**1. Introduction**

ABCB4 (ATP-binding cassette subfamily B member 4), also named MDR3 (MultiDrug Resistance 3), is one of the main biliary transporters [1]. It is expressed at the canalicular membrane of hepatocytes where it mediates phosphatidylcholine (PC) secretion into bile [2]. In the aqueous environment of bile, PC plays a critical role in cholesterol solubilization as well as bile acid neutralization [3]. Variations in the *ABCB4* gene are associated with

**Citation:** Ben Saad, A.; Vauthier, V.; Lapalus, M.; Mareux, E.; Bennana, E.; Durand-Schneider, A.-M.; Bruneau, A.; Delaunay, J.-L.; Gonzales, E.; Housset, C.; et al. RAB10 Interacts with ABCB4 and Regulates Its Intracellular Traffic. *Int. J. Mol. Sci.* **2021**, *22*, 7087. https://doi.org/ 10.3390/ijms22137087

Academic Editor: Jose J.G. Marin

Received: 27 May 2021 Accepted: 29 June 2021 Published: 30 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

several rare cholestatic diseases, including Progressive Familial Intrahepatic Cholestasis type 3 (PFIC3), Low Phospholipid-Associated Cholelithiasis (LPAC) syndrome, and Intrahepatic Cholestasis of Pregnancy (ICP) [4,5]. While treatment with ursodeoxycholic acid remains efficient for the majority of patients with milder forms of ABCB4-related diseases, this is not the case for patients with PFIC3, the most severe form of these diseases, who most often require liver transplantation [6,7], stressing the unmet need for new therapeutic options. In the frame of personalized medicine, new targeted pharmacotherapies for ABCB4-related diseases have been proposed from in vitro studies and include the potentiator Ivacaftor/VX-770 as well as structural analogues of roscovitine [8,9]. In addition, AAV8- or mRNA-mediated gene therapy constitute an interesting alternative to rescue ABCB4 deficiency, as recently described in mouse models by several research groups [10–13]. In order to better characterize and understand the mode of action of new therapies, a better understanding of the molecular mechanisms regulating ABCB4 traffic and function is required.

Over the last decade, proteomic studies have become a powerful tool to decipher molecular mechanisms at the subcellular level and to provide new insights into protein biology. In the last years, a limited number of ABCB4 interacting partners were identified by yeast two-hybrid screens, including HS1-Associated protein X-1 (HAX1), the motor protein Myosin II regulatory Light Chain 2 (MLC2), Receptor for Activated C-kinase 1 (RACK1), and ERM-Binding Phosphoprotein of 50 kDa (EBP50) [14–18]. Aiming at identifying novel molecular partners of ABCB4, we used an immunoprecipitation approach combined with HPLC-coupled tandem mass spectrometry analyses (usually defined as "Affinity Purification-Mass Spectrometry" or AP-MS). This allowed us to identify the small GTPase RAB10 as a potential ABCB4 interactor. RAB10 belongs to the Ras-related in brain (Rab) family of proteins, which are well known as master regulators of intracellular traffic and sorting processes [19]. RAB proteins are implicated in the majority of vesicular transport steps, including vesicle formation, motility, and tethering, as well as their fusion with target membranes [19]. To date, more than 60 RAB proteins have been identified in mammals. These soluble proteins are ubiquitously expressed and can be membrane-associated with many subcellular compartments thanks to their post-translational geranyl-geranylation [20]. RAB10 was first cloned from Madin-Darby Canine Kidney cells [21]. It is mainly involved in protein trafficking from the Golgi apparatus to the plasma membrane [22]. Its implication in GLUT4 traffic as well as TLR4 exocytosis is well documented [23,24]. RAB10 also plays a key role in ciliogenesis, neuronal development, and basolateral recycling [22].

In the present study, we used biochemical and morphological approaches in cell models, namely HEK and HeLa cells. These cells allow high transfection rate and high expression level of transgenes, and they are validated models for investigating the cell biology of ABC transporters [25,26]. We found that the overexpression of RAB10-wild type (WT) or its constitutively active mutant increases ABCB4 membrane expression and function, whereas RAB10 silencing attenuates ABCB4 cell surface expression, as well as its PC secretion function. Taken together, our results indicate that RAB10 is an important regulator of ABCB4 traffic, by promoting its transport from the Golgi apparatus to the plasma membrane.
