Improved mammalian retromer cryo-EM structures reveal a new assembly interface

Area of Science:

• Cell Biology
• Structural Biology
• Molecular Mechanisms

Background:

• Retromer (VPS26/VPS35/VPS29) mediates endosomal recycling of transmembrane proteins and is involved in mitochondrial homeostasis, nutrient sensing, autophagy, and fission.
• Mechanisms of mammalian retromer assembly and cargo sorting to various destinations by sorting nexins are not fully understood.
• Previous structural studies of mammalian retromer oligomers were limited by poor resolution.

Purpose of the Study:

• To elucidate the mechanisms of mammalian retromer assembly and oligomerization.
• To determine high-resolution structures of retromer oligomers, particularly focusing on novel assembly interfaces.
• To understand how retromer utilizes different sorting nexins for cargo sorting.

Main Methods:

• Single-particle cryo-electron microscopy (cryo-EM) with advanced data processing techniques, including data from tilted specimens and a 3D starting model for particle picking in RELION.
• Utilized a 3KE retromer mutant to disrupt known VPS35-mediated interfaces and facilitate the discovery of new assembly sites.
• Structural analysis of the retromer complex to identify and characterize novel protein-protein interactions.

Main Results:

• Achieved improved resolution of mammalian retromer oligomer structures using advanced cryo-EM methods.
• Identified and determined the structure of a new retromer assembly interface formed by the N-termini of VPS26A and VPS35.
• This novel interface involves an N-terminal VPS26A arrestin saddle linking retromer chains via interaction with a neighboring VPS35 N-terminus, distinct from the known C-VPS26/N-VPS35 interaction.

Conclusions:

• The newly identified VPS26A-VPS35 N-terminal interface provides a substantial buried surface area, suggesting a role in retromer chain formation.
• This discovery indicates that metazoan retromer can function as an adaptable scaffold, potentially linking multiple retromer complexes.
• The findings offer new insights into the structural plasticity and assembly mechanisms of the retromer complex in mammalian cells.