Background

A large number of transmembrane proteins are formed through the assembly of multiple subunits leading to complicated oligomeric structures that can often exist in multiple stoichiometries. Understanding how changes in assembly alter trafficking and localization within different organelles is essential to determining a protein’s physiological role and the connection to diseases associated with maturation and transport. Single molecule approaches can provide a better understanding of the assembly of oligomeric proteins by directly measuring their stoichiometry (Ulbrich and Isacoff, 2007; Richards et al., 2012). This approach avoids ensemble averaging which provides the average state of all the stoichiometries (Walter and Bustamante, 2014). Single molecule studies have recently been employed to understand the structural and functional properties of macromolecules including conformational dynamics (Tan et al., 2014), ion channel gating (Wang et al., 2016), ligand-receptor interaction (Moonschi et al., 2015), and stoichiometric assembly (Ulbrich and Isacoff, 2007; Moonschi et al., 2015). To conduct single molecule studies, it is imperative to isolate single receptors in a supporting bilayer. Separation from the cellular environment is often necessary because the endogenous concentration of membrane proteins in mammalian cells is typically much higher than conditions compatible with single molecule studies (Richards et al., 2012). Additionally, live cell single molecule studies suffer from a number of disadvantages including high levels of auto-fluorescence, limited fluorophore brightness and the mobility of membrane proteins on the cell surface (Andersson et al., 1998; Lippincott-Schwartz et al., 1999). Isolation strategies using artificial bilayers such as liposomes are also problematic as they include an intermediate step where the protein is stabilized into a detergent solution which poses a threat to the structural integrity of the transmembrane protein. Membrane-derived vesicles enable the protein to remain embedded in its physiological membrane maintaining its structural and functional integrity. These nanoscale vesicles have been employed to study stoichiometric assembly of multimeric proteins, to probe ligand receptor interactions, and to understand the effect of ligands on the assembly of nicotinic receptors (Fox et al., 2015; Moonschi et al., 2015; Fox-Loe et al., 2017).

Transmembrane proteins are synthesized and assembled in the endoplasmic reticulum and then trafficked to the plasma membrane. Oligomeric proteins with multiple non-identical subunits can often be assembled with different subunit stoichiometries potentially leading to different trafficking and functional properties (Grady et al., 2010). For instance, α4β2 nicotinic receptors are pentameric receptor with two possible stoichiometric assemblies: (α4)₂(β2)₃ and (α4)₃(β2)₂. It has been hypothesized that cellular machinery preferentially traffics the high sensitivity isoform (α4)₂(β2)₃ over the other isoform from the ER to the plasma membrane. It has also been hypothesized that nicotine alters the assembly of this receptor from the low sensitivity to high sensitivity isoform in the ER leading to higher levels of the preferentially trafficked assembly in both the ER and plasma membrane (Lester et al., 2009; Henderson and Lester, 2015). Single molecule studies of proteins from specific organelles enable the correlation of changes in structural assembly of the nicotinic receptors to changes in trafficking and assembly.

Here we present a method which can isolate single transmembrane proteins into the ER and plasma membrane derived vesicles using nitrogen cavitation and an OptiPrep gradient. The ER and plasma membrane derived vesicles exhibit different densities because they contain different phospholipids and associated proteins. ER originated vesicles are much denser than those obtained from the plasma membrane. The OptiPrep gradient was selected because of its superior ability to maintain isosmotic pressure independent of the density of the gradient used to isolate cellular organelles and subcellular vesicles (Graham et al., 1994). We applied this method to study ligand induced changes in the assembly of nicotinic receptors in the ER as well as the plasma membrane and validated our method with Western blotting and single molecule step-wise photobleaching. We believe this method can be applied for virtually any type of transmembrane proteins to conduct single molecule studies and to understand organelle-specific structural and functional properties.