To ensure that newly synthesized proteins do not misfold and aggregate, the E. coli cell contains molecular chaperones. These chaperones have an important function in protein transport and prevent preproteins from folding into stable tertiary structures that are incompatible with translocation (Kumamoto, 1991). The Gram-negative bacterial protein SecB is a translocation-dedicated molecular chaperone that exists as a homotetramer of 16 kDa subunits (Fekkes and Driessen, 1999). It binds to the mature part of preproteins and facilitates their targeting to the translocase because of its affinity for the SecA protein, but is released immediately after the initiation of translocation (Hartl et al., 1990; Fekkes et al., 1997). SecB has also been implicated in the post-translational translocation of some extracellular proteins that are secreted by dedicated ABC (ATP binding cassette) transport systems (Delepelaire and Wandersman, 1998).
An alternative targeting route to the cytoplasmic membrane for precursor proteins is mediated by the signal recognition particle (SRP). The constituents of this ribonucleoprotein, a 48 kDa protein termed p48 or Ffh (fifty-four homologue) and a 4.5S RNA molecule, were identified on the basis of homology with subunits from the eukaryotic SRP involved in the translational arrest and targeting of ribosome–nascent chain complexes (RNCs) to the ER membrane (Poritz et al., 1988; Römisch et al., 1989). The E. coli SRP recognizes preprotein signal sequences and hydrophobic regions of nascent membrane proteins (Luirink et al., 1992; Valent et al., 1997). Together with its receptor, the GTPase FtsY, it is a functional substitute of the eukaryotic SRP-targeting pathway (Powers and Walter, 1997). Membrane lipid association activates FtsY for GTPase activity (De Leeuw et al. 2000), and this may regulate the SRP-mediated targeting of the RNCs. Upon the FtsY- and GTP-dependent release from SRP, RNCs can be cross-linked to SecA and the SecYEG complex present in isolated inner membranes, suggesting that the SecB- and SRP-mediated targeting routes converge at the translocase (Valent et al., 1998). The cross-linking of RNCs to SecA may result from their mutual affinity for the SecYEG complex and not represent a functional interaction, as targeting and transfer of the nascent chain from SRP to SecYEG also occurs in SecA-free membrane fractions or SecYEG proteoliposomes (Koch et al., 1999; Scotti et al., 1999). In contrast, for at least one SRP substrate, the AcrB protein, it has been demonstrated that the in vivo membrane targeting is dependent on SecA (Qi and Bernstein, 1999). These paradoxical results suggest that targeting to the cytoplasmic membrane is mediated by several, possibly overlapping interactions between preproteins, targeting factors, membrane receptors and translocase subunits. Physicochemical properties of the substrates may determine their preferred targeting route and dependency on the SecA protein for targeting and/or translocation. Several secretory proteins and, especially, cytoplasmic membrane proteins use the SRP-mediated pathway of targeting to the cytoplasmic membrane (Phillips and Silhavy, 1992; Luirink et al., 1994; Ulbrandt et al., 1997; Beck et al., 2000). Owing to their high hydrophobicity, cytoplasmic membrane proteins are more prone to aggregation and may therefore be more dependent on a co-translational targeting pathway (De Gier et al., 1997), although it should be remarked that a translational arrest of nascent polypeptide synthesis has not been demonstrated for the E. coli SRP. Direct binding of the ribosome to the SecYEG complex (Prinz et al., 2000) will also add to the proper targeting of membrane proteins. Nascent secretory proteins, on the other hand, interact with trigger factor, a cis-trans proline isomerase that competes for binding with SRP, and are routed into the SecA/SecB-mediated post-translational targeting pathway (Valent et al., 1995; 1997; Beck et al., 2000).