Dissecting the molecular organization of the translocon-associated protein complex

Stefan Pfeffer,Johanna Dudek,Richard Zimmermann,Friedrich Förster,Sahradha Albert,Wolfgang Baumeister,Benjamin D Engel,Jürgen M Plitzko,Miroslava Schaffer,Bobby G Ng,Hudson H Freeze

doi:10.1038/ncomms14516

Abstract

In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. The translocon-associated protein (TRAP) complex is an integral component of the translocon, assisting the Sec61 protein-conducting channel by regulating signal sequence and transmembrane helix insertion in a substrate-dependent manner. Here we use cryo-electron tomography (CET) to study the structure of the native translocon in evolutionarily divergent organisms and disease-linked TRAP mutant fibroblasts from human patients. The structural differences detected by subtomogram analysis form a basis for dissecting the molecular organization of the TRAP complex. We assign positions to the four TRAP subunits within the complex, providing insights into their individual functions. The revealed molecular architecture of a central translocon component advances our understanding of membrane protein biogenesis and sheds light on the role of TRAP in human congenital disorders of glycosylation.

Highlights

In eukaryotic cells, one-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon
In the high-resolution subtomogram average of the ER-associated ribosome (EMD-3068)[25], mammalian translocon-associated protein (TRAP) is represented by three topological segments (Fig. 1b, Supplementary Movie 1): (I) a cytosolic domain bound to the ribosome via large subunit ribosomal RNA expansion segments and ribosomal protein L38, (II) a bundle of a
Based on the set of difference densities obtained from the TRAPd-deficient human translocon and the algal translocon (Fig. 5a, Supplementary Movie 2), the individual TRAP subunits can be assigned in the overall density of the mammalian TRAP complex (Fig. 5b)

Summary

Introduction

One-third of all proteins must be transported across or inserted into the endoplasmic reticulum (ER) membrane by the ER protein translocon. Cryo-electron tomography (CET) in conjunction with subtomogram analysis can resolve the structures of membraneembedded and -associated complexes within a natural membrane environment[21] This technique enabled the first insights into the structure and molecular organization of the native translocon complex in rough ER vesicles derived from mammalian cells[18,22] and within undisturbed vitrified HeLa cells thinned by focused ion beam (FIB) milling[23]. These studies established the positions of three major translocon constituents: Sec[61], TRAP and the oligosaccharyl–transferase (OST) complex. We assign positions to the four TRAP subunits in the assembled mammalian complex, providing new insights into membrane protein biogenesis and the role of TRAP in human congenital disorders of glycosylation

Methods

Results

Conclusion