Cell-Free Expression to Probe Co-Translational Insertion of an Alpha Helical Membrane Protein

Abstract

The majority of alpha helical membrane proteins fold co-translationally during their synthesis on the ribosome. In contrast, most mechanistic folding studies address refolding of full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation of membrane proteins is emerging as a useful tool to address folding during translation by a ribosome. We summarise the benefits of this approach and show how it can be successfully extended to a membrane protein with a complex topology. The bacterial leucine transporter, LeuT can be synthesised and inserted into lipid membranes using a variety of in vitro transcription translation systems. Unlike major facilitator superfamily transporters, where changes in lipids can optimise the amount of correctly inserted protein, LeuT insertion yields are much less dependent on the lipid composition. The presence of a bacterial translocon either in native membrane extracts or in reconstituted membranes also has little influence on the yield of LeuT incorporated into the lipid membrane, except at high reconstitution concentrations. LeuT is considered a paradigm for neurotransmitter transporters and possesses a knotted structure that is characteristic of this transporter family. This work provides a method in which to probe the formation of a protein as the polypeptide chain is being synthesised on a ribosome and inserting into lipids. We show that in comparison with the simpler major facilitator transporter structures, LeuT inserts less efficiently into membranes when synthesised cell-free, suggesting that more of the protein aggregates, likely as a result of the challenging formation of the knotted topology in the membrane.

Improving cell-free expression of membrane proteins by tuning ribosome co-translational membrane association and nascent chain aggregation

“…Synthetic membranes, in the form of liposomes and nanodiscs, have been used in CFE systems to improve expression of membrane proteins [17]- [20]. Experiments have established that membrane composition, available membrane area, and formation of co-translational membrane-bound ribosome complexes are crucial for successful CFE of membrane proteins [18], [21], [22]. However, each of the properties must be tuned to effectively produce properly folded, functional membrane proteins, limiting the ready adoption of membrane proteins into CFE systems [17], [22], [23].…”

Methods to study folding of alpha-helical membrane proteins in lipids

“…For example, the E. coli SecYEG translocon has been shown to require cardiolipin (CL) to function optimally in vivo [ 119 ]. It has recently been demonstrated that the insertion yield of the bacterial leucine transporter (LeuT) expressed in vitro is significantly improved when both SecYEG and CL are present, but not when either CL or the translocon are included alone [ 120 ]. This study highlights that particularly when moving towards more ‘native-like’ IVTT systems, the effects of lipid composition on chaperone function, as well as on insertion and folding, must be considered.…”

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

“…The PURE system has the advantage of being composed of well-defined components, and it has thus found wide application in bottom-up synthetic biology. ,, However, membrane proteins synthesized in vitro by transcription-translation with PURE tend to display low activities, partly due to limitations intrinsic to PURE (Box ) and partly due to the lack of control over protein insertion and folding. A number of studies have reported that polytopic membrane proteins self-insert into membrane bilayers when expressed cotranslationally, provided that specific phospholipid requirements are met. ,,, However, little or no quantitative information about the activity of the self-inserted membrane proteins is available (Box ). Thus, the synthesis of membrane proteins in vitro should be inspired by living cells, which couple the translation of membrane proteins to their insertion (i.e., transertion) into the lipid bilayer.…”

“…A number of studies have reported that polytopic membrane proteins self-insert into membrane bilayers when expressed cotranslationally, provided that specific phospholipid requirements are met. 68 , 69 , 220 , 221 However, little or no quantitative information about the activity of the self-inserted membrane proteins is available ( Box 3 ). Thus, the synthesis of membrane proteins in vitro should be inspired by living cells, which couple the translation of membrane proteins to their insertion (i.e., transertion) into the lipid bilayer.…”