Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli.
Reconstitution of the translocation machinery for secretory proteins from purified constituents was performed. SecY was solubilized from SecY/SecE-overproducing Escherichia coli cells and purified by chromatography on ion-exchange and size-exclusion columns. Proteoliposomes active in protein translocation were reconstituted from the purified preparations of SecY and SecE. The reconstituted translocation activity was SecA- and ATP-dependent. Although the purified preparations of SecY and SecE were still contaminated with minute amounts of other proteins, the elution profiles of SecY and SecE on column chromatographies coincided with the elution profiles of reconstituted translocation activity, indicating that SecY and SecE are the indispensable components in these preparations. We conclude that SecY, SecE, and SecA are essential components of the protein secretion machinery and that translocation activity can be reconstituted from only these three proteins and phospholipids.
Akimaru
J
J
Institute of Applied Microbiology, University of Tokyo, Japan.
Matsuyama
S
S
Tokuda
H
H
Mizushima
S
S
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D008081:Liposomes; D026901:Membrane Transport Proteins; D008970:Molecular Weight; D010743:Phospholipids / Q000378:metabolism*; D014176:Protein Biosynthesis; D011510:Proteolipids / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008081:Liposomes; D026901:Membrane Transport Proteins; D010743:Phospholipids; D011510:Proteolipids; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; C020485:proteoliposomes; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins