical membrane potential is necessary for the transloca
tion of both the wild type and the mutant Pf3 coat proteins, suggestin
g that membrane insertion is driven by electrophoretic forces.