How will the GET pathway grow up?

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The emergence of the GET pathway as the dominant mechanism for targeting TA proteins to the ER raises a nagging question: why is the GET pathway not essential in budding yeast (nota bene: mice lacking TRC40 die during early embryogenesis [32])? One obvious interpretation is that other targeting pathways work redundantly with the GET pathway to either help carry the load, or split the clientele. This could be mediated through tail anchor interactions with the SRP, protein folding chaperones, and/or calmodulin, which have all been observed in vitro [33,34]. It has been difficult, however, to mechanistically build alternative pathways from these observations or establish their physiological significance. A related possibility is that get mutants turn on the heat shock response, similar to the yeast srp mutants [35], to compensate by providing an inefficient TA protein delivery mechanism to Sec61. Conversely, disruption of the GET pathway might indirectly perturb other ER targeting pathways (or more generally, protein homeostasis) by engendering a competition between TA proteins and other hydrophobic substrates. Recent technological advances in ribosome profiling [36] point a way out of this morass, at least in mammalian cells. Specifically, because the Bag6 complex is selectively recruited to ribosomes that are synthesizing TA proteins, footprinting of ‘Bag6-marked’ ribosomes should reveal the natural flux of substrates through the GET pathway in unperturbed cells.

Our mechanistic understanding of the GET pathway has gotten sophisticated over the past five years. Biochemical reconstitution approaches carried out in yeast and mammalian cell-free systems have lead to a coherent view of how newly synthesized TA proteins hop from a pre-targeting complex to Get3 or TRC40, respectively. In yeast, we even know the structures of many of the pre-targeting components and have a basic understanding of how they work. More broadly, substrate handoff between two chaperones is a recurring mechanism in biology. For example, the folding of many regulatory proteins in eukaryotes depends on their transfer from Hsp70 to Hsp90 as part of a single complex organized by a scaffolding protein [37]. The two significant technical challenges associated with studying this mechanism are the biochemically complex nature of Hsp90 and the poorly characterized and multivalent hydrophobic epitopes on the substrate. In comparison, the tail anchor is a well-defined substrate that moves through a more elementary chaperone machine. Nonetheless, it is remarkable that Sgt2 and Get3 do not apparently incur the energetic cost of exposing empty substrate-binding sites to the cytosol during substrate transfer. Future structural and single-molecule studies of the role of Get4/5 during TA protein handoff should extend our understanding of chaperone trapeze acts in cell biology.

Arguably, the hardest conceptual problem in all membrane insertion pathways is visualizing TMD insertion into the lipid bilayer (Box 2). Structural studies of the Sec61 translocon have revealed an elegant solution to this: a protein translocation channel with a lateral gate [1]. Nonetheless, this insertion mechanism is difficult to probe further because both Sec61 and ribosome-associated substrates are biochemically complex and staging the insertion reaction for detailed kinetic analysis is challenging. By contrast, the insertion step of the GET pathway is specialized for insertion of a single hydrophobic epitope without coupling to biosynthesis and extensive protein translocation. Consequently, it has relatively few moving parts and they can all be made recombinantly. Staging will still be a challenge but the accessibility of the TMD C terminus offers hope that conditional roadblocks can be attached to it. As such, structural and functional studies of the Get1/2 complex are promising to move us one step closer towards a fundamental understanding of how cells exploit chaperones to put transmembrane domains into lipid bilayers.