3.A.19.  The Guided Entry of Tail Anchored Protein (GET) Family, formerly the TMS Recognition/Insertion Complex (TRC) Family

An important class of proteins in eukaryotic cells includes tail-anchored (TA) membrane proteins, which include cytochrome b₅ (the founding member of the TA family), the SNARE proteins involved in vesicle trafficking, proteins involved in apoptosis (Bcl-2 family), and several subunits of the mitochondrial and endoplasmic reticulum (ER) protein translocation channels.  Membrane insertion of the exocytic SNARE protein, synaptobrevin, requires ATP hydrolysis and one or more protease-sensitive ER membrane proteins (Kutay et al., 1995). The yeast genome encodes 55 tail-anchored membrane proteins (Beilharz et al., 2003) that are ultimately localized to the nuclear envelope, the outer mitochondrial membrane, the peroxisome, and all membranes within the exocytic and endocytic pathways (Mandon and Gilmore, 2007).  In plants such as Arabidopsis thaliana, the GE pathway is involed in root hair growth and SNARE abundance (Xing et al. 2017). ER membrane receptors of the GET pathway seem to be conserved throughout eukaryotes (Asseck et al. 2021). TA protein insertion with a special focus on plants has been reviewed (Mehlhorn et al. 2021). Distinct features of Get3d include an incomplete active site, a 'closed' conformation in the apo-state, and a hydrophobic chamber. It has ATPase activity and is capable of binding TA proteins, supporting a potential role in TA protein targeting. Get3d is first found with the development of photosynthesis and conserved across 1.2 billion years into the chloroplasts of higher plants across the evolution of photosynthesis, suggesting a role in the homeostasis of the photosynthetic machinery (Barlow et al. 2023).

Tail-anchored (TA) proteins serve numerous essential roles in cells. TRC40/Asna-1 interacts posttranslationally with TA proteins in a TMS-dependent manner for delivery to a proteinaceous receptor. Subsequent release from TRC40/Asna-1 and insertion into the membrane depends on ATP hydrolysis. Consequently, an ATPase-deficient mutant of TRC40/Asna-1 dominantly inhibited TA protein insertion selectively without influencing other translocation pathways (Stefanovic and Hegde, 2007).  The GET pathway selects TA proteins destined for the endoplasmic reticulum (ER), utilizing distinct molecular steps, including differential binding by the co-chaperone Sgt2 and kinetic proofreading after ATP hydrolysis by the targeting factor Get3 (Rao et al. 2016). The molecular basis of tail-anchored integral membrane protein recognition by the cochaperone Sgt2 has been studied (Lin et al. 2021), showing that Sgt2 binds to the hydrophobic transmembrane domain of the TA protein.

TA proteins are post-translationally targeted to and inserted into the ER membrane through their single C-terminal transmembrane domain. Membrane insertion of TA proteins in mammalian cells is mediated by the ATPase TRC40/Asna1 (Get3 in yeast) and a receptor in the ER membrane. Vilardi et al. (2011) identified the tryptophan-rich basic protein (WRB), also known as congenital heart disease protein 5 (CHD5), as the ER membrane receptor for TRC40/Asna1. WRB shows sequence similarity to Get1, a subunit of the membrane receptor complex for yeast Get3. It is an ER-resident membrane protein that interacts with TRC40/Asna1 and recruits it to the ER membrane. A coiled-coil domain of WRB is the binding site for TRC40/Asna1. A soluble form of the coiled-coil domain interferes with TRC40/Asna1-mediated membrane insertion of TA proteins (Vilardi et al., 2011), revealing that the.

Entry of newly synthesized TA proteins into the GET pathway in Saccharomyces cerevisiae begins with efficient TMS capture by Sgt2 (a small glutamine-rich tetratricopeptide repeat-containing protein) (Denic 2012). This chaperone shields the TMS after it is released from the ribosome to prevent TA protein aggregation in the cytosol or mistargeting to mitochondria. Sgt2 is in a complex with Get4 and Get5, two pathway components that facilitate TA protein transfer from Sgt2 to Get3, a dimeric/tetrameric ATPase that is the ER membrane targeting factor of the GET pathway. This is achieved, first, when ATP stimulates binding of Get3 to Get4, and this increases the local concentration of Get3 near the TA protein because of the Get4-Get5-Sgt2 bridge. Second, Get4 increases the intrinsic rate of Get3-TA protein complex formation, most likely by making Get3 receptive for TMS binding. ATP binding converts Get3 from an open to a semi-closed state; ATP hydrolysis fully closes the Get3 conformation, creating a composite, hydrophobic groove that cradles the TMS. Tail anchors are sandwiched inside the dimeric Get3, which has a head-to-head arrangement of hydrophobic grooves (Denic 2012).

