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Alignment of Conduits for the Nascent Polypeptide Chain in the Ribosome-Sec61 Complex

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Science  19 Dec 1997:
Vol. 278, Issue 5346, pp. 2123-2126
DOI: 10.1126/science.278.5346.2123

Abstract

An oligomer of the Sec61 trimeric complex is thought to form the protein-conducting channel for protein transport across the endoplasmic reticulum. A purified yeast Sec61 complex bound to monomeric yeast ribosomes as an oligomer in a saturable fashion. Cryo–electron microscopy of the ribosome-Sec61 complex and a three-dimensional reconstruction showed that the Sec61 oligomer is attached to the large ribosomal subunit by a single connection. Moreover, a funnel-shaped pore in the Sec61 oligomer aligned with the exit of a tunnel traversing the large ribosomal subunit, strongly suggesting that both structures function together in the translocation of proteins across the endoplasmic reticulum membrane.

The existence of a protein-conducting channel (PCC) for protein transport across the endoplasmic reticulum (ER) was proposed in 1975 (1). Electrophysiological experiments in 1991 provided the first direct evidence for the existence of the PCC (2). Moreover, fluorescently labeled nascent chains in membrane-bound ribosomes remain in an aqueous environment sealed from the cytoplasm and accessible to fluorescence quenching from the lumen of the ER (3). An aqueous pore with a diameter of 40 to 60 Å during cotranslational translocation is suggested by similar experiments (4).

The Sec61 trimeric complex is a strong candidate for the PCC of the ER in yeast and mammalian cells (5, 6). The Sec61 complex provides the principal binding site for ribosomes at the ER during protein translocation (7, 8) and, together with other membrane proteins, is associated with ribosomes after solubilization of rough microsomes with digitonin (6). A two-dimensional map of the purified Sec61 complex obtained by electron microscopy has revealed a quasi-pentagonal, circular structure with a central depression (9).

The three-dimensional (3D) structure of monomeric ribosomes is currently known at various resolutions for Escherichia coli(10), wheat germ (11), and yeast (12). Among the structural features recognized is a tunnel that traverses the large ribosomal subunit and has been considered a candidate for the nascent chain conduit. Here, we present a 3D reconstruction of the ribosome-Sec61 complex.

For purification of the trimeric Sec61 complex (13) containing the Sec61α, Sec61β, and Sec61γ subunits (Sec61p, Sbh1p, and Sss1p), a heptameric complex (14) was isolated first with protein A– tagged Sec63 protein, followed by elution of the trimeric Sec61 complex with Triton X-100 (Fig.1A). To determine whether the trimeric Sec61 complex could bind to ribosomes (15) in a membrane-free system, we incubated the purified Sec61 complex with ribosomes and analyzed the incubation mixture by sucrose density-gradient centrifugation (16). The Sec61 complex incubated without ribosomes remained in the top fraction of the gradient. In the presence of ribosomes, however, the Sec61 complex migrated with ribosomes as determined by immunoblotting (16) with antibodies to Sec61β (anti-Sec61β) (Fig. 1B) and Sec61α (anti-Sec61α) (17). In agreement with the known salt sensitivity of the Sec61-ribosome interaction, there was no binding at 1 M KOAc (OAc, acetate) (17). Incubation of a fixed amount of ribosomes with increasing amounts of Sec61 complex resulted in saturation of ribosome-binding sites (Fig. 1, C and D). On the basis of the amount of Sec61α and ribosomes, we estimate that, at saturation, two to four Sec61 trimers were bound per ribosome and that the dissociation constant K d is about 10 nM.

Figure 1

Purification and binding of the trimeric Sec61 complex to ribosomes. (A) Purification of the Sec61 complex. Crude nuclear envelopes from Sec63prAcells were extracted with digitonin, and the digitonin extract was incubated with IgG-Sepharose. The trimeric Sec61 complex was eluted with Triton X-100 and analyzed by SDS-PAGE followed by Coomassie blue staining. Lane 1, crude nuclear envelopes (1/30,000); lane 2, digitonin extract (1/30,000); lane 3, IgG-Sepharose flow through (1/30,000); and lane 4, Triton X-100 eluate (1/10). Ten to 15 micrograms of Sec61α [by comparison with a bovine serum albumin standard] were typically purified from 80 g of packed cells. Molecular mass is given in kilodaltons at the left. (B) The Sec61 complex binds to ribosomes. The Sec61 complex (0.5 μg) was incubated with and without ribosomes (0.5 A 260), and the incubation mixture was analyzed by sucrose density-gradient centrifugation followed by SDS-PAGE analysis and immunoblotting of gradient fractions. Lanes 1 to 6 represent top to bottom fractions probed with a peptide antibody to Sec61β. For the ribosomes, the Coomassie-stained proteins were quantitated with NIH image (arb. units, arbitrary units). (C) Binding of the Sec61 complex to ribosomes is saturable. Increasing amounts of the Sec61 complex (0 μg of Sec61α in lane 1 to 0.5 μg of Sec61α in lane 6) were incubated with a fixed amount of ribosomes (0.125A 260) and analyzed by density-gradient centrifugation followed by SDS-PAGE and SYPRO Red staining. Asterisks denote Sec61α. (D) Quantitation of data in (C). The amount of bound Sec61α was calculated according to the calibration shown in the inset.

The ribosome-Sec61 complex formed under saturating conditions was examined by cryo–electron microscopy (18). In the electron micrographs (Fig. 2A), the ribosome-Sec61 complex appears in random orientations (Fig. 2B). This distribution allowed an artifact-free 3D reconstruction by means of a 3D projection alignment procedure (19) with an existing reconstruction of the ribosome from yeast (12) as a reference. In side views of the ribosome (marked by arrows in Fig. 2B), an ∼100 Å–long ellipsoidal mass of density appears at the surface of the large subunit. This location on the ribosome is the same as the site where, in projection, the exiting polypeptide chain was located on both eubacterial and eukaryotic ribosomes by immuno–electron microscopy (20).

Figure 2

(A) Cryo–electron micrograph showing a field of yeast ribosome-Sec61 complexes. Scale bar, 200 Å. (B) Averaged projections of the ribosome-Sec61 complex obtained by classification. Particles marked with arrowheads show the ribosome in side view, with the Sec61 complex visible as a 100 Å–long mass lying parallel to the ribosome surface. Scale bar, 200 Å.

In the resulting reconstruction (Fig. 3), which has a resolution of 26 Å (21), the Sec61 complex appears as a slightly pentagonally shaped toroidal structure with an outer diameter of 95 Å, an inner diameter ranging from 15 to 35 Å depending on depth, and an overall thickness of 40 Å supported by a single stem attached to the base of the large ribosomal subunit. A single site of rigid attachment may facilitate lateral opening of the channel, at the opposite site, to allow the release of nascent transmembrane segments into the lipid bilayer. The surface of the Sec61 oligomer facing the ribosome is parallel to the surface of the large subunit. The distance between these surfaces ranges from 15 to 20 Å. Hence, the attachment of the Sec61 oligomer to the ribosome does not appear to form the tight seal that was implicated in nascent chain fluorescence-quenching experiments. Seal-forming proteins could be missing, or, more likely, the signal sequence may be necessary for seal formation (2, 3, 8, 22).

Figure 3

Three-dimensional reconstruction of the ribosome-Sec61 complex in a surface representation. (A) Front view, with the Sec61 oligomer shown in red. (B) Front view, with the Sec61 oligomer shown as transparent, to demonstrate the alignment of the Sec61 oligomer pore with the tunnel exit of the large ribosomal subunit indicated with the yellow arrow. (C) Side view, with the Sec61 oligomer shown in red. (D) Ribosome-Sec61 complex lying in the same orientation as in (C), cut along a plane that cross sections the pore of the Sec61 oligomer and the ribosome tunnel. The arrow indicates the stem connecting the ribosome with the Sec61 oligomer; the space between the two ribosomal subunits is indicated by an asterisk. The ribosomal tunnel and its alignment with the Sec61 pore is indicated by a broken yellow line. Scale bar, 100 Å.

The central pore of the Sec61 oligomer aligns precisely with an opening in the large ribosomal subunit that represents the exit of a tunnel (Fig. 3B). This tunnel runs from the interface canyon (12) to the lower portion of the large subunit (Fig. 3D). At the threshold level chosen (23), a small segment of the tunnel is blocked because of the limiting resolution, but it appeared to be open at increased threshold levels (17). A similar effect has been observed in the reconstruction of the 70S E. coli ribosome (10). These tunnels, seen in cryo–electron microscopy maps of the ribosome from E. coli (10) and Saccharomyces cerevisiae (12), have been proposed as exit pathways of the nascent polypeptide chain, although the evidence for that is indirect (20). The precise alignment between the pore of the Sec61 oligomer and the tunnel (Fig.3, C and D) provides strong support for this hypothesis. Conversely, this structural arrangement also implies that the Sec61 oligomer indeed constitutes the PCC.

Detailed analysis of the Sec61 oligomer (Fig.4, A to C) shows that the azimuthal distribution of mass is irregular, with the bulk of the mass lying on the side attached to the stem. Again, as speculated above, the thinner wall opposite the attachment stem might facilitate lateral opening of the channel. There is a further asymmetry in the Sec61 oligomer: the pore is funnel-shaped, with a diameter of 15 Å at the lumenal site of the ER, widening toward the ribosome, so that a small vestibule with a diameter of 35 Å is formed. Consistent with the estimate from kinetic data, the measured volume of the Sec61 oligomer would accommodate two trimeric Sec61 complexes. The reasons for the asymmetric appearance of the ribosome-bound Sec61 oligomer are presently not clear.

Figure 4

Three closeup views of the Sec61 oligomer. (A) Surface facing the ribosome. There is a vestibule (diameter of 35 Å) formed by the funnel-like structure and the pore (diameter of 15 Å). (B) Surface facing away from the ribosome. (C) View of the side opposite the attachment site. The ribosome would be located underneath the channel. The wall opposite the attachment site is thinner and more irregular. Arrows indicate the attachment site. Scale bar, 50 Å.

It is likely that the Sec 61 oligomer bound to the ribosome represents the PCC in its inactive and closed conformation. Future structural analyses of an in vitro–assembled complex containing a ribosome, a nascent chain, and the Sec61 complex will lead to information regarding the active state of the PCC.

  • * To whom correspondence should be addressed. E-mail: beckmar{at}rockvax.rockefeller.edu

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