Similarities between S. cerevisiae SEc61p and E. coli SecY Suggest a Common Origin for Protein Translocases of the Eukaryotic ER and the Bacterial Plasma Membrane

The first stage in the eukaryotic secretory pathway is the translocation of polypeptides across the endoplasmic reticulum (ER) membrane. This process has been extensively studied in mammalian systems using an in vitro assay which faithfully reproduces the cotranslational translocation of specific precursor proteins into the ER lumen (Blobel, & Dobberstein, 1975). Such biochemical analyses have revealed that translocation requires both cytosolic and membrane-associated factors, including the cytosolic ribonucleoprotein complex, signal recognition particle (SRP), and its cognate membrane-bound receptor, SRP-receptor (or “Docking protein”; Walter & Blobel, 1980: Meyer et al., 1982). Evidence suggests that SRP binds to the signal sequence of a nascent secretory protein as it emerges from the ribosome, and in doing so effects a reduction in the rate of polypeptide chain elongation (“elongation arrest”, Walter and Blobel, 1981). The arrested complex is then targeted to the ER membrane via an interaction between SRP and the integral membrane protein SRP-receptor (Gilmore et al., 1982; Meyer et al., 1982). SRP-receptor then mediates the GTP-dependent displacement of SRP from the signal sequence/ribosome complex (Connolly & Gilmore, 1989), thus releasing elongation arrest, and enabling the co-translational translocation of the targeted precursor across the.ER membrane. Whilst the SRP-dependent targeting cycle is relatively well characterised, the mechanism by which a targeted polypeptide actually penetrates the lipid bilayer remains obscure. There exists a wealth of data implicating integral membrane proteins in the translocation process, however, the identification and characterisation of these proteins has proven problematic. Several candidates for components of the mammalian ER translocase have been identified in a series of crosslinking studies in which a translocating polypeptide is first trapped in the membrane, and then cross-linked to those proteins in closest proximity. Such analyses have identified an ER membrane protein termed signal sequence receptor (SSR), which interacts directly with the signal peptide of nascent pre-proteins (Weidmann et al., 1987). Current evidence suggest that SSR exists as an oligomeric complex comprising at least two integral membrane glycoproteins, namely SSRα (34K), and SSRβ (22K) (Rapoport, 1990). Moreover, SSRα remains in close proximity to the mature portion of a translocating pre-protein, and may therefore represent a constituent of the translocon per se (Weidmann et al., 1989; Prehn et al., 1990; Rapoport, 1990). More recent cross-linking studies have identified a 37K ER protein (P37) which can be cross-linked to the signal anchor sequence of a type I membrane protein during its insertion into the membrane (High et al., 1991). However, despite the identification of these putative translocon components, their actual contribution to the translocation process remains speculative. A more potent case can be made for the recently identified integral membrane glycoprotein TRAM (for translocating chain-associating membrane protein; Gorlich et al., 1992). TRAM represents the major species found crosslinked to short nascent chains, and may therefore be involved in the early stages of translocation. TRAM is an abundant ER-protein, present at a level at least equivalent to the number of membrane-bound ribosomes, raising the possibility that TRAM represents a constitutive component of the active translocase. A direct role for TRAM in translocation is supported by the observation that addition of the purified protein stimulates the translocation of preprolactin into reconstituted microsomes to levels approaching those of native ER microsomes (Gorlich et al., 1992).