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- PDB-8r8q: Lysosomal peptide transporter -

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Basic information

Entry
Database: PDB / ID: 8r8q
TitleLysosomal peptide transporter
Components
  • Glycosylated lysosomal membrane protein
  • Major facilitator superfamily domain-containing protein 1
KeywordsMEMBRANE PROTEIN / MFS transporter / MFSD family / nutrition transporter / lysosomal transporter / outward open
Function / homology
Function and homology information


dipeptide transmembrane transport from lysosomal lumen to cytosol / dipeptide uniporter activity / protein localization to lysosome / lysosome / protein stabilization / lysosomal membrane / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / nucleus / membrane / cytosol
Similarity search - Function
: / Lysosomal transcription factor, NCU-G1 / Lysosomal transcription factor, NCU-G1 / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Lysosomal dipeptide transporter MFSD1 / Glycosylated lysosomal membrane protein
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.08 Å
AuthorsJungnickel, K.E.J. / Loew, C.
Funding support Germany, 1items
OrganizationGrant numberCountry
Other government05K18YEA Germany
CitationJournal: Nat Cell Biol / Year: 2024
Title: MFSD1 with its accessory subunit GLMP functions as a general dipeptide uniporter in lysosomes.
Authors: Katharina Esther Julia Jungnickel / Océane Guelle / Miharu Iguchi / Wentao Dong / Vadim Kotov / Florian Gabriel / Cécile Debacker / Julien Dairou / Isabelle McCort-Tranchepain / Nouf N ...Authors: Katharina Esther Julia Jungnickel / Océane Guelle / Miharu Iguchi / Wentao Dong / Vadim Kotov / Florian Gabriel / Cécile Debacker / Julien Dairou / Isabelle McCort-Tranchepain / Nouf N Laqtom / Sze Ham Chan / Akika Ejima / Kenji Sato / David Massa López / Paul Saftig / Ahmad Reza Mehdipour / Monther Abu-Remaileh / Bruno Gasnier / Christian Löw / Markus Damme /
Abstract: The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator ...The lysosomal degradation of macromolecules produces diverse small metabolites exported by specific transporters for reuse in biosynthetic pathways. Here we deorphanized the major facilitator superfamily domain containing 1 (MFSD1) protein, which forms a tight complex with the glycosylated lysosomal membrane protein (GLMP) in the lysosomal membrane. Untargeted metabolomics analysis of MFSD1-deficient mouse lysosomes revealed an increase in cationic dipeptides. Purified MFSD1 selectively bound diverse dipeptides, while electrophysiological, isotope tracer and fluorescence-based studies in Xenopus oocytes and proteoliposomes showed that MFSD1-GLMP acts as a uniporter for cationic, neutral and anionic dipeptides. Cryoelectron microscopy structure of the dipeptide-bound MFSD1-GLMP complex in outward-open conformation characterized the heterodimer interface and, in combination with molecular dynamics simulations, provided a structural basis for its selectivity towards diverse dipeptides. Together, our data identify MFSD1 as a general lysosomal dipeptide uniporter, providing an alternative route to recycle lysosomal proteolysis products when lysosomal amino acid exporters are overloaded.
History
DepositionNov 29, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jun 19, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Jul 24, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.4Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Major facilitator superfamily domain-containing protein 1
B: Glycosylated lysosomal membrane protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,9747
Polymers98,8682
Non-polymers1,1065
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area3390 Å2
ΔGint-9 kcal/mol
Surface area35260 Å2
MethodPISA

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Components

#1: Protein Major facilitator superfamily domain-containing protein 1


Mass: 55765.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Mfsd1 / Production host: Homo sapiens (human) / References: UniProt: Q9DC37
#2: Protein Glycosylated lysosomal membrane protein / Lysosomal protein NCU-G1


Mass: 43102.711 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Glmp / Production host: Homo sapiens (human) / References: UniProt: Q9JHJ3
#3: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationY

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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: GLMP-MFSD1 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHis-Ala1
2150 mMsodium chlorideNaCl1
30.015 %DDM1
40.0015 %CHS1
SpecimenConc.: 3.33 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: PROPANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3193

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4CTFFINDCTF correction
7ISOLDE1.0b3model fitting
8Coot0.9.8.1model fitting
10PHENIX1.20.1-4487model refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.08 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 400191 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035855
ELECTRON MICROSCOPYf_angle_d0.6327971
ELECTRON MICROSCOPYf_dihedral_angle_d4.5801
ELECTRON MICROSCOPYf_chiral_restr0.04922
ELECTRON MICROSCOPYf_plane_restr0.004988

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