Cook et al. 2026

Identifcation of lipid specifcity in membrane organizing protein complexes

Authors

Katelyn C. Cook1, Karen Palacio-Rodriguez2, Kristin Böhlig1, H. Mathilda Lennartz1, Swantje Lenz1,3, Andrej Shevchenko1, Alf Honigmann4,5, Gerhard Hummer2,6, Alexander von Appen1,5, André Nadler1,5

Affiliations

1 Max Planck Institute of Molecular Cell Biology and Genetics (Dresden, Germany) 2 Department of Theoretical Biophysics, Max Planck Institute of Biophysics (Frankfurt am Main, Germany) 3 Center for Systems Biology (Dresden, Germany) 4 Biotechnologisches Zentrum, Center for Molecular and Cellular Bioengineering, Technische Universität Dresden (Dresden, Germany) 5 Cluster of Excellence Physics of Life, TU Dresden (Dresden, Germany) 6 Institute of Biophysics, Goethe University Frankfurt (Frankfurt am Main, Germany)

Journal

Abstract

Organelle membrane identity is encoded by protein and lipid composition. Human cells produce thousands of distinct lipid species for this purpose. Yet, the precise molecular functions of most lipids remain unknown, owing to limited methods for in situ investigations of individual lipid species. Here, we use minimally modified bifunctional lipid probes to detect and functionally characterize lipid-protein interactions across time and subcellular compartments. We leverage this approach to map time-resolved protein interactomes of individual lipids representing major membrane lipid classes as they are transported through the organelle system of human cells. We identify hundreds of unique lipid-protein interaction candidates, measure lipid-induced protein abundance changes, and distinguish between directly and indirectly interacting subunits of associated protein complexes. Informed by this multimodal dataset, we interrogate the lipid specificity of membrane organizing complexes, in particular the selective co-association of phosphatidylethanolamine with the ancestral subunits of the nuclear pore (NDC1) and MICOS (MIC60) complexes. Using super-resolution microscopy and molecular dynamics simulations, we demonstrate that phosphatidylethanolamine stabilizes both NDC1 and MIC60 in highly curved membrane nanodomains, indicating that major membrane organizing complexes are positioned by selective lipid-protein interactions. Altogether, our experimental strategy provides a blueprint for discovering and characterizing cellular functions of individual lipids.

Lipid probes utilized

Bifunctional Sphingomyelin (18:1/Y16)

Bifunctional Phosphatidylethanolamine (18:1/Y16)

Bifunctional Phosphatidylcholine (18:1/Y16)

Bifunctional Phosphatidylcholine (20:0/Y16)

Bifunctional Phosphatidylcholine (14:0/Y14)

Probe notation is Headgroup(acyl chain sn-1/acyl chain sn-2), with Yx denoting the bifunctional chain and x = number of carbons

Cell line analyzed

U2OS

Species: Homo sapiens

Cell type: Epithelial (immortalized osteosarcoma cell line)

Protocol Overview

Identification of False Lipid-binding Unspecific Background (FLUB) proteins
Uncaging & Crosslinking timeline

Mass spectrometry quantification method

Label-free protein quantification (LFQ)

Data visualization

Volcano plots depict the relative enrichment of each protein versus control (x-axis) and the statistical significance of each protein following a Student’s t-test (y-axis, -log10 transformed). The left panel depicts the results of a pulldown using trifunctionalized phosphatidic acid; the right panel depicts the results of the pulldown using trifunctionalized phosphatidylethanolamine. Black proteins are unenriched or depleted in the presence of probe, Purple proteins are enriched to the probe but fall below a FDR or p-value threshold of significance, and Orange proteins surpass the threshold of significance (need details of threshold).

Ranked-order plots depict the relative enrichment of each protein versus control (y-axis) from lowest to highest. The left panel depicts the results of a pulldown using trifunctionalized phosphatidic acid; the right panel depicts the results of the pulldown using trifunctionalized phosphatidylethanolamine. Black proteins are unenriched or depleted in the presence of probe, Purple proteins are enriched to the probe but fall below a FDR or p-value threshold of significance, and Orange proteins surpass the threshold of significance (need details of threshold).

MA plots

Gene Ontology Analysis

In beta: GO analysis still under development

(No BP pathways identified among enriched hits and candidates.)

Figure 1: GO Dot plots display the enrichment of GO terms among the proteins enriched to the probe. For these analyses, only proteins categorized as “enriched candidates” and “enriched hits” were subject to GO analysis. The Cell Compartment analysis assesses whether the list of enriched proteins contains a statistically significant number of proteins in the same cellular region; appropriately, the Molecular Function and Biological Process analyses does the same for molecular function and biological process, respectively. Click here for more information about Gene Ontology analysis.

Data exploration

Check the boxes below to filter the dataset by which lipid probe was used in the pulldown and by significance thresholds.

Data download