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.
Highlights
Membrane proteins constitute roughly one third of all gene products in any given organism, and over half of all current pharmaceutical targets [1,2]
We show that ADP/ATP Carrier (AAC) translated in a wheat germ lysate-based system integrates into small unilamellar vesicles (SUVs) in the absence of translocons and that it is functional as an adenine nucleotide transporter
We adopted the recently-described procedure for spontaneous insertion of in vitro translated membrane proteins into liposomes [7,8,9,10,11,29] to test whether AAC would properly integrate into SUVs included in our wheat germ lysate system
Summary
Membrane proteins constitute roughly one third of all gene products in any given organism, and over half of all current pharmaceutical targets [1,2]. Cell-free protein synthesis is conducted in the presence of detergents to maintain the solubility of the translation product before reconstitution into liposomes [5,6]. Examples include proteins translated in cell-free systems based on E.coli lysates (i.e. bacteriorhodopsin [7], connexin-43 [8] and the Fo/F1 ATP synthase [10]) as well as wheat germ lysates (i.e. stearoyl-CoA desaturase [9] and sphingolipid synthase [11]). By this experimental approach, some polypeptides require a specific lipid composition for unassisted integration. In this study we have investigated the cell-free spontaneous integration of the mitochondrial ADP/ATP Carrier (AAC)
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