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- PDB-8ezj: Cryo-EM structure of the S. cerevisiae Arf-like protein Arl1 boun... -

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Basic information

Entry
Database: PDB / ID: 8ezj
TitleCryo-EM structure of the S. cerevisiae Arf-like protein Arl1 bound to the Arf guanine nucleotide exchange factor Gea2
Components
  • ADP-ribosylation factor-like protein 1
  • ARF guanine-nucleotide exchange factor 2
KeywordsLIPID TRANSPORT / small GTPase / guanine nucleotide exchange factor / membrane trafficking / lipid flippase / trans-Golgi network / protein transport
Function / homology
Function and homology information


Retrograde transport at the Trans-Golgi-Network / secretory granule organization / VxPx cargo-targeting to cilium / trans-Golgi network membrane organization / Golgi cis cisterna / trans-Golgi Network Vesicle Budding / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / regulation of ARF protein signal transduction / cytoplasm to vacuole targeting by the Cvt pathway ...Retrograde transport at the Trans-Golgi-Network / secretory granule organization / VxPx cargo-targeting to cilium / trans-Golgi network membrane organization / Golgi cis cisterna / trans-Golgi Network Vesicle Budding / COPI-dependent Golgi-to-ER retrograde traffic / COPI-mediated anterograde transport / regulation of ARF protein signal transduction / cytoplasm to vacuole targeting by the Cvt pathway / protein localization to Golgi apparatus / Golgi to plasma membrane protein transport / intra-Golgi vesicle-mediated transport / protein localization to phagophore assembly site / protein targeting to vacuole / protein-containing complex localization / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / endoplasmic reticulum to Golgi vesicle-mediated transport / vesicle-mediated transport / response to endoplasmic reticulum stress / guanyl-nucleotide exchange factor activity / macroautophagy / intracellular protein transport / trans-Golgi network / endocytosis / actin cytoskeleton organization / endosome membrane / Golgi membrane / GTPase activity / GTP binding / Golgi apparatus / cytosol
Similarity search - Function
Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily / Sec7 domain superfamily / Sec7 domain / SEC7 domain profile. / Sec7 domain / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases ...Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily / Sec7 domain superfamily / Sec7 domain / SEC7 domain profile. / Sec7 domain / small GTPase Arf family profile. / Sar1p-like members of the Ras-family of small GTPases / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / ARF-like small GTPases; ARF, ADP-ribosylation factor / Rab subfamily of small GTPases / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / ADP-ribosylation factor-like protein 1 / ARF guanine-nucleotide exchange factor 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsDuan, H.D. / Li, H.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)CA231466 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural insight into an Arl1-ArfGEF complex involved in Golgi recruitment of a GRIP-domain golgin.
Authors: H Diessel Duan / Bhawik K Jain / Hua Li / Todd R Graham / Huilin Li /
Abstract: Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates ...Arl1 is an Arf-like (Arl) GTP-binding protein that interacts with the guanine nucleotide exchange factor Gea2 to recruit the golgin Imh1 to the Golgi. The Arl1-Gea2 complex also binds and activates the phosphatidylserine flippase Drs2 and these functions may be related, although the underlying molecular mechanism is unclear. Here we report high-resolution cryo-EM structures of the full-length Gea2 and the Arl1-Gea2 complex. Gea2 is a large protein with 1459 residues and is composed of six domains (DCB, HUS, SEC7, HDS1-3). We show that Gea2 assembles a stable dimer via an extensive interface involving hydrophobic and electrostatic interactions in the DCB and HUS region. Contrary to the previous report on a Gea2 homolog in which Arl1 binds to the dimerization surface of the DCB domain, implying a disrupted dimer upon Arl1 binding, we find that Arl1 binds to the outside surface of the Gea2 DCB domain, leaving the Gea2 dimer intact. The interaction between Arl1 and Gea2 involves the classic FWY aromatic residue triad as well as two Arl1-specific residues. We show that key mutations that disrupt the Arl1-Gea2 interaction abrogate Imh1 Golgi association. This work clarifies the Arl1-Gea2 interaction and improves our understanding of molecular events in the membrane trafficking.
History
DepositionNov 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 15, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 13, 2024Group: Database references / Structure summary / Category: citation / citation_author / struct
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ARF guanine-nucleotide exchange factor 2
B: ARF guanine-nucleotide exchange factor 2
C: ADP-ribosylation factor-like protein 1
D: ADP-ribosylation factor-like protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)379,1486
Polymers378,1024
Non-polymers1,0462
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ARF guanine-nucleotide exchange factor 2


Mass: 168590.703 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: GEA2, YEL022W / Production host: Escherichia coli (E. coli) / References: UniProt: P39993
#2: Protein ADP-ribosylation factor-like protein 1 / Arf-like GTPase 1


Mass: 20460.168 Da / Num. of mol.: 2 / Fragment: N-terminal 17 residues deleted / Mutation: Q72L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ARL1, ARF3, YBR164C, YBR1216 / Production host: Escherichia coli (E. coli) / References: UniProt: P38116
#3: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Gea2 homodimer in complex with two Arl1 monomers / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.379 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1700 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 69 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 712548 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00423604
ELECTRON MICROSCOPYf_angle_d0.5331915
ELECTRON MICROSCOPYf_dihedral_angle_d11.3218871
ELECTRON MICROSCOPYf_chiral_restr0.043764
ELECTRON MICROSCOPYf_plane_restr0.0044011

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