EndoMAP.v1, a Structural Protein Complex Landscape of Human Endosomes

Overview

Journal bioRxiv

Date 2025 Feb 20

PMID 39975243

Authors

Miguel A Gonzalez-Lozano

Ernst W Schmid

Enya Miguel Whelan

Yizhi Jiang

Joao A Paulo

Johannes C Walter

J Wade Harper

Affiliations

Soon will be listed here.

Abstract

Early/sorting endosomes are dynamic organelles that play key roles in proteome control by triaging plasma membrane proteins for either recycling or degradation in the lysosome. These events are coordinated by numerous transiently-associated regulatory complexes and integral membrane components that contribute to organelle identity during endosome maturation. While a subset of the several hundred protein components and cargoes known to associate with endosomes have been studied at the biochemical and/or structural level, interaction partners and higher order molecular assemblies for many endosomal components remain unknown. Here, we combine cross-linking and native gel mass spectrometry of purified early endosomes with AlphaFold and computational analysis to create a systematic human endosomal structural interactome. We present dozens of structural models for endosomal protein pairs and higher order assemblies supported by experimental cross-links from their native subcellular context, suggesting structural mechanisms for previously reported regulatory processes. Using induced neurons, we validate two candidate complexes whose interactions are supported by crosslinks and structural predictions: TMEM230 as a subunit of ATP8/11 lipid flippases and TMEM9/9B as subunits of CLCN3/4/5 chloride-proton antiporters. This resource and its accompanying structural network viewer provide an experimental framework for understanding organellar structural interactomes and large-scale validation of structural predictions.

References

Pons V, Ustunel C, Rolland C, Torti E, Parton R, Gruenberg J . SNX12 role in endosome membrane transport. PLoS One. 2012; 7(6):e38949. PMC: 3376135. DOI: 10.1371/journal.pone.0038949. View

Farrer M . Doubts about TMEM230 as a gene for parkinsonism. Nat Genet. 2019; 51(3):367-368. DOI: 10.1038/s41588-019-0354-6. View

Merkley E, Rysavy S, Kahraman A, Hafen R, Daggett V, Adkins J . Distance restraints from crosslinking mass spectrometry: mining a molecular dynamics simulation database to evaluate lysine-lysine distances. Protein Sci. 2014; 23(6):747-59. PMC: 4093951. DOI: 10.1002/pro.2458. View

Wandinger-Ness A, Zerial M . Rab proteins and the compartmentalization of the endosomal system. Cold Spring Harb Perspect Biol. 2014; 6(11):a022616. PMC: 4413231. DOI: 10.1101/cshperspect.a022616. View

Bartolec T, Vazquez-Campos X, Norman A, Luong C, Johnson M, Payne R . Cross-linking mass spectrometry discovers, evaluates, and corroborates structures and protein-protein interactions in the human cell. Proc Natl Acad Sci U S A. 2023; 120(17):e2219418120. PMC: 10151615. DOI: 10.1073/pnas.2219418120. View

Thanou E, Koopmans F, Pita-Illobre D, Klaassen R, Ozer B, Charalampopoulos I . Suspension TRAPping Filter (sTRAP) Sample Preparation for Quantitative Proteomics in the Low µg Input Range Using a Plasmid DNA Micro-Spin Column: Analysis of the Hippocampus from the 5xFAD Alzheimer's Disease Mouse Model. Cells. 2023; 12(9). PMC: 10177283. DOI: 10.3390/cells12091242. View

Giurgiu M, Reinhard J, Brauner B, Dunger-Kaltenbach I, Fobo G, Frishman G . CORUM: the comprehensive resource of mammalian protein complexes-2019. Nucleic Acids Res. 2018; 47(D1):D559-D563. PMC: 6323970. DOI: 10.1093/nar/gky973. View

Lin D, Conibear E . ABHD17 proteins are novel protein depalmitoylases that regulate N-Ras palmitate turnover and subcellular localization. Elife. 2015; 4:e11306. PMC: 4755737. DOI: 10.7554/eLife.11306. View

Itzhak D, Tyanova S, Cox J, Borner G . Global, quantitative and dynamic mapping of protein subcellular localization. Elife. 2016; 5. PMC: 4959882. DOI: 10.7554/eLife.16950. View

10.

HaileMariam M, Eguez R, Singh H, Bekele S, Ameni G, Pieper R . S-Trap, an Ultrafast Sample-Preparation Approach for Shotgun Proteomics. J Proteome Res. 2018; 17(9):2917-2924. DOI: 10.1021/acs.jproteome.8b00505. View

11.

Henne W, Buchkovich N, Emr S . The ESCRT pathway. Dev Cell. 2011; 21(1):77-91. DOI: 10.1016/j.devcel.2011.05.015. View

12.

Vietri M, Radulovic M, Stenmark H . The many functions of ESCRTs. Nat Rev Mol Cell Biol. 2019; 21(1):25-42. DOI: 10.1038/s41580-019-0177-4. View

13.

Muraleedharan A, Vanderperre B . The Endo-lysosomal System in Parkinson's Disease: Expanding the Horizon. J Mol Biol. 2023; 435(12):168140. DOI: 10.1016/j.jmb.2023.168140. View

14.

Stenmark H, Vitale G, Ullrich O, Zerial M . Rabaptin-5 is a direct effector of the small GTPase Rab5 in endocytic membrane fusion. Cell. 1995; 83(3):423-32. DOI: 10.1016/0092-8674(95)90120-5. View

15.

Huttlin E, Bruckner R, Navarrete-Perea J, Cannon J, Baltier K, Gebreab F . Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell. 2021; 184(11):3022-3040.e28. PMC: 8165030. DOI: 10.1016/j.cell.2021.04.011. View

16.

Elias J, Gygi S . Target-decoy search strategy for mass spectrometry-based proteomics. Methods Mol Biol. 2009; 604:55-71. PMC: 2922680. DOI: 10.1007/978-1-60761-444-9_5. View

17.

Chambers M, MacLean B, Burke R, Amodei D, Ruderman D, Neumann S . A cross-platform toolkit for mass spectrometry and proteomics. Nat Biotechnol. 2012; 30(10):918-20. PMC: 3471674. DOI: 10.1038/nbt.2377. View

18.

Tremel S, Ohashi Y, Morado D, Bertram J, Perisic O, Brandt L . Structural basis for VPS34 kinase activation by Rab1 and Rab5 on membranes. Nat Commun. 2021; 12(1):1564. PMC: 7946940. DOI: 10.1038/s41467-021-21695-2. View

19.

Kofler N, Corti F, Rivera-Molina F, Deng Y, Toomre D, Simons M . The Rab-effector protein RABEP2 regulates endosomal trafficking to mediate vascular endothelial growth factor receptor-2 (VEGFR2)-dependent signaling. J Biol Chem. 2018; 293(13):4805-4817. PMC: 5880142. DOI: 10.1074/jbc.M117.812172. View

20.

Liu F, Rijkers D, Post H, Heck A . Proteome-wide profiling of protein assemblies by cross-linking mass spectrometry. Nat Methods. 2015; 12(12):1179-84. DOI: 10.1038/nmeth.3603. View