The structure of the Sgt2/Get5 complex is known (Simon et al. 2013) as is that of Get3 bound to different TA proteins.  These structures revealed the α-helical TMS occupying the hydrophobic groove that spans the Get3 homodimer (Mateja et al. 2015). The heterotetrameric Get4/Get5 complex (Get4/5), tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Crystal structures of the Get3·Get4/5 complex have also been solved (Gristick et al. 2015). The core of the GET insertase is conserved within structures of the ER membrane protein complex (EMC), which acts in parallel to insert a different subset of TA proteins. Structures of the dislocases Spf1 and Msp1 show how they remove mislocalised TA proteins from the ER and outer mitochondrial membranes, respectively (Sinning and McDowell 2022).

The metazoan protein BCL2-associated athanogene cochaperone 6 (Bag6) forms a hetero-trimeric complex with ubiquitin-like 4A and transmembrane domain recognition complex 35 (TRC35). This Bag6 complex is involved in tail-anchored protein targeting and various protein quality-control pathways in the cytosol Mock et al. 2017 presented a crystal structure of Bag6 and its cytoplasmic retention factor TRC35, revealing that TRC35 is conserved throughout the opisthokont lineage except at the C-terminal Bag6-binding groove, which evolved to accommodate Bag6, a unique metazoan factor. While TRC35 and its fungal homolog, guided entry of tail-anchored protein 4 (Get4), utilize a conserved hydrophobic patch to bind their respective partners, Bag6 wraps around TRC35 on the opposite face relative to the Get4-5 interface. Mock et al. 2017 also demonstrated that TRC35 binding is critical for occluding the Bag6 nuclear localization sequence from karyopherin alpha to retain Bag6 in the cytosol.

A large number of TA proteins engage with TRC40 when other targeting machineries are fully operational. Coy-Vergara et al. 2019 used a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduced interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. They confirmed known TRC40 substrates and identified many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, they showed that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40 (Coy-Vergara et al. 2019).

The ER delivery of endogenous mitochondrial transmembrane proteins, especially those belonging to the SLC25A mitochondrial carrier family, is dependent on the guided entry of tail-anchored proteins (GET) complex. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. Loss of the GET pathway is detrimental to yeast cells experiencing mitochondrial import failure and prevents re-import of mitochondrial proteins from the ER via the ER-SURF pathway (Xiao et al. 2021). Close coordination between chaperones is essential for protein biosynthesis, including the delivery of tail-anchored (TA) proteins containing a single C-terminal transmembrane domain to the endoplasmic reticulum (ER) by the conserved GET pathway. For successful targeting, nascent TA proteins must be promptly chaperoned and loaded onto the cytosolic ATPase Get3 through a transfer reaction involving the chaperone SGTA and bridging factors Get4, Ubl4a and Bag6. Keszei et al. 2021 reported cryo-EM structures of metazoan pretargeting GET complexes at 3.3-3.6 Å resolution. The structures revealed that Get3 helix 8 and the Get4 C terminus form a composite lid over the Get3 substrate-binding chamber that is opened by SGTA. Another interaction with Get4 prevents formation of Get3 helix 4, which links the substrate chamber and ATPase domain. Both interactions facilitate TA protein transfer from SGTA to Get3. These findings show how the pretargeting complex primes Get3 for coordinated client loading and ER targeting (Keszei et al. 2021).

Axonal proteins contain specific axon-targeting motifs that permit access to the axonal compartment as well as downstream targeting to the axonal membrane (Steele-Nicholson and Andrews 2022). These motifs target proteins to the axonal compartment by a variety of mechanisms including: promoting segregation into axon-targeted secretory vesicles, increasing interaction with axonal kinesins and enhancing somatodendritic endocytosis. Axon-targeting motifs within the context of established neuron trafficking mechanisms are discussed, and examples of how these motifs have been applied to target proteins to the axonal compartment of neurons are presented (Steele-Nicholson and Andrews 2022).

TA proteins contain a single C-terminal transmembrane domain that must be post-translationally recognized, guided to, and ultimately inserted into the correct cellular compartment. The majority of TA proteins begin their biogenesis in the ER and utilize two parallel strategies for targeting and insertion: the guided-entry of tail-anchored proteins (GET) and ER-membrane protein complex (EMC) pathways. Guna et al. 2022 described how these two sets of machinery target, transfer, and insert TAs into the lipid bilayer in close collaboration with quality control machinery. They highlighted the unifying features of the insertion process as revealed by structures of the GET and EMC membrane protein complexes.  The core of the GET insertase is conserved within structures of the ER membrane protein
complex (EMC), which acts in parallel to insert a different subset of TA proteins. Structures of the dislo-
cases, Spf1 and Msp1, show how they remove mislocalised TA proteins from the ER and outer mitochondrial membranes, respectively (Sinning and McDowell 2022).

The reaction mediated by the TRC40/GET pathway is:

Tail-anchored (TA) protein (cytosol) + ATP → TA protein (endomembrane; integrated) + ADP + Pi

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