Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates preprotein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.

In Escherichia coli, components of a signal recognition particle (SRP) and its receptor have been identified which appear to be essential for efficient translocation of several proteins. In this study we use cross‐linking to demonstrate that E. coli SRP interacts with a variety of nascent presecretory proteins and integral inner membrane proteins. Evidence is presented that the interaction is correlated with the hydrophobicity of the core region of the signal sequence and thereby with its ability to promote transport in vivo. A second E. coli component, which is identified as trigger factor, can be efficiently cross‐linked to all tested nascent chains derived from both secreted and cytosolic proteins. We propose that SRP and trigger factor act as secretion‐specific and general molecular chaperone respectively, early in protein synthesis.

SummaryThe Escherichia coli signal recognition particle (SRP) and trigger factor are cytoplasmic factors that interact with short nascent polypeptides of presecretory and membrane proteins produced in a heterologous in vitro translation system. In this study, we use an E. coli in vitro translation system in combination with bifunctional cross-linking reagents to investigate these interactions in more detail in a homologous environment. Using this approach, the direct interaction of SRP with nascent polypeptides that expose particularly hydrophobic targeting signals is demonstrated, suggesting that inner membrane proteins are the primary physiological substrate of the E. coli SRP. Evidence is presented that the overproduction of proteins that expose hydrophobic polypeptide stretches, titrates SRP. In addition, trigger factor is efficiently cross-linked to nascent polypeptides of different length and nature, some as short as 57 amino acid residues, indicating that it is positioned near the nascent chain exit site on the E. coli ribosome.

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...

Assembly of several inner membrane proteins-leader peptidase (Lep), a Lep derivative (Lep-inv) that inserts with an inverted topology compared with the wild-type protein, the phage M13 procoat protein, and a procoat derivative (H1-procoat) with the hydrophobic core of the signal peptide replaced by a stretch from the first transmembrane segment in Lep-has been studied in vitro and in Escherichia coli strains that are conditional for the expression of either the 54 homologue (Ffh) or 4.5S RNA, which are the two components of the E. coli signal recognition particle (SRP), or SecE, an essential core component of the E. coli preprotein translocase. Membrane insertion has also been tested in a SecB null strain. Lep, Lep-inv, and H1-procoat require SRP for correct assembly into the inner membrane; in contrast, we find that wild-type procoat does not. Lep and, surprisingly, Lep-inv and H1-procoat fail to insert properly when SecE is depleted, whereas insertion of wild-type procoat is unaffected under these conditions. None of the proteins depend on SecB for assembly. These observations indicate that inner membrane proteins can assemble either by a mechanism in which SRP delivers the protein at the preprotein translocase or by what appears to be a direct integration into the lipid bilayer. The observed change in assembly mechanism when the hydrophobicity of the procoat signal peptide is increased demonstrates that the assembly of an inner membrane protein can be rerouted between different pathways.

Targeting and assembly of the Escherichia coli inner membrane protein leader peptidase (Lep) was studied using a homologous in vitro targeting/translocation assay. Assembly of full-length Lep was efficient in the co-translational presence of membrane vesicles and hardly occurred when membranes were added post-translationally. This is consistent with the signal recognition particle-dependent targeting of Lep. Crosslinking experiments showed that the hydrophilic region P1 of nascent membrane-inserted Lep 100-mer was in the vicinity of SecA and SecY, whereas the first transmembrane domain H1 was in the vicinity of YidC. These results suggested that YidC, together with the Sec translocase, functions in the assembly of Lep. YidC might be a more generic component in the assembly of inner membrane proteins. ß

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitro cross-linking studies have demonstrated that short nascent chains exposing a highly hydrophobic targeting signal interact with the SRP. This SRP, assisted by its receptor, FtsY, mediates the transfer to a common translocation site in the inner membrane that contains SecA, SecG, and SecY. Here we describe a further in vitro reconstitution of SRP-mediated membrane insertion in which purified ribosomenascent chain-SRP complexes are targeted to the purified SecYEG complex contained in proteoliposomes in a process that requires the SRP-receptor FtsY and GTP. We found that in this system SecA and ATP are dispensable for both the transfer of the nascent inner membrane protein FtsQ to SecY and its stable membrane insertion. Release of the SRP from nascent FtsQ also occurred in the absence of SecYEG complex indicating a functional interaction of FtsY with lipids. These data suggest that SRP/FtsY and SecB/SecA constitute distinct targeting routes.Going across or integrating into the inner membrane presents two different challenges to proteins synthesized in the cytosol of a prokaryotic cell, and this is reflected by the existence of two main targeting routes. The SecB pathway is specialized for the targeting of periplasmic and outer membrane proteins. SecB is a cytosolic chaperone that binds to the mature region of a subset of preproteins (1). The SecB-preprotein complex is targeted to SecA, which is bound with high affinity to the membrane-embedded SecYEG complex (for review, see Ref.2).Both in vivo and in vitro experiments indicate that the signal recognition particle (SRP) 1 pathway is primarily used for the targeting of integral inner membrane proteins (3-7). This pathway resembles SRP-mediated targeting of proteins to the membrane of the endoplasmic reticulum (ER) in eukaryotes (for review, see Ref. 8). The eukaryotic SRP interacts with nascent membrane and secreted proteins and targets them to the SRP receptor SR␣ at the ER membrane. The eukaryotic SRP is a complex consisting of six proteins arranged on an RNA scaffold, the 7 S RNA. Escherichia coli contains a smaller SRP composed of the P48 protein and the 4.5 S RNA, which are homologous to the eukaryotic SRP54 and the 7 S RNA, respectively (for review, see Refs. 9 and 10). In addition, an SR␣ homologue has been identified in E. coli, designated FtsY (7, 11). Both P48 and FtsY (and their eukaryotic counterparts) are GTPases, and GTP binding and hydrolysis regulate the targeting cycle in a mechanism that has not yet been fully defined (12)(13)(14). In vivo overproduction of polytopic inner membrane proteins titrated out the limited amount of endogenous E. coli SRP (5), whereas depletion of essential SRP components affected membrane targeting of both polytopic and bitopic inner membrane proteins (3, 4).We have developed an in vitro cross-linking approach to dissect subsequent stages in SRP-mediated protein targeting. Short nascent inner m...

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23-and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences. The signal recognition particle (SRP)1 of mammalian cells is a ribonucleoprotein complex which promotes the signal sequence-dependent targeting of nascent precursor proteins to the endoplasmic reticulum (1-3). Mammalian SRP is composed of six polypeptides and a 7 S RNA, although only one of the polypeptides, the 54-kDa subunit (SRP54), binds to the hydrophobic endoplasmic reticulum (ER)-targeting signals (1-2). Functional homologues of SRP54 have been identified in many organisms and appear relatively conserved during evolution (4, 5). These proteins are usually found complexed with a 7 S-like RNA and the minimum requirement for SRP-dependent protein targeting seems to be a ribonucleoprotein particle composed of an SRP54-like protein and a 7 S-like RNA (6 -8), together with a cognate receptor for the SRP-precursor protein complex (3, 9, 10). Perturbation of SRP-dependent targeting pathways often leads to the accumulation of secretory proteins. However, in many cases only a subset of precursors accumulate while other proteins continue to be secreted normally (9,(11)(12)(13). This suggests that a discrete population of precursors preferentially utilize an SRP-dependent targeting pathway (1, 4, 13).The delivery of precursor proteins to the thylakoid membrane of chloroplasts is governed by thylakoid-targeting signals. These signals are clearly related to those which target proteins to the ER membrane of eukaroytes and the cytoplasmic membrane of prokaryotes....