Assembling Retromer-coated membrane tubules for biochemical and structural studies

Area of Science:

• Cell Biology
• Structural Biology
• Biochemistry

Background:

• The Retromer complex is crucial for endosomal cargo retrieval, involving Vps29, Vps26, and Vps35.
• Retromer collaborates with sorting nexin (SNX) proteins to form coated tubulovesicular carriers for cargo sorting and trafficking.
• In vitro reconstitution of these membrane assemblies is challenging but vital for structural and functional studies.

Purpose of the Study:

• To describe protocols for producing multilamellar vesicles (MLVs) for Retromer membrane binding studies.
• To detail the assembly of the yeast Retromer-Vps5-Vps17 complex for reconstituting membrane tubulation.
• To provide methods for imaging Retromer-coated tubules using cryoelectron tomography (CryoET).

Main Methods:

• Utilizing multilamellar vesicles (MLVs) for generating uniform tubules.
• Employing cryoelectron tomography (CryoET) for high-resolution imaging of membrane assemblies.
• Developing protocols for in vitro reconstitution of Retromer-membrane interactions.

Main Results:

• Successful production of MLVs suitable for Retromer binding studies.
• Reconstitution of membrane tubulation mediated by the yeast Retromer-Vps5-Vps17 complex.
• Observation of both ordered and disordered Retromer coats in the experimental setup.

Conclusions:

• The described protocols facilitate in vitro studies of Retromer function and structure.
• CryoET imaging of reconstituted membrane tubules provides insights into Retromer coat organization.
• This work offers a foundation for further structural and mechanistic investigations of the Retromer complex.