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.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3846719 | PMC |
| http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081534 | PLOS |
Publication Analysis
Top Keywords
Similar Publications
Evolutionary origins of the lysosome-related organelle sorting machinery reveal ancient homology in post-endosome trafficking pathways.
Proc Natl Acad Sci U S A
October 2024
Division of Infectious Diseases, Department of Medicine, University of Alberta, Edmonton, AB T6G 2N8, Canada.
The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis.
View Article and Find Full Text PDFHypomyelinated Mutant Zebrafish Exhibit Systemic and Neurodevelopmental Pathologies.
Int J Mol Sci
July 2024
Department of Ophthalmology, Visual and Anatomical Sciences, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human have been associated with MPS and dystonia.
View Article and Find Full Text PDFAn initial HOPS-mediated fusion event is critical for autophagosome transport initiation from the axon terminal.
Autophagy
October 2024
Department of Integrative Biology, University of Wisconsin-Madison, Madison, WI, USA.
In neurons, macroautophagy/autophagy is a frequent and critical process. In the axon, autophagy begins in the axon terminal, where most nascent autophagosomes form. After formation, autophagosomes must initiate transport to exit the axon terminal and move toward the cell body via retrograde transport.
View Article and Find Full Text PDFStructure of the endosomal CORVET tethering complex.
Nat Commun
June 2024
Department of Biology/Chemistry, Structural Biology Section, Osnabrück University, 49076, Osnabrück, Germany.
Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS.
View Article and Find Full Text PDFCORVET-specific subunit levels determine the balance between HOPS/CORVET endosomal tethering complexes.
Sci Rep
May 2024
Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University (ELTE), Pázmány Péter sétány 1/C, Budapest, 1117, Hungary.
The closely related endolysosomal tethering complexes HOPS and CORVET play pivotal roles in the homo- and heterotypic fusion of early and late endosomes, respectively, and HOPS also mediates the fusion of lysosomes with incoming vesicles including late endosomes and autophagosomes. These heterohexameric complexes share their four core subunits that assemble with additional two, complex-specific subunits. These features and the similar structure of the complexes could allow the formation of hybrid complexes, and the complex specific subunits may compete for binding to the core.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!