ER translocation intermediates are adjacent to a nonglycosylated 34-kD integral membrane protein.

Abstract

Abstract. We have used the homobifunctional crosslinking reagent disuccinimidyl suberate (DSS) to identify proteins that are adjacent to nascent polypeptides undergoing translocations across mammalian rough ER . Translocation intermediates were assembled by supplementing cell free translations of truncated mRNAs with the signal recognition particle (SRP) and microsomal membrane vesicles . Two prominent crosslinked products of 45 and 64 kD were detected . The 64-kD product was obtained when the cell free translation contained SRP, while formation of the 45-kD product required both SRP and translocation competent microsomal membrane vesicles . In agreement with previous investigators, we suggest that the 64-kD T HE signal sequence of proteins destined for translocatlon across or integration into the rough ER (RER)' contains the information that initiates sequential interactions with cytoplasmic and membrane bound components of the translocation apparatus . The cytoplasmic ribonucleoprotein signal recognition particle (SRP) cotranslationally selects proteins with RER signal sequences by binding to the nascent polypeptide when the signal sequence emerges from the large ribosomal subunit (Walter and Blobel, 19ß1b ;Walter and Blobel, 1982;Walter et al ., 1981) . The signal sequence recognition activity of SRP has been localized to the 54-kD subunit (SRP54) by photo-cross-linking experiments (Krieg et al., 1986;Kurzchalia et al., 1986) . The affinity of the SRP-ribosome complex for the membrane-bound SRP receptor (or docking protein) results in the selective delivery of the ribosome to the membrane surface (Gilmore et al., 19ß2b ; Meyer et al ., 19ß2a ; Walter and Blobel, 198 la), SRP receptor displaces SRP from the signal sequence in a GTPdependent reaction that allows the subsequent membrane insertion of the nascent polypeptide Gilmore and Blobel, 1983) . Although translocation of the polypeptide across the membrane bilayer is a poorly understood process, the preponderance of current experimental evidence suggests that nascent polypeptides undergoing transport are in contact with integral membrane proteins that 1. Abbreviations used in this paper: NEM, N-ethylmaleimide ; RER, rough endoplastnic reticulum; SRP, signal recognition particle .

The Sec61 complex is essential for the insertion of proteins into the membrane of the endoplasmic reticulum

“…We have used a photocross-linking approach to identify these components and found that the major photocross-linking partner of nascent type I and type II signalanchor proteins is an integral membrane protein denoted Sec61~ [7]. Other groups have identified non-glycosylated proteins of between 34 and 39 kDa as being adjacent to nascent membrane proteins during insertion into the ER [8,9]. Although formal identification of these proteins remains to be made it seems likely that these proteins are Sec61~.…”

Sec61alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F9 teratocarcinoma cells after retinoic acid treatment

“…However, several investigators have provided evidence that the ER membrane glycoproteins TRAM and the Sec61p complex are components of the mammalian ER translocon that participates in protein translocation (2). These translocon proteins may associate with ER proteins during the translocation of secretory proteins.…”

“…This protein-conducting channel consists of ER-associated proteins forming a translocon, which includes the translocon-associated protein, the translocating chain-associating membrane protein (TRAM) and the Sec61p complex (1,2). TRAM and Sec61p are tightly associated with membrane-bound ribosomes, suggesting that polypeptide nascent chains pass directly from the ribosome into a protein-conducting channel (3,4).…”

“…Secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel (1,2). This protein-conducting channel consists of ER-associated proteins forming a translocon, which includes the translocon-associated protein, the translocating chain-associating membrane protein (TRAM) and the Sec61p complex (1,2).…”

Glycoprotein Folding in the Endoplasmic Reticulum

“…Sec61α is the largest component of the complex, spanning the membrane 10 times. The use of photo-crosslinking in tandem with modified mRNAs encoding cross-linkable and prematurely truncated polypeptides has shown that Sec61 is intimately associated with the translocating polypeptide (Mothes et al, 1994) (Kellaris et al, 1991) (High et al, 1993). Diffusion studies have suggested that the pore has a size of 40 to 60 Å (Hamman et al, 1997).…”