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Principal Investigator: FREDERICK M HUGHSON
Organization: PRINCETON UNIVERSITY
Fiscal Year: 2024
Award: $375,270
Funding agency: National Institute of General Medical Sciences
PROJECT SUMMARY / ABSTRACT
The traffic patterns established by transport vesicles are of fundamental importance for protein localization,
modification, and function within eukaryotic cells. Cargo transported by these vesicles is delivered through the
fusion of the vesicle with the membrane of a target organelle or, in the case of exocytosis, the plasma
membrane. Membrane fusion is executed by SNARE complexes that bridge the vesicle and target
membranes. The formation of these complexes requires that four different SNARE proteins, anchored in two
different membranes, undergo a coupled folding and assembly reaction during which the SNARE motifs zipper
up into a parallel four-helix bundle. This complicated process is inefficient in vitro, and is certain to be even
more challenging in vivo, where it must compete with the formation of various non-cognate and off-pathway
SNARE complexes. We hypothesize that SNARE complex assembly reactions in the cell are orchestrated by
`topologically aware' chaperones called multisubunit tethering complexes (MTCs). We furthermore propose
that the key task of catalyzing four-helix bundle formation falls to the Sec1/Munc18 (SM) proteins, working
together with—and sometimes as integral subunits of—the MTCs. Therefore, the overarching goal of this
proposal is to achieve an improved structural and mechanistic understanding of MTC and SM function,
especially as they relate to one another, in the assembly of membrane fusogenic SNARE complexes. Aim 1 is
focused on SM proteins with the goal of characterizing their precise catalytic role in SNARE complex
assembly. Principally through the use of X-ray crystallography and complementary single-molecule optical
tweezers experiments, we will determine the structures and thermodynamic stabilities of SM-bound SNARE
assembly intermediates. In Aims 2 and 3, we broaden our focus to include MTCs. In Aim 2, we will investigate
the simplest known MTC, the yeast Dsl1 complex, and its interactions with SNAREs and the SM protein Sly1.
Cryo-EM studies of arrested SNARE assembly intermediates in complex with both the Dsl1 complex and Sly1
are designed to reveal how the Dsl1 complex and Sly1 collaborate. In Aim 3, we will turn our attention to the
homotypic fusion and vacuole protein sorting (HOPS) complex, a well-studied MTC that is required for fusion at
late endosomes and lysosomes/vacuoles. Importantly, HOPS contains an SM protein as an integral subunit,
making it an ideal system for studying MTC–SM collaboration. In order to elucidate how HOPS organizes
SNAREs for assembly, we will expand our ongoing cryo-EM studies of HOPS to include bound SNAREs and
SNARE assembly intermediates. Overall, this research program has the potential to revolutionize our
mechanistic understanding of chaperoned SNARE complex assembly, with potentially profound implications for
elucidating diverse biological processes and their subversion during infection and disease. While the proposed
work is more fundamental than applied, it will lay a foundation for efforts to manipulate trafficking and other
processes entailing membrane fusion, with potential future applications to therapeutic intervention.
Terms: <Architecture><Attention><Awareness><Binding><Biological Function><Biological Process><Carrier Proteins><Cell Body><Cell membrane><Cells><Chaperone><Chimera Protein><Chimeric Proteins><Clampings><Closure by clamp><Coated vesicle><Collaborations><Complex><Coupled><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasmic Membrane><Disease><Disorder><Electron Cryomicroscopy><Engineering / Architecture><Eukaryotic Cell><Exocytosis><Foundations><Fusion Protein><Future><Genetic Alteration><Genetic Change><Genetic defect><Goals><In Vitro><Infection><Lasso><Lipids><Lysosomes><Mediating><Membrane><Membrane Fusion><Modification><Molecular Chaperones><Molecular Interaction><Munc-18 protein><Munc18 protein><Mutation><NSF attachment protein receptor><Nerve Cells><Nerve Unit><Neural Cell><Neurocyte><Neurons><Organelles><Pathway interactions><Pattern><Plasma Membrane><Process><Productivity><Protein Sortings><Proteins><Proteomics><Reaction><Reporting><Research><Role><SNAP receptor><SNARE><Single Crystal Diffraction><Sorting><Structure><Surface><Synapses><Synaptic><System><Therapeutic Intervention><Thermodynamic><Thermodynamics><Transport Protein Gene><Transport Proteins><Transporter Protein><Vacuole><Vesicle><Work><X Ray Crystallographies><X-Ray Crystallography><X-Ray Diffraction Crystallography><X-Ray/Neutron Crystallography><Xray Crystallography><Yeasts><base><bases><biochemical tools><biochemistry tools><cryo-EM><cryoEM><cryogenic electron microscopy><design><designing><experiment><experimental research><experimental study><experiments><falls><flexibility><flexible><genome mutation><human disease><improved><in vivo><intervention therapy><laser tweezer><late endosome><membrane assembly><membrane structure><nano machine><nanomachine><neuronal><neurotransmitter release><optic tweezer><optical tweezers><pathway><plasmalemma><programs><protein function><reconstitute><reconstitution><single molecule><social role><soluble N-ethylmaleimide-sensitive-factor attachment protein receptor><synapse><syntaxin binding protein 1><syntaxin binding protein Munc18><trafficking><vesicle transport><vesicular transport><virtual>