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# Interactome characterisation of a protein involved in lipid transport across the mycobacterial membrane: an XL-AP-MS approach

Marion Beaupere, Eve Randrianaridera, Emmanuelle Mouton-Barbosa, Alexandre Stella, Odile Burlet-Schiltz, Christian Chalut, Manuelle Ducoux-Petit

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## ► To cite this version:

Marion Beaupere, Eve Randrianaridera, Emmanuelle Mouton-Barbosa, Alexandre Stella, Odile Burlet-Schiltz, et al. Interactome characterisation of a protein involved in lipid transport across the mycobacterial membrane: an XL-AP-MS approach. EuPA, Jun 2025, Saint Malo, France. hal-05404491

HAL Id: hal-05404491

https://hal.science/hal-05404491v1

Submitted on 8 Dec 2025

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# Interactome characterisation of a protein involved in lipid transport across the mycobacterial membrane : an XL-AP-MS approach

Marion Beaupere¹, Eve Randrianaridera², Emmanuelle Mouton-Barbosa¹, Alexandre Stella¹, Odile Burlet-Schiltz¹, Christian Chalut¹, Manuelle Ducoux-Petit¹

¹ Proteomics and Mass Spectrometry of biomolecules lab, Proteotoul facility, Institut de Pharmacologie et de Biologie Structurale (IPBS), CNRS, Université de Toulouse, France.

² Institut de Science des Matériaux de Mulhouse, CNRS, 15 Rue Jean Starcky, 68057 Mulhouse Cedex, France.

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## INTRODUCTION

**Context:** Mycobacteria are a large family of bacteria, including the well-known representatives *Mycobacterium tuberculosis* and *Mycobacterium leprae*, responsible for tuberculosis and leprosy, respectively. The genus name "Mycobacteria" is due to the presence of a specific mycomembrane mainly composed of long-chain fatty acids: the mycolic acids. These mycolic acids are the support of the integrity of the mycomembrane, which contributes significantly to the virulence of the pathogenic mycobacteria and their ability to resist broad-spectrum antibiotics (Chiaradia *et al.* 2017). The biosynthesis of the mycomembrane is therefore a perfect target for new therapeutic research against diseases such as tuberculosis, especially proteins involved in the lipid transport and their interactions. Unfortunately, these proteins remain poorly characterised (Bories *et al.* 2024).

**Objectives:** Deciphering the interactome of MmpL10, a protein known to be involved in the transport of lipid chains to the mycomembrane (Thouvenel *et al.* 2020) will improve the knowledge of the proteins partners responsible for membrane assembly and integrity. Experiments will be carried out on the non-pathogenic and fast-growing strain *Mycobacterium smegmatis*.

**Strategy: XL-AP-MSMS of MmpL10**

XL : stabilisation of protein interactions within complexes involving MmpL10 by *in vivo* cross-linking.

AP : affinity purification of the complexes including the recombinant Twin-Strep tag-MmpL10.

MSMS : differential LC-MSMS analysis to identify and quantify the partners of MmpL10 compared to the control condition.

**Results:** This approach allowed us to identify and quantify more than 1,500 proteins. Of these, 48 were significantly enriched in the experimental condition compared to the control condition. These 48 proteins are then potential candidates involved in the lipid transport across the mycomembrane and could provide insights into the understanding of the pathogenic mycomembrane virulence.

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## STRATEGY

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