Detergent extracts of canine pancreas rough microsomal membranes were depleted of either the signal recognition particle receptor (SR), which mediates the signal recognition particle (SRP)-dependent targeting of the ribosome/nascent chain complex to the membrane, or the signal sequence receptor (SSR), which has been proposed to function as a membrane bound receptor for the newly targeted nascent chain and/or as a component of a multi-protein translocation complex responsible for transfer of the nascent chain across the membrane. Depletion of the two components was performed by chromatography of detergent extracts on immunoaffinity supports. Detergent extracts lacking either SR or SSR were reconstituted and assayed for activity with respect to SR dependent elongation arrest release, nascent chain targeting, ribosome binding, secretory precursor translocation, and membrane protein integration. Depletion of SR resulted in the loss of elongation arrest release activity, nascent chain targeting, secretory protein translocation, and membrane protein integration, although ribosome binding was unaffected. Full activity was restored by addition of immunoaffinity purified SR before reconstitution of the detergent extract. Surprisingly, depletion of SSR was without effect on any of the assayed activities, indicating that SSR is either not required for translocation or is one of a family of functionally redundant components.
Proteoliposomes were reconstituted by detergent dialysis of a sodium cholate extract of inverted vesicles derived from Escherichia coli plasma membrane. The translocation of precursor proteins into reconstituted vesicles occurred at high efficiency and was SecB dependent. The protein composition of the reconstituted vesicles differed markedly from that of native vesicles. Immunoblot analysis of the sodium cholate extract and of the reconstituted vesicles indicated that PrlA (SecY) protein remained largely unsolubilized under the described conditions and was virtually absent from the reconstituted vesicles, suggesting that PrlA may not be required for in vitro translocation.A variety of mutant selection schemes have been devised to identify the proteins that mediate, either directly or indirectly, protein export in Escherichia coli. Through genetic analysis five genes, secA (1, 2), secB (3, 4), secD (5), secE (6, 7), and priA (secY) (8-11) have been identified as essential for protein translocation. The products of the genes secA and secB are soluble proteins of molecular mass 102 kDa (12, 13) and 16 kDa (14-16), respectively. Purified SecA has been shown to restore translocation activity to vesicles derived from a secA amber mutant and thus appears required for protein export (12). Purified SecB has been observed to bind to the signal sequence of precursor proteins (17), although other reports have suggested that SecB binds to the mature portion (18,19).SecD, SecE, and PrlA (SecY) are integral membrane proteins of as yet unknown function. priA was originally identified as a suppressor of signal sequence mutations of pre-LamB (8, 9) and, therefore, was suggested to interact directly with the signal sequence (8,9,20,21). Moreover, data from cell-free translocation systems (22-24) strongly suggest a role for PrLA in protein export. Recently, signal sequence mutation suppressors were shown to be tightly linked to the secE gene, suggesting direct interaction between SecE and signal sequences (25). No explicit function has been ascribed to the secD product.A biochemical description of the protein-mediated events occurring at and within the membrane during translocation requires solubilization and reconstitution of translocationcompetent vesicles. Using procedures similar to those we had developed for reconstitution of translocation-competent vesicles from cholate-solubilized canine rough microsomes (26), we report here reconstitution of translocation-competent vesicles from cholate-solubilized E. coli inverted vesicles (INV). MATERIALS AND METHODSPreparation of INV. INV were prepared from E. coli strain MRE600 according to Schnaitman (27). To remove peripheral proteins, INV were diluted with an equal volume of high-salt buffer (0.25 M sucrose/50 mM triethanolamine acetate (pH 7.5)/2 M KOAc/1 mM dithiothreitol) and incubated on ice for 1 hr. Washed vesicles were collected by centrifugation through a cushion consisting of 0.5 M sucrose/50 mM triethanolamine acetate (pH 7.5)/1 M KOAc/1 mM dithiothreitol for 2 hr ...
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