(l to r) Vlad Denic, Fei Wang, Charlene Chan, and Nicholas Weir
Membrane protein insertion is a fundamental process in cell biology. Consider a membrane protein with an elaborate topology, like an ABC transporter. It is a zigzag of hydrophobic amino acid sequences embedded in the phospholipid bilayer, which deposits hydrophilic regions on alternating sides of the bilayer. How do hydrophilic regions penetrate through the hydrophobic core of the bilayer during the process of membrane protein insertion? A rudimentary knowledge of molecular cell biology has most likely already evoked in your mind the elegant solution to this problem: a translating ribosome feeding a channel its nascent polypeptide. A hazier recollection of the textbook might also bring images of the channel’s lateral gate that captures hydrophobic sequences as they are moving through the channel and lets them slip into the surrounding lipid bilayer.
Now consider the simplest membrane protein topology, that of a tail-anchored protein (TA). Most of the sequence resides in the cytosol and only a single hydrophobic sequence at the C-terminal end embeds the protein in the membrane. TA insertion occurs after protein synthesis is completed, thus excluding the possibility of ribosomes feeding them to the aforementioned protein translocation channel. Instead, we have previously shown that membrane delivery of TAs depends on the GET (Guided Entry of TAs) pathway: TAs are captured by a specialized TA chaperone (Get3) that delivers them to the insertion membrane by forming interactions with a specialized receptor (Get1/2). This led to a debate in the field about the process by which the hydrophobic sequence of TAs is inserted into the lipid bilayer: is the insertion step spontaneous or facilitated by the receptor? Since TA insertion doesn’t involve movement of hydrophilic polypeptide regions across the membrane, the prevailing view was that spontaneous interactions between the hydrophobic tail anchor and the phospholipids would enable TAs to insert themselves.
In this work, we disproved the spontaneous insertion hypothesis by discovering that the Get1/2 transmembrane domain is an insertase. In one key experiment we made proteoliposomes containing a Get1/2 insertase mutant and showed that they still recruit Get3-TA complexes to their surface. Despite the proximity to the lipids, the TAs preferred to stay bound to Get3 even though when we attached an artificial TA trap to the surface of the mutant proteoliposomes, we could drive dissociation of TAs from Get3. In a separate line of experiments, we arrested the TA insertion process by fusing an epitope tag to the C-terminus of a TA bound to Get3 and adding the epitope-binding protein. Using this tool, we observed that Get1/2 has an intra-membrane pocket for the hydrophobic tail anchor. The formation of a Get1/2-TA complex is an essential intermediate that enables TA dissociation from Get3 while helping usher the anchor into the lipid bilayer.
In summary, our work reveals that membrane protein insertion machines-be they protein translocation channels or insertases-use a universal machine language: an intra-membrane binding site for hydrophobic sequences that helps reduce the kinetic barrier that stands in the way of spontaneous membrane insertion.