The Cell-Free Integration of a Polytopic Mitochondrial Membrane Protein into Liposomes Occurs Cotranslationally and in a Lipid-Dependent Manner

Abstract

The ADP/ATP Carrier (AAC) is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

From Nanodiscs to Isotropic Bicelles: A Procedure for Solution Nuclear Magnetic Resonance Studies of Detergent-Sensitive Integral Membrane Proteins

“…Unassisted helical insertion has been demonstrated to have similar free energies as MP insertion by the Sec61 translocon (Ulmschneider et al, 2014). Furthermore, direct expression into bilayers is gaining recognition as a method to produce functional MPs in lipid bilayers that have never been in contact with detergent (Goren et al, 2009; Henrich et al, 2016; Kalmbach et al, 2007; Long et al, 2012; Yildiz et al, 2012). …”

Advances in the use of nanoscale bilayers to study membrane protein structure and function

“…For example, the MraY translocase, containing 10 transmembrane helices, required larger diameter nanodiscs and the presence of anionic phospholipids for efficient integration and activity. In the absence of protein integration machinery, the mechanism by which MPs are incorporated into such pre-assembled model membranes remains elusive, although the cotranslational mode of integration and key lipids are likely to be important factors, as we discuss in length elsewhere (Long, O'Brien, & Alder, 2012). In the case of nanodiscs, the observed accelerated desorption of lipids in comparison to lipid vesicles (Nakano et al, 2009) may be a mechanistic underpinning for the ability of transmembrane segments to cotranslationally displace existing lipids.…”

Hydrophobic supplements in cell‐free systems: Designing artificial environments for membrane proteins

“…DOPC and other lipids are also tolerated by wheat germ extracts, and the cotranslational membrane insertion of connexins and apo cytochrome b5 in wheat germ CF systems are some examples. Effects of individual lipids on MP insertion and MP quality can systematically be screened with synthetic liposomes, and some lipids such as cardiolipin or cholesterol have shown beneficial chaperone effects on specific MP targets .…”

“…DOPC and other lipids are also tolerated by wheat germ extracts, and the cotranslational membrane insertion of connexins [55] and apo cytochrome b5 [56] in wheat germ CF systems are some examples. Effects of individual lipids on MP insertion and MP quality can systematically be screened with synthetic liposomes, and some lipids such as cardiolipin or cholesterol have shown beneficial chaperone effects on specific MP targets [45,57]. Besides synthetic liposomes, also vesicles prepared out of complex lipid mixtures or isolated from natural membranes are interesting as supplements in L-CF expression reactions [12,16,21,44,47,54,58,59].…”