
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.20.608660
preprint
2
Article
Lipid- and protein-directed photosensitizer proximity labeling captures the cholesterol interactome
Becker Andrew P. http://orcid.org/0000-0002-1654-0912

Biletch Elijah http://orcid.org/0000-0002-3723-536X

Kennelly John Paul http://orcid.org/0000-0002-4399-3976

Julio Ashley R. http://orcid.org/0000-0002-6258-2849

Villaneuva Miranda http://orcid.org/0000-0002-4892-8356

Nagari Rohith T. http://orcid.org/0000-0002-0677-3237

Turner Daniel W.
Burton Nikolas R. http://orcid.org/0000-0003-2887-8846

Fukuta Tomoyuki
Cui Liujuan http://orcid.org/0000-0001-6186-1558

Xiao Xu
Hong Soon-Gook http://orcid.org/0000-0002-1385-4430

Mack Julia J. http://orcid.org/0000-0002-9239-8436

Tontonoz Peter http://orcid.org/0000-0003-1259-0477

Backus Keriann M. http://orcid.org/0000-0001-8541-1404

24 8 2024
2024.08.20.608660https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.20.608660
nihpp-2024.08.20.608660.pdf
Abstract

The physical properties of cellular membranes, including fluidity and function, are influenced by protein and lipid interactions. In situ labeling chemistries, most notably proximity-labeling interactomics are well suited to characterize these dynamic and often fleeting interactions. Established methods require distinct chemistries for proteins and lipids, which limits the scope of such studies. Here we establish a singlet-oxygen-based photocatalytic proximity labeling platform (POCA) that reports intracellular interactomes for both proteins and lipids with tight spatiotemporal resolution using cell-penetrant photosensitizer reagents. Using both physiologically relevant lipoprotein-complexed probe delivery and genetic manipulation of cellular cholesterol handling machinery, cholesterol-directed POCA captured established and unprecedented cholesterol binding proteins, including protein complexes sensitive to intracellular cholesterol levels and proteins uniquely captured by lipoprotein uptake. Protein-directed POCA accurately mapped known intracellular membrane complexes, defined sterol-dependent changes to the non-vesicular cholesterol transport protein interactome, and captured state-dependent changes in the interactome of the cholesterol transport protein Aster-B. More broadly, we find that POCA is a versatile interactomics platform that is straightforward to implement, using the readily available HaloTag system, and fulfills unmet needs in intracellular singlet oxygen-based proximity labeling proteomics. Thus, we expect widespread utility for POCA across a range of interactome applications, spanning imaging to proteomics.
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pmc
