Quido A. Valent

The Escherichia coli SRP and SecB targeting pathways converge at the translocon

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 pre‐protein 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.

Early events in preprotein recognition in E. coli: interaction of SRP and trigger factor with nascent polypeptides.

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.

Nascent membrane and presecretory proteins synthesized in Escherichia coli associate with signal recognition particle and trigger factor

The 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.

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

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 Ffh 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. coli.

SecA is not required for signal recognition particle-mediated targeting and initial membrane insertion of a nascent inner membrane protein

In Escherichia coli, signal recognition particle (SRP)-dependent targeting of inner membrane proteins has been described. In vitrocross-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 vitroreconstitution of SRP-mediated membrane insertion in which purified ribosome-nascent 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.

Chloroplast SRP54 Interacts with a Specific Subset of Thylakoid Precursor Proteins

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 E. coli SRP: preferences of a targeting factor

Research on the targeting of proteins to the cytoplasmic membrane of E. coli has mainly focused on the so‐called ‘general secretory pathway’ (GSP) which involves the Sec‐proteins. Recently, evidence has been obtained for an alternative targeting pathway in E. coli which involves the signal recognition particle (SRP). The constituents of this SRP pathway in E. coli are homologous to those of the well‐characterized eukaryotic SRP pathway, which is the main targeting pathway for both proteins translocated across and inserted into the endoplasmic reticulum membrane. However, until recently, no clear function could be assigned to the SRP in E. coli. New studies point to an important role of the E. coli SRP in the assembly of inner membrane proteins.

Subcellular localization and topology of the K88 usher FaeD in Escherichia coli.

The subcellular localization of the K88 usher FaeD was studied in Escherichia coli whole ceils by using iso‐pycnic sucrose density gradient centrifugation of isolated membranes, the detergents Triton X‐100 and sodium lauryl sarcosinate and immunoblotting with a specific FaeD antiserum. Cells containing the complete K88 operon, as well as cells containing the sub‐cloned faeD gene in various expression vectors, were used. Most of the FaeD was present in the outer membranes in a detergent‐resistant form. Agglutination experiments with E coli cells expressing FaeD confirmed an outer membrane localization and indicated the presence of FaeD at the cell surface. Automated Edman degradation indicated that the mature FaeD contained 777 amino acid residues and confirmed that FaeD is synthesized with a rather long signal sequence of 35 amino acid residues. Twelve different FaeD–PhoA fusion proteins were prepared and characterized by nucleotide sequencing and immuno‐blotting. Most of these fusion sites were located in the amino‐terminal and carboxyl‐terminal regions of FaeD. Six amino‐terminal fusion proteins were soluble proteins in the peripiasm, whereas the other fusion proteins were associated with the outer membrane. The protease accessibility of FaeD and of the six outer membrane‐bound FaeD–PhoA fusion proteins was studied using whole cells, cells with permeabilized outer membranes, and isolated membranes. Collagenase H, kallikrein, trypsin and proteinase K were used. Based on the results of these experiments and computer predictions, a model for the membrane topology of FaeD was developed in which FaeD contains a large central domain containing 24 membrane‐spanning segments and two relatively large periplasmic regions, at the amino‐terminal and carboxyl‐terminal end of the protein, respectively.

Stability and function of the signal peptide of the pCloDF13-derived bacteriocin release protein.

The pCloDF13-derived bacteriocin release protein (BRP) is synthesized as a prelipoprotein with a signal peptide which remains stable after processing. This signal peptide accumulates in the cytoplasmic membrane and is, together with the mature BRP, required for efficient release of cloacin DF13. We investigated the structural requirements for stability of the BRP signal peptide by constructing hybrid signal peptides consisting of parts of the BRP and Lpp signal peptides. Signal peptide stability was investigated by pulse-labelling and pulse-chase experiments. To study the functioning of the BRP signal peptide, the hybrid constructs were tested for their ability to promote BRP-mediated cloacin DF13-release and their ability to affect the viability of the host cells. The results obtained suggest that the N-terminal part of the BRP signal peptide together with the C-terminal alanine residue are important for stability. When expressed as a separate entity, all mutant signal peptides that contain a part of the BRP signal peptide are capable of affecting cell viability. The results indicated a possible correlation between stability of the BRP signal peptide and cloacin DF13-release.