Dissecting the molecular organization of the translocon-associated protein complex

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.

The atomic structure of a eukaryotic oligosaccharyltransferase complex

“…Biochemical studies suggested that Ost1 and Wbp1 recognize acceptor and donor substrates, respectively 8,21,22 . The structures of the eukaryotic OST have been limited to low-resolution EM reconstructions, hindering a mechanistic understanding of protein N-glycosylation in eukaryotes 23–26 .…”

“…Recent cryo-electron tomographic studies showed the relative positions of mammalian translocons and OSTs bound to the ribosomes 24,26 . However, the detailed interface—i.e., which subunits mediate the OST/translocon interaction—was unclear.…”

Charting Molecular Landscapes Using Cryo-Electron Tomography

“…These three-dimensional views revealed novel protein arrays within the Golgi cisternae [1], shed light on the structures of individual nuclear pore complexes [2], and visualized polyribosomes attached to the endoplasmic reticulum [3]. All of these macromolecular complexes were captured in their native environment using cryo-electron tomography (CET).…”

Cryo-FIB Lift-out Sample Preparation Using a Novel Cryo-gripper Tool

“…Cryo-FIB has enabled cryoelectron tomography (cryo-ET) of plunge-frozen hydrated cells, revealing the cellular interior with sufficient resolution and contrast to study membrane-bound macromolecules in their native state [4][5][6][7].…”

“…Cryo-FIB has enabled cryoelectron tomography (cryo-ET) of plunge-frozen hydrated cells, revealing the cellular interior with sufficient resolution and contrast to study membrane-bound macromolecules in their native state [4][5][6][7].In the current standard cryo-FIB preparation technique, lamellas are milled on plunge-frozen TEM grids by completely removing material over tens of micrometers on both sides of the lamella, while the lamella itself remains connected to the bulk material on the grid. Several lamellas can be prepared on a single grid, which is then imaged directly by cryo-ET [8].…”