Membrane Insertion and Topology of the Translocating Chain-Associating Membrane Protein (TRAM)

doi:10.1016/j.jmb.2011.01.009

Journal of Molecular Biology

Volume 406, Issue 4, 4 March 2011, Pages 571-582

https://doi.org/10.1016/j.jmb.2011.01.009 Get rights and content

Abstract

The translocating chain-associating membrane protein (TRAM) is a glycoprotein involved in the translocation of secreted proteins into the endoplasmic reticulum (ER) lumen and in the insertion of integral membrane proteins into the lipid bilayer. As a major step toward elucidating the structure of the functional ER translocation/insertion machinery, we have characterized the membrane integration mechanism and the transmembrane topology of TRAM using two approaches: photocross-linking and truncated C-terminal reporter tag fusions. Our data indicate that TRAM is recognized by the signal recognition particle and translocon components, and suggest a membrane topology with eight transmembrane segments, including several poorly hydrophobic segments. Furthermore, we studied the membrane insertion capacity of these poorly hydrophobic segments into the ER membrane by themselves. Finally, we confirmed the main features of the proposed membrane topology in mammalian cells expressing full-length TRAM.

Graphical Abstract

Research Highlights

► TRAM is recognized by the SRP and inserted into the ER membrane cotranslationally through translocon components. ► The protein crosses the membrane eight times and orients both the N- and C-termini toward the cytosol. ► Membrane assembly requires the assistance of preceding TM segments for the insertion of poorly hydrophobic segments.

Introduction

Protein insertion into or translocation across eukaryotic and prokaryotic membranes is a vital event in the biosynthesis of more than a third of the proteins in all living organisms. These processes are initiated by the signal recognition particle (SRP) targeting of secreted and integral membrane proteins to sites in the membrane, termed translocons, where both translocation and integration occur. The translocons therefore function as two-way gates that direct hydrophilic protein regions across the membrane and hydrophobic transmembrane (TM) segments laterally into the lipid bilayer.

The translocon is a multi-protein complex composed of the Sec61 α, β, and γ subunits and the translocating chain-associating membrane protein (TRAM)¹ in eukaryotic cells. Since translocon activity can be reproduced by ab initio reconstitution of these four membrane proteins in pure lipids,² these proteins are considered to be the core components of the mammalian translocon.³ The determination of the topology of the translocon components is essential for any understanding of the structure–function relationships of the translocon during translocation or integration. The membrane topology of the Sec61α subunit was first determined in yeast using C-terminal reporter domain fusions and protease digestions, which suggested the presence of 10 TM segments.⁴ The crystal structure of the archeal homologue showed that the Sec61α subunit consists of two domains of five TM segments each,⁵ which form a ‘clam shell’ structure that would provide a lateral gate for TM segments of nascent membrane proteins to partition into the lipid phase. Less information is available regarding the other translocon component TRAM.

TRAM is a polytopic (multi-spanning) integral glycoprotein with an apparent size of 37 kDa, which is involved early in the translocation of secreted proteins into the endoplasmic reticulum (ER) lumen1, 6 and in the insertion of integral membrane proteins into the lipid bilayer.2, 7, 8 On the basis of a hydrophobicity analysis, TRAM is thought to span the ER membrane eight times with both the N- and C-termini facing the cytosol,¹ although this model has not been experimentally verified.

In this study, we have used a photocross-linking approach to determine the mechanism of TRAM insertion into the ER membrane. Our results establish that TRAM insertion involves SRP and the translocon. In addition, we report an experimental determination of the topology that TRAM acquires in ER membranes. In vitro translation of a series of TRAM truncations containing an N-linked glycosylation reporter tag identified the topological orientation of eight TM segments. Four of these TM segments are predicted to insert poorly into the membrane. In fact, two of these poorly hydrophobic TM segments failed to insert into the ER membrane by themselves, and their presence in the membrane suggests a functional role for TRAM during membrane protein biogenesis at the translocon. Finally, the main features of our in vitro experiments were confirmed by experiments using HEK293 cells expressing full-length TRAM.

Section snippets

Cotranslational insertion of TRAM

Integration of membrane proteins into ER-derived membranes can be monitored by glycosylation. This modification is performed by the oligosaccharyl transferase (OST) enzyme, which is adjacent to the translocon.⁹ OST adds sugar residues cotranslationally to a consensus sequence after the protein emerges from the translocon pore. Glycosylation of a protein region translated in vitro in the presence of microsomal membranes therefore shows that this region of the nascent protein is exposed to the

Discussion

To examine the biogenesis of a primary component of the ER translocation/insertion machinery, we have investigated the targeting and insertion of TRAM in eukaryotic membranes. TRAM does not have a cleaved amino-terminal signal sequence, but we have demonstrated here that the first TM segment functions as a signal sequence that must emerge from the ribosome to bind SRP and thereby enable nascent TRAM targeting to the membrane (Fig. 2). Site-directed photocross-linking assays using TRAM nascent

Enzymes and chemicals

All enzymes as well as plasmid pGEM1, the RiboMAX SP6 RNA polymerase system, and rabbit reticulocyte lysate were purchased from Promega (Madison, WI). ER RMs from dog pancreas, SRP, and wheat germ translation extracts were obtained from tRNA Probes (College Station, TX). [³⁵S]Met/Cys and ¹⁴C-methylated markers were purchased from GE Healthcare. Restriction enzymes and endoglycosidase H were purchased from Roche Molecular Biochemicals. DNA plasmid, RNA Clean-up, and PCR purification kits were

Acknowledgements

This work was supported by grants PR2008-0051 (Mobility Program) and BFU2009-08401 (BMC) from the Spanish Ministry of Science and Innovation (MICINN, ERDF supported) and PROMETEO/2010/005 from the Generalitat Valenciana (to I.M.), by the ISCIII-MICINN starting grant MPY1413-09 (to M.V.), and by NIH grant GM26494 and Robert A. Welch Foundation chair grant BE-0017 (to A.E.J.). S.T. was the recipient of a predoctoral fellowship from the University of Valencia (V Segles program). We are grateful to

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Journal of Molecular Biology

Membrane Insertion and Topology of the Translocating Chain-Associating Membrane Protein (TRAM)

Abstract

Graphical Abstract

Research Highlights

Introduction

Section snippets

Cotranslational insertion of TRAM

Discussion

Enzymes and chemicals

Acknowledgements

Cell

J. Biol. Chem.

J. Biol. Chem.

Cell

Mol. Cell

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Cell

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Cell

J. Biol. Chem.

Virology

A protein of the endoplasmic reticulum involved early in polypeptide translocation

Nature