A positively charged region is a determinant of the orientation of cytoplasmic membrane proteins in Escherichia coli.
Basic amino acid residues were introduced into an extracellular (periplasmic) domain, preceding a membrane-spanning hydrophobic domain, of SecY, an integral cytoplasmic membrane protein. The localization of the domain was monitored as to the alkaline phosphatase activity of TnPhoA fused adjacent to the domain. The alkaline phosphatase activity of such Escherichia coli cells drastically decreased when positive charges were introduced, indicating that on the introduction the SecY domain showed a change in localization from the periplasm to the cytoplasm. In another experiment, positive charges were introduced to the same periplasmic domain of another SecY-PhoA fusion protein, in which PhoA is fused to the cytoplasmic domain of SecY following the particular hydrophobic domain. The alkaline phosphatase activity increased drastically when positive charges were introduced, indicating that the SecY domain fused to PhoA showed a change in localization from the cytoplasm to the periplasm. In both experiments, the removal of a large amino-terminal portion of the SecY domain did not alter the effect of the positive charge introduction. Changes in localization of SecY domains thus demonstrated were also supported by a protease accessibility test on spheroplasts. It is proposed that a positively charged region adjacent to a membrane-embedded hydrophobic region tends to be stabilized on the cytoplasmic surface of the membrane, which in turn endows the hydrophobic region with the ability to act as a stop-transfer sequence or a signal sequence and consequently determines the orientation of the hydrophobic region in the membrane.
Yamane
K
K
Institute of Applied Microbiology, University of Tokyo, Japan.
Akiyama
Y
Y
Ito
K
K
Mizushima
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008961:Models, Structural; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins