Architecture of the Ribosome–Channel Complex Derived from Native Membranes

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The mammalian Sec61 complex forms a protein translocation channel whose function depends upon its interaction with the ribosome and with membrane proteins of the endoplasmic reticulum (ER). To study these interactions, we determined structures of “native” ribosome–channel complexes derived from ER membranes. We find that the ribosome is linked to the channel by seven connections, but the junction may still provide a path for domains of nascent membrane proteins to move into the cytoplasm. In addition, the native channel is significantly larger than a channel formed by the Sec61 complex, due to the presence of a second membrane protein. We identified this component as TRAP, the translocon-associated protein complex. TRAP interacts with Sec61 through its transmembrane domain and has a prominent lumenal domain. The presence of TRAP in the native channel indicates that it may play a general role in translocation. Crystal structures of two Sec61 homologues were used to model the channel. This analysis indicates that there are four Sec61 complexes and two TRAP molecules in each native channel. Thus, we suggest that a single Sec61 complex may form a conduit for translocating polypeptides, while three copies of Sec61 play a structural role or recruit accessory factors such as TRAP.

Introduction

In all cells, secretory and membrane proteins must be translocated across or integrated into endoplasmic reticulum (ER) membranes, with the help of a protein-conducting channel. The central component of the conserved channel is a heterotrimer of three membrane proteins, termed the SecY complex in bacteria and archaebacteria, and the Sec61 complex in eukaryotes. The largest subunit of this heterotrimer (termed SecY or Sec61α) is a multi-spanning membrane protein that forms the pore of the channel.1, 2 Evidence from prokaryotes and eukaryotes suggests that three to four copies of either the SecY or Sec61 complex may associate to form channels.3, 4, 5, 6, 7, 8 These data have been interpreted to indicate that a translocation pore is formed at the confluence of multiple subunits. However, a recent crystal structure of the archael SecY complex suggests that the pore may reside within a single heterotrimer.9 In 2D membrane crystals, SecY complexes form a dimer with their essential SecE subunits facing each other.10 This SecY/Sec61 dimer is thought to form the building block of prokaryotic and eukaryotic protein translocation channels.5, 9, 11, 12

SecY and Sec61 complexes must associate with partners that provide a driving force for polypeptide transport. In bacteria, post-translational translocation requires a cytosolic ATPase, termed SecA, while in eukaryotes the partners are a tetrameric Sec62/63 membrane protein complex and BiP, an ATPase located in the lumen of the endoplasmic reticulum.13 Co-translational translocation in all organisms requires the ribosome as the major channel partner. In this pathway, nascent chains exit from a tunnel in the large ribosomal subunit and are transferred into the translocation channel. Co-translational translocation is also responsible for the integration of most membrane proteins. In this process, transmembrane (TM) segments move into the lipid phase through a lateral gate in the channel.14 In addition, a cytosolic domain that follows a TM segment must exit through the ribosome–channel junction and fold in the cytoplasm. Importantly, the membrane barrier for small molecules should be maintained during all steps of translocation.

The eukaryotic channel associates with additional membrane proteins during co-translational translocation. These include the signal peptidase complex (SPC), the oligosacharryl transferase (OST), the translocating chain associated membrane protein (TRAM) and the translocon-associated protein complex (TRAP). The SPC catalyzes signal sequence cleavage,14 while the nine subunit OST complex (290 kDa) is responsible for the Asn-linked glycosylation of nascent chains.15 TRAM is an abundant membrane protein that has been shown to enhance the translocation of some secretory proteins and is located in close proximity to nascent chains.16, 17, 18, 19 The TRAP complex is comprised of four membrane protein subunits. The α, β, and δ-subunits are single-spanning proteins with prominent lumenal domains, while the γ-subunit crosses the membrane four times.20 TRAP enhances the translocation of nascent proteins that have prolonged access to the cytoplasm21 and can be crosslinked to nascent chains.17, 22, 23, 24 Sequence-based analysis has identified TRAP-related proteins in higher eukaryotes but not in budding yeast. Currently, little is known about the way in which SPC, OST, TRAM and TRAP associate with the Sec61 channel.

Previous studies of ribosome–channel complexes (RCCs) by electron microscopy (EM) provided evidence that Sec61 channels isolated from ER membranes associate with additional membrane proteins. These native channels are noticeably larger than channels containing Sec61 alone, have an elliptical rather than cylindrical cross-section in the plane of the membrane, and contain a prominent lumenal domain.6, 8 Biochemical analyses demonstrated that “native” RCC preparations used for these structures contain several membrane proteins in addition to the Sec61 complex, but only TRAP and OST are abundant enough to contribute to the maps.6

We now provide an improved picture of the RCC and identify TRAP within the native channel. TRAP has a tripartite architecture and its prominent lumenal domain is connected to the channel by two stalk-like features. We then modeled the general arrangement of the Sec61 and TRAP complexes in the native channel. Based on this analysis, the native channel in our maps is comprised of four Sec61 and two TRAP complexes. Thus, OST may be disordered in this specimen. Since TRAP is present in nearly all copies of the RCC, this component may play a general role in protein translocation within higher eukaryotes. The pattern of connections between the ribosome and channel suggests that several Sec61 complexes may play a structural role, while at the same time creating docking sites for accessory factors such as TRAP. Thus, only one Sec61 may provide an active pore for protein translocation, as suggested by a recent crystal structure of an archaebacterial SecY complex.9

Section snippets

Maps of the native ribosome–channel complex

To generate native RCCs in a defined state, we used a procedure developed previously.8 In this approach, translating ribosomes were removed from rough microsomes with puromycin and high salt. These stripped ER membranes were then incubated with ribosomes which contained a tRNA in the exit-site,8 and floated in a sucrose gradient. The RCCs were then solubilized from the membranes in deoxy-BigCHAP (DBC) containing buffer, isolated by centrifugation, and flash frozen on a carbon-coated grid in

Discussion

The present work provides 3D maps of the RCC that show new features of the junction and the protein translocation channel. We also identified TRAP as a ubiquitous component of the mammalian translocon. We suggest that the translocation channel may be created by the lateral association of two Sec61 dimers and in higher eukaryotes, the channel may contain two molecules of TRAP. When taken together, these results have implications for our understanding of protein translocation.

Preparation of ribosome–channel complexes

A total extract of canine ER membranes was prepared using DBC (pH 7.5).21 TRAP was depleted from canine ER extracts using Q-Sepharose under high salt conditions, as described.16, 21 Purification of porcine TRAP, replenishment of canine ER detergent extracts, proteoliposome reconstitution, immuno-blots and translocation assays were carried out as described.21 Native RCCs were made by adding canine ribosomes with an E-site tRNA to ribosome-stripped proteoliposomes.8 The complexes were then

Acknowledgements

We thank A. Neuhof for help in preparing native RCCs, D. Centrella for scanning and particle selection and W. Clemons Jr and E. Hartmann for helpful discussions. T.A.R. is an investigator of the Howard Hughes Medical Institute. Work in the T.A.R. and C.W.A. laboratories was supported by NIH grants.

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