title: Purification of SecE and reconstitution of SecE-dependent protein translocation activity.
abstract: SecE was solubilized from SecE-overproducing E. coli cells and purified through ion exchange and size exclusion chromatographies. When the solubilized membrane containing overproduced amounts of SecY and SecE was fractionated by means of size exclusion chromatography, the two proteins were eluted in different fractions with slight overlapping. Proteoliposomes active in protein translocation were reconstituted from these fractions only when both SecE and SecY were present. When reconstitution was carried out with the purified SecE and fractions containing SecY but only a small amount of SecE, the resultant proteoliposomes exhibited appreciable translocation activity, indicating that SecE is essential for protein translocation. The translocation activity of proteoliposomes was proportional to the amount of purified SecE used for reconstitution. SecE-dependent protein translocation absolutely required ATP and SecA.
authors.affiliation: Institute of Applied Microbiology, University of Tokyo, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tokuda
authors.forename: J
authors.initials: J
authors.lastname: Akimaru
authors.forename: S
authors.initials: S
authors.lastname: Matsuyama
authors.forename: K
authors.initials: K
authors.lastname: Nishiyama
authors.forename: S
authors.initials: S
authors.lastname: Mizushima
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000032:analysis* / Q000235:genetics; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D000069816:SEC Translocation Channels
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli