Assembly of a cytoplasmic membrane protein in Escherichia coli is dependent on the signal recognition particle

Abstract

Targeting of the cytoplasmic membrane protein leader peptidase (Lep) and a Lep mutant (Lep-inv) that inserts with an inverted topology compared to the wild-type protein was studied in Escherichia coli strains that are conditional for the expression of either Fill or 4.5S RNA, the two components of the E. coli SRP. Depletion of either component strongly affected the insertion of both Lep and Lep-inv into the cytoplasmic membrane. This indicates that SRP is required for the assembly of cytoplasmic membrane proteins in E. coil K, y words: Escherichia coli; Signal recognition particle; Membrane protein; Leader peptidase; Protein targeting ln~oducfionTargeting of secretory proteins to the cytoplasmic memb~ ane of E. coli can follow different pathways that probably c~nverge at the membrane embedded translocation machinery, the translocon [1][2][3]. The so-called SRP pathway involves the signal recognition particle (SRP), a complex of a 4.5S R NA and the Ffh protein which are homologous to the 7S R NA and 54 kDa constituents of the mammalian SRP, respectively [2,3]. FtsY, an E. coli homologue of the mammalian SRP receptor, has been identified on the basis of sequence similarity [4] and has been found to have affinity for the E. c~li SRP in vitro [5]. The SRP subunits 4.5S RNA and Ffh as ~' 11 as their receptor FtsY are essential for viability and their depletion results in defective protein secretion [6][7][8][9].In E. coli there seems to be a correlation between the affi~Lity of a signal sequence for the SRP-targeting pathway and tl~e hydrophobicity of the signal sequence core region [10]. Tfis seems to hold also for the SRP-targeting pathways in 5tceharomyces cerevisiae [11] and chloroplasts (S. High, pers~,nal communication). Whether the SRP is also involved in the targeting and assembly of cytoplasmic membrane proteins fi E. coli is not known, however. Based on the relatively strong hydrophobicity of signal anchor domains and their ability to be cross-linked to SRP in vitro, it has been proposed tl at, like in mammalian cells, membrane proteins are targeted v a the SRP pathway [10]. A role for the SRP in the targeting *~ 7orresponding author. Fax: (46)(8) 153679. E mail: gunnar@biokemi.su.seAbbreviations." Ffh, Fifty-four homologue; SRP, signal recognition particle; Lep, leader peptidase; LacY, lactose permease; IPTG, isopropyl-l-thio-~-D-galactopyranoside; PMSF, phenylmethylsulfonyl flaoride; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel el zetrophoresis of the cytoplasmic membrane protein lactose permease (LacY) has been suggested using an indirect approach [12]. In addition, overproduction of another cytoplasmic membrane protein, leader peptidase (Lep), results in reduced levels of free SRP and a SRP depletion secretion phenotype (our unpublished data).In this study, the membrane assembly of wild-type Lep and a Lep mutant (Lepqnv) [13] that inserts with an inverted topology compared to the wild-type protein has been studied in E. coli strains that are conditional for the expression of either Ff...

Association of Escherichia coli ribosomes with the inner membrane requires the signal recognition particle receptor but is independent of the signal recognition particle

“…Consistent with the conclusion that ribosomes do not bind nonspecifically to the membrane, previous studies (27) on the interaction between eukaryotic ribosomes and target membranes demonstrate that under physiological conditions, most nonspecific membrane binding of ribosomes is lost. Thus, we favor the hypothesis that the lethal effect of Ffh depletion is not due to defective ribosome targeting to the membrane but is caused possibly by a role of Ffh in assembly of membrane proteins (9)(10)(11).…”

Structure of TatA Paralog, TatE, Suggests a Structurally Homogeneous Form of Tat Protein Translocase That Transports Folded Proteins of Differing Diameter

“…9, center panel). Control experiments demonstrate that permeabilization of the spheroplast membrane with the detergent Triton X-100 renders both TatE and TatA susceptible to digestion by proteinase K. As a control for the efficacy of the proteinase K treatment, we tested for degradation of the periplasmic loop of leader peptidase as demonstrated previously (37). The data in Fig.…”

Biogenesis of MalF and the MalFGK2 Maltose Transport Complex in Escherichia coli Requires YidC

“…OmpA, outer membrane protein A, served as a positive control for spheroplast formation by monitoring the proteinase K sensitivity of its periplasmic domain (42). Band X, a cytoplasmic protein, is a negative control.…”