Visualizing membrane trafficking through the electron microscope: cryo-tomography of coat complexes.

Evgenia A Markova,Giulia Zanetti

doi:10.1107/s2059798319005011

Abstract

Coat proteins mediate vesicular transport between intracellular compartments, which is essential for the distribution of molecules within the eukaryotic cell. The global arrangement of coat proteins on the membrane is key to their function, and cryo-electron tomography and subtomogram averaging have been used to study membrane-bound coat proteins, providing crucial structural insight. This review outlines a workflow for the structural elucidation of coat proteins, incorporating recent developments in the collection and processing of cryo-electron tomography data. Recent work on coat protein I, coat protein II and retromer performed on in vitro reconstitutions or in situ is summarized. These studies have answered long-standing questions regarding the mechanisms of membrane binding, polymerization and assembly regulation of coat proteins.

Highlights

Recent advances in cryo-electron microscopy have enabled numerous high-resolution studies which have addressed long-standing structural biology questions
This tightly controlled intercompartmental exchange is enabled by vesicular transport, in which coat proteins promote the formation of cargo-carrying vesicles from a donor compartment, and the budded vesicles travel towards a target compartment and fuse with it, delivering their cargo (Caro & Palade, 1964)
We briefly describe the basic steps for coat protein II (COPII) budding reconstitution in vitro (Fig. 4)

Summary

Introduction

Recent advances in cryo-electron microscopy (cryo-EM) have enabled numerous high-resolution studies which have addressed long-standing structural biology questions. Eukaryotic cells are separated into membrane-bound compartments that constantly exchange molecules while retaining their own biochemical identity This tightly controlled intercompartmental exchange is enabled by vesicular transport, in which coat proteins promote the formation of cargo-carrying vesicles from a donor compartment, and the budded vesicles travel towards a target compartment and fuse with it, delivering their cargo (Caro & Palade, 1964). Different coat protein families are responsible for the transport between specific intracellular compartments, mediating local vesicular trafficking (Barlowe et al, 1994). Clathrin-coated vesicles mediate the transport of endocytic cargo from the plasma membrane, as well as trafficking from the trans-Golgi network to endosomal compartments (Roth & Porter, 1964; Robinson, 1994).

Sample preparation

Data collection

Challenges and future perspectives