Related Experiment Video
Updated: May 5, 2026

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro
Published on: January 11, 2017
Subunit organisation of in vitro reconstituted HOPS and CORVET multisubunit membrane tethering complexes
Zhong Guo1, Wayne Johnston, Oleksiy Kovtun
1Department of Cell and Molecular Biology, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
Abstract:
Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.
Related Concept Videos
Protein Complexes with Interchangeable Parts
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order...
Protein Complexes with Interchangeable Parts
Protein Complex Assembly
Many viruses self-assemble into a fully functional unit using the infected host cell to...
Protein Complex Assembly
Vesicular Tubular Clusters
With the help of motor proteins such...
Protein Translocation Machinery on the ER Membrane
Sec61 protein conducting channel
In eukaryotes, the translocon complex comprises a core heterotrimeric translocator channel called the Sec61 complex. This channel includes three transmembrane proteins, Sec61α, Sec61β, and Sec61γ, and is the largest subunit of the...

