A tetrameric complex of membrane proteins in the endoplasmic reticulum

Abstract

The translocation site (translocon), at which nascent polypeptides pass through the endoplasmic reticulum membrane, contains a component previously called 'signal sequence receptor' that is now renamed as 'translocon-associated protein' (TRAP). Tbo glycosylated subunits of the TRAP complex have been identified before (a and P subunits). We now show that the TRAP complex is actually comprised of four membrane proteins (a, P, y, 4, present in a stoichiomemc relation, which are genuine neighbours in intact microsomes. The amino acid sequences of the additional, nonglycosylated subunits were deduced from cloning of the corresponding cDNAs. The 6 subunit spans the membrane only once and has its major portion, containing a disulfide bridge, at the lumenal side. The y subunit is predicted to span the membrane four times.Proteins are translocated across the endoplasmic reticulum (ER) membrane at specific sites (translocons [l]), probably through protein-conducting channels (for review see 121). To identify membrane proteins located in the vicinity of translocating polypeptides, crosslinking methods have been applied [3 -91. Short translocating polypeptide chains, representing an early phase of the translocation process, can be crosslinked through their signal sequence to an integral, glycosylated membrane protein that is about 35 kDa and has a cytoplasmic tail of about 5 kDa [3]. Based on these properties, the 'signal sequence receptor' (a subunit) was purified [lo] and received its name on the assumption that it was identical with the major crosslinking partner of short nascent chains. However, it is now clear that both this protein and the recently discovered 'translocating-chain-associating membrane' (TRAM) protein [8] (TRAP) since the protein, although not a signal sequence receptor, seems to be located at the translocation site. The a subunit of TRAP (TRAPa) can be crosslinked to a minor extent to various translocating chains, and the proportion of TRAPa among glycoprotein crosslinks appears to increase as chain length increases [8]. TRAPa is segregated to the rough portion of the ER [12], it can be crosslinked to membranebound ribosomes [13] and TRAP is associated in part with ribosomes after solubilization of rough microsomes with detergents [9]. The protein is a major constituent of the ER membrane of various species [lo]. Finally, antibodies against TRAPa and Fab fragments prepared from the antibodies inhibit the in vitro translocation of several secretory proteins [lo].The amino acid sequence of TRAPa was deduced from cloning of its cDNA and indicates that it is a single-spanning membrane protein [14]. Employing Nonidet P-40 as a detergent to solubilize canine pancreatic microsomes, it was found that TRAPa is tightly and stoichiometrically associated with a second protein, called TRAPP {previously SSRB) [6]. The cDNA of TRAPD has also been cloned and codes for a glycosylated, single-spanning membrane protein [6].In the present study, we have used digitonin as a milder detergent to solubilize the membranes a...

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of “ER-resident” molecular chaperones

“…2C), all of which are associated with a macromolecular complex (translocon) involved in the translocation of nascent proteins from the cytosol to the ER lumen or their cotranslational modification (44)(45)(46). We could clearly detect surface expression of both ribophorin I and ribophorin II, although detection of ribophorin I expression required longer exposure times than that for ribophorin II.…”

Substrate-specific regulation of the ribosome– translocon junction by N-terminal signal sequences

“…However, variations on the above paradigm seem likely given the enormously diverse set of sequences that serve as signals for targeting and translocation (20), and the complex interactions of these signals with both cytosolic and ER proteins (10,15,17,(21)(22)(23). In this study, we have focused on the critical, but poorly understood, posttargeting steps of signal sequence function.…”

The Topogenic Contribution of Uncharged Amino Acids on Signal Sequence Orientation in the Endoplasmic Reticulum

“…In reconstitution assays, TRAM was found to be particularly important for signals with relatively short N-terminal hydrophilic segments (25). The TRAP (translocon-associated protein) complex has also been shown to be in proximity to translocating signal sequences (28,29), and although it is not essential for translocation, its true role is yet to be elucidated. Recognition of the signal sequence by a binding site in or closely associated with the translocon is likely to be a key event in defining the orientation of the signal and thus whether the N terminus or the C terminus of the protein is translocated across the membrane.…”