Integrative structure and function of the yeast exocyst complex.
EM
SNAREs
chemical cross-linking mass spectrometry
exocytosis
integrative modeling
membrane fusion
protein cross-linking
structural models
yeast exocyst complex
Journal
Protein science : a publication of the Protein Society
ISSN: 1469-896X
Titre abrégé: Protein Sci
Pays: United States
ID NLM: 9211750
Informations de publication
Date de publication:
06 2020
06 2020
Historique:
received:
04
03
2020
revised:
25
03
2020
accepted:
27
03
2020
pubmed:
3
4
2020
medline:
24
2
2021
entrez:
3
4
2020
Statut:
ppublish
Résumé
Exocyst is an evolutionarily conserved hetero-octameric tethering complex that plays a variety of roles in membrane trafficking, including exocytosis, endocytosis, autophagy, cell polarization, cytokinesis, pathogen invasion, and metastasis. Exocyst serves as a platform for interactions between the Rab, Rho, and Ral small GTPases, SNARE proteins, and Sec1/Munc18 regulators that coordinate spatial and temporal fidelity of membrane fusion. However, its mechanism is poorly described at the molecular level. Here, we determine the molecular architecture of the yeast exocyst complex by an integrative approach, based on a 3D density map from negative-stain electron microscopy (EM) at ~16 Å resolution, 434 disuccinimidyl suberate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride cross-links from chemical-crosslinking mass spectrometry, and partial atomic models of the eight subunits. The integrative structure is validated by a previously determined cryo-EM structure, cross-links, and distances from in vivo fluorescence microscopy. Our subunit configuration is consistent with the cryo-EM structure, except for Sec5. While not observed in the cryo-EM map, the integrative model localizes the N-terminal half of Sec3 near the Sec6 subunit. Limited proteolysis experiments suggest that the conformation of Exo70 is dynamic, which may have functional implications for SNARE and membrane interactions. This study illustrates how integrative modeling based on varied low-resolution structural data can inform biologically relevant hypotheses, even in the absence of high-resolution data.
Identifiants
pubmed: 32239688
doi: 10.1002/pro.3863
pmc: PMC7255525
doi:
Substances chimiques
Protein Subunits
0
Saccharomyces cerevisiae Proteins
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
1486-1501Subventions
Organisme : NIGMS NIH HHS
ID : T32 GM008284
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM083960
Pays : United States
Organisme : NIGMS NIH HHS
ID : P41 GM103314
Pays : United States
Organisme : NIH HHS
ID : GM068803
Pays : United States
Organisme : NIH HHS
ID : R01 GM112108
Pays : United States
Organisme : NIH HHS
ID : P41 GM109824
Pays : United States
Organisme : NIH HHS
ID : GM103314
Pays : United States
Organisme : NIH HHS
ID : R01GM083960
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM068803
Pays : United States
Organisme : NIGMS NIH HHS
ID : R01 GM112108
Pays : United States
Organisme : NIGMS NIH HHS
ID : P41 GM109824
Pays : United States
Organisme : NIH HHS
ID : S10 OD021596
Pays : United States
Informations de copyright
© 2020 The Protein Society.
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