No abstract

The heterotrimeric Sec61p complex is a major component of the protein-conducting channel of the endoplasmic reticulum (ER) membrane, associating with either ribosomes or the Sec62/63 complex to perform co- and posttranslational transport, respectively. We show by electron microscopy that purified mammalian and yeast Sec61p complexes in detergent form cylindrical oligomers with a diameter of approximately 85 A and a central pore of approximately 20 A. Each oligomer contains 3-4 heterotrimers. Similar ring structures are seen in reconstituted proteoliposomes and native membranes. Oligomer formation by the reconstituted Sec61p complex is stimulated by its association with ribosomes or the Sec62/63p complex. We propose that these cylindrical oligomers represent protein-conducting channels of the ER, formed by ligands specific for co- and posttranslational transport.

Abstract. The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61ot, a multi-spanning membrane protein that is associated with two additional membrane proteins (Sec61/~ and 3') to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p-complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes.To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the proteaseresistance of Sec61ot which is shielded by these ribosomes. In contrast, p34 and plS0 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes.These data provide further evidence that the Sec61p-complex is responsible for the membrane-anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process. MANY proteins are targeted to the mammalian ER membrane by means of the signal recognition particle (SRP) t and its membrane receptor (SRPreceptor or docking protein) (for review see Rapoport, 1992). They are subsequently transported across the membrane, presumably through a protein-conducting channel. The main component of this channel may be Sec61p (now named Sec61ot), a multi-spanning membrane protein, originally discovered in S. cerevisiae by genetic screening for translocation defects (Deshaies and Schekman, 1987;Stifling et al., 1992). Sec61, is adjacent to polypeptide chains passing through the ER membrane of yeast and mammals, it is essenAddress all correspondence to Dr. Tom Rapoport, Max-Delbriick-Center for Molecular Medicine, Robert-R6ssle-Street 10, 13125 Berlin-Buch, Federal Republic of Germany.The present address of Dr. Dirk G6rlich, Wellcome/Cancer Research Campaign Institute of Cancer and Developmental Biology, Tennis Court Road, CB2 1QR Cambridge, England.1. Abbreviations used in this paper: eq., equivalents; PK-RM, puromycin/high salt-stripped rough microsomes; RM, rough microsomes; SLR, S-labeling reagent; SRP, signal recognition particle; TRAM protein, translocating chain-associating membrane protein.tial for prote...

In yeast, efficient protein transport across the endoplasmic reticulum (ER) membrane may occur co-translationally or post-translationally. The latter process is mediated by a membrane protein complex that consists of the Sec61p complex and the Sec62p-Sec63p subcomplex. In contrast, in mammalian cells protein translocation is almost exclusively co-translational. This transport depends on the Sec61 complex, which is homologous to the yeast Sec61p complex and has been identified in mammals as a ribosome-bound pore-forming membrane protein complex. We report here the existence of ribosome-free mammalian Sec61 complexes that associate with two ubiquitous proteins of the ER membrane. According to primary sequence analysis both proteins display homology to the yeast proteins Sec62p and Sec63p and are therefore named Sec62 and Sec63, respectively. The probable function of the mammalian Sec61-Sec62-Sec63 complex is discussed with respect to its abundance in ER membranes, which, in contrast to yeast ER membranes, apparently lack efficient post-translational translocation activity.

