Background

Membrane proteins interact with their lipid environment with the hydrophobic and hydrophilic patches of their transmembrane domains via a specific binding site (non-annular lipids) or the physiochemical properties of the lipid bilayer (annular lipids) (Contreras et al., 2011; Stangl and Schneider, 2015). These protein–lipid interactions can alter the activity, stability, folding, or localization of the target protein, leading to various cellular signaling events (Engelman, 2005; van Meer et al., 2008; Corradi et al., 2019a;). The characterization of these signaling mechanisms and the corresponding lipid–membrane protein interaction is a constant challenge due to the inherent phase difference imposed on the system from the presence of a mixture of detergent/lipid, lipid/protein, and detergent/lipid/protein (Sych et al., 2022). Therefore, research into the mechanisms used by lipids to regulate protein function and structure is restricted to a small number of membrane protein systems, often because of the technical difficulties as well as complexity associated with handling these systems in a laboratory setting (Fahy et al., 2009). Hence, understanding the mechanism of how lipids can modulate the activity of a specific membrane protein and the identification of the lipids that interact with the membrane protein of interest is often still an open and fundamental research question (Corradi et al., 2019b).

Phospholipids are the most abundant lipids in mammalian membranes and are characterized by their hydrophilic head group and their hydrophobic tail, which is built by two fatty acid chains. These fatty acid chains can vary in length and saturation, creating an immense variety (Horvath and Daum, 2013; Cockcroft, 2021). The chemically diverse hydrophilic head groups define the lipid classes (Table 1), whereas the different lengths and saturation subdivide the class in different lipid species (Liebisch et al., 2020). The lipid composition of the lipid bilayer is specific to the tissue and the cellular compartment, which comprises a distinct set of phospholipids (Table 1) (Ernst et al., 2016).

Table 1. Overview of phospholipids, their chemical characteristic, and the abundance of subcellular fractions [plasma membrane, mitochondria, and endoplasmic reticulum (ER)) of rat liver (Horvath and Daum, 2013)]

Nowadays, different membrane-mimicking systems are available to solvate membrane proteins. The most common method for working with membrane proteins in vitro is to solubilize them in detergent. The detergent forms a micelle layer around the hydrophobic surface patches of the protein, leading to the stabilization of the membrane (or membrane-associated) protein outside the natural lipid bilayer. Over the last years, alternatives to detergent-based methods were developed, such as nanodiscs (Pettersen et al., 2023), styrene–maleic acid copolymers (SMAs) (Dörr et al., 2016), or liposomes (Verchère et al., 2017). However, these methods often need a detergent-solubilized membrane protein as the initial step and optimization is often labor intensive. Here, we describe two easy-to-establish and straightforward protocols to examine the interaction and effect of different lipid classes on the activity and stability of purified and detergent-solubilized MARCH5 in vitro. The described protocols were initially used to test lipid stability in connection with the human integral membrane protein SERINC5 (Pye et al., 2020) and, since then, tested against a broader spectrum of membrane proteins (Cecchetti et al., 2021).