Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 4 de 4
Filter
Add more filters










Database
Language
Publication year range
1.
J Biol Chem ; 297(5): 101334, 2021 11.
Article in English | MEDLINE | ID: mdl-34688652

ABSTRACT

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.


Subject(s)
Golgi Apparatus/metabolism , Multiprotein Complexes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Biological Transport, Active , Golgi Apparatus/genetics , Multiprotein Complexes/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics
2.
EMBO J ; 39(20): e105117, 2020 10 15.
Article in English | MEDLINE | ID: mdl-32840906

ABSTRACT

Heterotetrameric adapter (AP) complexes cooperate with the small GTPase Arf1 or lipids in cargo selection, vesicle formation, and budding at endomembranes in eukaryotic cells. While most AP complexes also require clathrin as the outer vesicle shell, formation of AP-3-coated vesicles involved in Golgi-to-vacuole transport in yeast has been postulated to depend on Vps41, a subunit of the vacuolar HOPS tethering complex. HOPS has also been identified as the tether of AP-3 vesicles on vacuoles. To unravel this conundrum of a dual Vps41 function, we anchored Vps41 stably to the mitochondrial outer membrane. By monitoring AP-3 recruitment, we now show that Vps41 can tether AP-3 vesicles to mitochondria, yet AP-3 vesicles can form in the absence of Vps41 or clathrin. By proximity labeling and mass spectrometry, we identify the Arf1 GTPase-activating protein (GAP) Age2 at the AP-3 coat and show that tethering, but not fusion at the vacuole can occur without complete uncoating. We conclude that AP-3 vesicles retain their coat after budding and that their complete uncoating occurs only after tethering at the vacuole.


Subject(s)
ADP-Ribosylation Factors/metabolism , Cytoplasmic Vesicles/metabolism , GTPase-Activating Proteins/metabolism , Mitochondrial Membranes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Vesicular Transport Proteins/metabolism , ADP-Ribosylation Factors/genetics , Biological Transport, Active/genetics , Casein Kinase I/genetics , Casein Kinase I/metabolism , Cytoplasmic Vesicles/ultrastructure , GTPase-Activating Proteins/genetics , Gene Deletion , Golgi Apparatus/metabolism , Mass Spectrometry , Membrane Fusion , Microscopy, Electron , Mitochondrial Membranes/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/ultrastructure , Vesicular Transport Proteins/genetics
3.
Mol Biol Cell ; 28(2): 322-332, 2017 01 15.
Article in English | MEDLINE | ID: mdl-27852901

ABSTRACT

Membrane fusion at endomembranes requires cross-talk between Rab GTPases and tethers to drive SNARE-mediated lipid bilayer mixing. Several tethers have multiple Rab-binding sites with largely untested function. Here we dissected the lysosomal HOPS complex as a tethering complex with just two binding sites for the Rab7-like Ypt7 protein to determine their relevance for fusion. Using tethering and fusion assays combined with HOPS mutants, we show that HOPS-dependent fusion requires both Rab-binding sites, with Vps39 being the stronger Ypt7 interactor than Vps41. The intrinsic amphipathic lipid packaging sensor (ALPS) motif within HOPS Vps41, a target of the vacuolar kinase Yck3, is dispensable for tethering and fusion but can affect tethering if phosphorylated. In combination, our data demonstrate that a multivalent tethering complex uses its two Rab bindings to determine the place of SNARE assembly and thus fusion at endomembranes.


Subject(s)
Membrane Fusion/physiology , Saccharomyces cerevisiae Proteins/metabolism , rab GTP-Binding Proteins/metabolism , Binding Sites , Endosomes/metabolism , Phosphorylation , Protein Binding , Protein Transport/physiology , SNARE Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/physiology , Vacuoles/metabolism , Vesicular Transport Proteins/metabolism , rab GTP-Binding Proteins/physiology
4.
Proc Natl Acad Sci U S A ; 110(10): 3823-8, 2013 Mar 05.
Article in English | MEDLINE | ID: mdl-23417307

ABSTRACT

Membrane fusion along the endocytic pathway occurs in a sequence of tethering, docking, and fusion. At endosomes and vacuoles, the CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) tethering complexes require their organelle-specific Rabs for localization and function. Until now, despite the absence of experimental evidence, it has been assumed that CORVET is a membrane-tethering factor. To test this theory and understand the mechanistic analogies with the HOPS complex, we set up an in vitro system, and establish CORVET as a bona-fide tether for Vps21-positive endosome/vacuole membranes. Purified CORVET binds to SNAREs and Rab5/Vps21-GTP. We then demonstrate that purified CORVET can specifically tether Vps21-positive membranes. Tethering via CORVET is dose-dependent, stimulated by the GEF Vps9, and inhibited by Msb3, the Vps21-GAP. Moreover, CORVET supports fusion of isolated membranes containing Vps21. In agreement with its role as a tether, overexpressed CORVET drives Vps21, but not the HOPS-specific Ypt7 into contact sites between vacuoles, which likely represent vacuole-associated endosomes. We therefore conclude that CORVET is a tethering complex that promotes fusion of Rab5-positive membranes and thus facilitates receptor down-regulation and recycling at the late endosome.


Subject(s)
Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , rab GTP-Binding Proteins/chemistry , rab GTP-Binding Proteins/metabolism , rab5 GTP-Binding Proteins/chemistry , rab5 GTP-Binding Proteins/metabolism , Endocytosis , Endosomes/metabolism , Lysosomes/metabolism , Membrane Fusion , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , SNARE Proteins/chemistry , SNARE Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/metabolism , rab GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...