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the proteinconducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved. Keywords: protein translocation/ribosome/rRNA/SecY/ Sec61 IntroductionProtein translocation across the ER membrane can occur either co-or post-translationally. In the cotranslational pathway, a polypeptide chain is synthesized on a membrane-bound ribosome; the elongating chain is transferred from a tunnel in the ribosome through a channel in the membrane into the lumen of the ER (Crowley et al., 1994;Mothes et al., 1994;Beckmann et al., 1997). In the posttranslational pathway, the chain is ®rst completed in the cytosol before being transported across the membrane. In both cases the same membrane channel is used, formed by three or four copies of the evolutionarily conserved heterotrimeric Sec61p complex. In the cotranslational pathway, the Sec61p channel associates with the ribosome (Go Èrlich and Rapoport, 1993;Kalies et al., 1994;Hanein et al., 1996;Beckmann et al., 1997), and in the posttranslational pathway, at least in the yeast Saccharomyces cerevisiae, it associates with the tetrameric Sec62/63p membrane protein complex to form a heptameric Sec complex (Deshaies et al., 1991;Panzner et al., 1995;Hanein et al., 1996).How the ribosome binds to the Sec61p complex during cotranslational translocation is unknown, and it is unclear whether the interaction involves ribosomal RNA (rRNA), protein or both. Non-translating mammalian ribosomes can bind with high af®nity to the Sec61p complex in mammalian ER membranes (Rolleston, 1972;Borgese et al., 1974;Kalies et al., 1994). This interaction is salt sensitive, and the binding site is likely to be located in the large ribosomal subunit (Rolleston, 1972;Borgese et al., 1974;Unwin, 1977). The association of non-translating ribosomes with the Sec61p complex mimics early stages of cotranslational protein translocation (Jungnickel and Rapoport, 1995;Beckmann et al., 1997;Neuhof et al., 1998). When the signal sequence of a growing nascent chain has just emerged from the ribosome, the ribosome± nascent chain complex is targeted by the signal recognition particle (SRP) to the membrane (Walter and Blobel, 1981) and initially binds to the Sec61p complex in a saltsensitive manner (Jungnickel and Rapoport, 1995). After further chain elongation, the signal sequence is recognized by the channel and inserts into it, and the ribosome± membrane interaction becomes salt resistant (Jungnickel and Rapopor...

The Sec61 complex is the central component of the protein translocation apparatus of the ER membrane. We have addressed the role of the β subunit (Sec61β) during cotranslational protein translocation. With a reconstituted system, we show that a Sec61 complex lacking Sec61β is essentially inactive when elongation and membrane targeting of a nascent chain occur at the same time. The translocation process is perturbed at a step where the nascent chain would be inserted into the translocation channel. However, if sufficient time is given for the interaction of the nascent polypeptide with the mutant Sec61 complex, translocation is almost normal. Thus Sec61β kinetically facilitates cotranslational translocation, but is not essential for it.Using chemical cross-linking we show that Sec61β not only interacts with subunits of the Sec61 complex but also with the 25-kD subunit of the signal peptidase complex (SPC25), thus demonstrating for the first time a tight interaction between the SPC and the Sec61 complex. Interestingly, the cross-links between Sec61β and SPC25 and between Sec61β and Sec61α depend on the presence of membrane-bound ribosomes, suggesting that these interactions are induced when translocation is initiated. We propose that the SPC is transiently recruited to the translocation site, thus enhancing its activity.

A small chemical drug CADA specifically binds to the signal peptide of the membrane pre-protein CD4, disturbing its synthesis, impeding the routing to and expression on the cell surface.

Misfolded secretory proteins are transported across the endoplasmic reticulum (ER) membrane into the cytosol for degradation by proteasomes. A large fraction of proteasomes in a cell is associated with the ER membrane. We show here that binding of proteasomes to ER membranes is salt sensitive, ATP dependent, and mediated by the 19S regulatory particle. The base of the 19S particle, which contains six AAA-ATPases, binds to microsomal membranes with high affinity, whereas the 19S lid complex binds weakly. We demonstrate that ribosomes and proteasomes compete for binding to the ER membrane and have similar affinities for their receptor. Ribosomes bind to the protein conducting channel formed by the Sec61 complex in the ER membrane. We co-precipitated subunits of the Sec61 complex with ER-associated proteasome 19S particles, and found that proteoliposomes containing only the Sec61 complex retained proteasome binding activity. Collectively, our data suggest that the Sec61 channel is a principal proteasome receptor in the ER membrane.