In vitro membrane assembly of a polytopic, transmembrane protein results in an enzymatically active conformation.

Abstract

Abstract. In vitro integration of the polytopic, transmembrane lactose permease into membrane vesicles from Escherichia coli is demonstrated. To this end the enzyme was synthesized in a homologous, cell-free transcription-translation system. In this system, synthesis occurred in an essentially membrane-free environment leading to the formation of lactose permease aggregates, which were resistant to protease digestion and detergent solubilization. However, if inverted membrane vesicles from E. coli were included in the synthesis reaction, most de novo-synthesized lactose permease could be recovered from a membranecontaining subfraction (enriched in leader [signal] peptidase activity). This membrane association of lactose permease was Na2CO3 resistant, detergent sensitive, and yielded a distinct pattern of proteolytic cleavage peptides. Moreover, membrane vesicles when present cotranslationally during synthesis of lactose permease, acquired the capability to accumulate lactose, strongly suggesting a correct in vitro assembly of the enzyme.Because of the extensive aggregation of lactose permease synthesized in the absence of membranes, only low amounts originating from the soluble enzyme pool integrated posttranslationally into the membrane vesicles. Unlike the translocation of the outer membrane protein LamB into membrane vesicles, integration of lactose permease was found to be independent of the H+-motive force.

Secondary solute transport in bacteria

“…It has been suggested that hydrophobic proteins are prevented from aggregation in the cytoplasm in vivo by a mechanism which avoids the release of nascent polypeptides from the ribosome until the membrane insertion process has been initiated [159]. The mechanism, however, by which polytopic membrane proteins insert into the cytoplasmic membrane is far from understood.…”

“…In a study of the in vitro membrane assembly of the LacY protein it has been shown that the protein aggregates in the absence of membranes, but that it can integrate posttranslationally into membranes in an enzymatically active conformation independent of a Ap [159,165]. Membrane insertion independent of Ap in an in vitro assay has also been suggested for the IICBA protein (MtlA) of the mannitol PTS of E. coli [166].…”

Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY

“…Using a cell-free system, we previously demonstrated translocation across and integration into the lipid bilayer of INV from E. coli of a variety of periplasmic, outer-membrane (Miiller and Blobel, 1984;Miiller et al, 1987;Krishnabhakdi and Miiller, 1988) and inner-membrane proteins (Ahrem et al, 1989). As recently reported, translocation by membrane vesicles is inhibited by a previous exposure to trypsin due to loss of precursor targeting (Swidersky et al, 1990).…”

“…Preparation of INV, trypsin treatment of INV, subfractionation of translation products on a two-step sucrose gradient, translocation of the bound preLamB into membrane vesicles, SDS/PAGE (SecY-containing samples were denatured at 37°C) and fluorography, and quantitative determination of radioactivity contained in bands on SDS/ polyacrylamide gels were performed as previously described (Swidersky et al, 1990). Immunoprecipitation of [35S]methionine-labeled translation products were described in Ahrem et al (1989). The preparation of antibodies against the 22-amino-acid synthetic NH2-terminal peptide of SecY has been described elsewhere (Watanabe and Blobel, 1989).…”

“…After a 60-min incubation, aggregated material was removed by centrifugation in a tabletop microfuge, and the supernatant was incubated with 100 pl antiserum at 4 'C overnight. Antigen/antibody complexes were then bound to protein-A -Sepharose and further processed as described (Ahrem et al, 1989).…”