Biochemistry

Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome. The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion. They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation. In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12–ATG5-ATG16L1. However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction. In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8. This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation. It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation. Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout.

Key features

• Preparation of ATG7 E1 from insect cells (Sf9 cells).

• Preparation of ATG3 E2 from bacteria (E. coli).

• Preparation of LC3B S3C from bacteria (E. coli).

• Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.

Graphical overview

Experimental design to track the full reaction of ATG8 lipidation, described in this protocol

The lipid bilayers of the cell are composed of various lipid classes and species. These engage in cell signaling and regulation by recruiting cytosolic proteins to the membrane and interacting with membrane-embedded proteins to alternate their activity and stability. Like lipids, membrane proteins are amphipathic and are stabilized by the hydrophobic forces of the lipid bilayer. Membrane protein–lipid interactions are difficult to investigate since membrane proteins need to be reconstituted in a lipid-mimicking environment. A common and well-established approach is the detergent-based solubilization of the membrane proteins in detergent micelles. Nowadays, nanodiscs and liposomes are used to mimic the lipid bilayer and enable the work with membrane proteins in a more natural environment. However, these protocols need optimization and are labor intensive. The present protocol describes straightforward instructions on how the preparation of lipids is performed and how the lipid detergent mixture is integrated with the membrane protein MARCH5. The lipidation protocol was performed prior to an activity assay specific to membrane-bound E3 ubiquitin ligases and a stability assay that could be used for any membrane protein of choice.

Integral membrane proteins are an important class of cellular proteins. These take part in key cellular processes such as signaling transducing receptors to transporters, many operating within the plasma membrane. More than half of the FDA-approved protein-targeting drugs operate via interaction with proteins that contain at least one membrane-spanning region, yet the characterization and study of their native interactions with therapeutic agents remains a significant challenge. This challenge is due in part to such proteins often being present in small quantities within a cell. Effective solubilization of membrane proteins is also problematic, with the detergents typically employed in solubilizing membranes leading to a loss of functional activity and key interacting partners. In recent years, alternative methods to extract membrane proteins within their native lipid environment have been investigated, with the aim of producing functional nanodiscs, maintaining protein–protein and protein–lipid interactions. A promising approach involves extracting membrane proteins in the form of styrene maleic acid lipid particles (SMALPs) that allow the retention of their native conformation. This extraction method offers many advantages for further protein analysis and allows the study of the protein interactions with other molecules, such as drugs. Here, we describe a protocol for efficient SMALP extraction of functionally active membrane protein complexes within nanodiscs. We showcase the method on the isolation of a low copy number plasma membrane receptor complex, the nicotinic acetylcholine receptor (nAChR), from adult Drosophila melanogaster heads. We demonstrate that these nanodiscs can be used to study native receptor–ligand interactions. This protocol can be applied across many biological scenarios to extract the native conformations of low copy number integral membrane proteins.

G-protein coupled signaling pathways are organized into multi-protein complexes called signalosomes that are located within and on cellular membranes. We describe the use of silica nanoparticles coated with a unilamellar phospholipid bilayer (lipobeads) to reconstitute the activated photoreceptor G-protein α-subunit (Gtα*) with its cognate effector (phosphodiesterase-6; PDE6) for biochemical and structural studies of the activation mechanism regulating this GPCR signaling pathway. Lipobeads are prepared by resuspending dried-down phospholipid mixtures with monodisperse 70 nm silica particles, followed by extrusion through a 100 nm membrane filter. This uniform and supported liposomal preparation is easily sedimented, permitting the separation of soluble from membrane-associated proteins. Upon loading lipobeads with Gtα* and PDE6, we find that activation of PDE6 catalysis by Gtα* occurs much more efficiently than in the absence of membranes. Chemical cross-linking of membrane-confined proteins allows detection of changes in protein-protein interactions, resulting from G-protein activation of PDE6. The advantages of using lipobeads over partially purified membrane preparations or traditional liposomal preparations are generally applicable to the study of other membrane-confined signal transduction pathways.

The aim of this protocol is to generate COPII-coated procollagen I (PC1) carriers in a cell-free reaction. The COPII-coated PC1 carriers were reconstituted from donor membrane, cytosol, purified recombinant COPII proteins, and nucleotides. This protocol describes the preparation of donor membrane and cytosol, the assembly of the reaction, and the isolation and detection of reconstituted COPII-coated carriers. This cell-free reaction can be used to test conditions that stimulate or suppress the packaging of PC1 into COPII-coated carriers.

Proteins that bind to and disrupt cell membranes may target specific phospholipids. Here we describe a protocol to identify the lipid targets of proteins and biomolecules. First, we describe a screen to identify lipids in membranes that are specifically bound by the biomolecule of interest. Second, we describe a method for determining if the presence of these lipids within membranes is necessary for membrane disruption. The methods described here were used to determine that the malaria vaccine candidate CelTOS disrupts cell membranes by specifically targeting phosphatidic acid (Jimah et al., 2016). This protocol has a companion protocol: ‘Liposome disruption assay to examine lytic properties of biomolecules’ which can be applied to examine the ability of the biomolecule to disrupt membranes composed of the lipid target identified by following this protocol (Jimah et al., 2017).

A mitochondrion is a dynamic intracellular organelle that actively divides and fuses to control its size, number and shape in cells. A regulated balance between mitochondrial division and fusion is fundamental to the function, distribution and turnover of mitochondria (Roy et al., 2015). Mitochondrial division is mediated by dynamin-related protein 1 (Drp1), a mechano-chemical GTPase that constricts mitochondrial membranes (Tamura et al., 2011). Mitochondrial membrane lipids such as phosphatidic acid and cardiolipin bind Drp1, and Drp1-phospholipid interactions provide key regulatory mechanisms for mitochondrial division (Montessuit et al., 2010; Bustillo-Zabalbeitia et al., 2014; Macdonald et al., 2014; Stepanyants et al., 2015; Adachi et al., 2016). Here, we describe biochemical experiments that quantitatively measure interactions of Drp1 with lipids using purified recombinant Drp1 and synthetic liposomes with a defined set of phospholipids. This assay makes it possible to define the specificity of protein-lipid interaction and the role of the head group and acyl chains.

Phosphoinositides are rare membrane lipids involved in the control of the major cellular functions and signaling pathways. They are able to recruit specific effector proteins to the cytosolic face of plasma membrane and organelles to coordinate a vast variety of signaling and trafficking processes, as well to maintain specific identity of the different subcellular compartments (Di Paolo and De Camilli, 2006; Lemmon, 2003). Therefore, analysis of these effectors’ binding properties and specificity towards different phosphoinositides is crucial for the understanding of their cellular functions. This protocol describes a method to characterize the binding of proteins to different phosphoinositide-containing vesicles.

Examining the interaction of peptides with lipid bilayers to determine binding kinetics is often performed using surface plasmon resonance (SPR). Here we describe the technique of dual polarisation interferometry (DPI) that provides not only information on the kinetics of the peptide binding to the bilayer, but also how the peptide affects the lipid order of the bilayer.

Several membrane-perturbing agents extract lipids from membranes and, in some cases, this lipid efflux is lipid specific. In order to gain a better description of this phenomenon and to detail the intermolecular interactions that are involved, a method has been developed to characterize the extent and the specificity of membrane-lipid extraction by perturbing agents. A perturbing agent is incubated with model membranes existing as multilamellar vesicles (MLVs) and subsequently, the remaining MLVs and the small lipid/perturbing agent complexes resulting from the extraction are isolated and analysed to assess the extent and the specificity of the lipid extraction.