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).
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).
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).
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 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.
The reaction mediated by the TRC40/GET pathway is:
Tail-anchored (TA) protein (cytosol) + ATP → TA protein (endomembrane; integrated) + ADP + Pi
The ATP hydrolysis-dependent TRC receptor TRC40 (Asna-1) (Stefanovic and Hegde, 2007). (TRC40 is homologous to the ArsA ATPase of E. coli (TC# 3.A.4.1.1) and the GET3 ATPase of yeast (TC# 8.A.26.1.1)) Loss yields embryonic lethality. Tryptophan-rich basic protein (WRB) is the tail-anchored (TA) protein insertion receptor. Also called congenital heart disease protein-5 (CHD5). Related to the yeast Get1 protein in 3.A.21.1.1. Calcium-modulating cyclophilin ligand (CAML) is a mammal-specific receptor for TRC40, an ATPase targeting newly synthesized TA proteins. CAML mediates membrane insertion of TA proteins. TRC40 (Asna1) has been shown to mediate membrane insertion of two proteins, RAMP4 and Sec61beta, without the participation of other cytosolic proteins by a mechanism that depends on the presence of ATP or ADP and a protease-sensitive receptor in the ER membrane (Favaloro et al. 2010). TRC40 is required for release of Herpes simplex virus 1 (HSV1) virions (Ott et al. 2016). The functions and reciprocal interactions of the two subunits of the heteromeric TRC40 recpeptor, WBR and CAML (CAMLG), have revealed mutual dependencies for stability; CAML seems to normally be present in 5-fold excess over WBR (Colombo et al. 2016). CAMLG interacts with Classical Swine Fever Virus (CSFV) p7 and mediates calcium permeability in the ER (Gladue et al. 2018).
The TRC complex of Homo sapiens
WRB (CHO5) (O00258)
GET1 protein homologue
GET1 of Aspergillus niger (A2QHQ3)
Get3 ATPase homologue of 349 aas and 0 TMSs that drives the insertion of tail anchored (TA) proteins into the endoplasmic reticulum membrane. The 3-d structure of the tetramer has been solved (Suloway et al. 2012). The tetramer generates a hydrophobic chamber that probably binds the single C-terminal TMS of the TA protein.
Get3 ATPase of Methanocaldococcus jannaschii (Methanococcus jannaschii)
ArsA1 of 777 aas. ATPase required for the post-translational delivery of tail-anchored (TA) proteins to the chloroplast. It is required for the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon (TOC) complex (Formighieri et al. 2013, Maestre-Reyna et al. 2017). ArsA1 recognizes and selectively binds the transmembrane domain of TA proteins in the cytosol. This complex then targets the protein to the chloroplast, where the tail-anchored protein is released for insertion. This process is regulated by ATP binding and hydrolysis (Maestre-Reyna et al. 2017). Both ArsA proteins exhibit oxyanion-independent ATPase activity, but co-expression of ArsA proteins with TA-transmembrane regions showed not only that the former interact with the latter, but that ArsA1 does not share the same ligand specificity as ArsA2. ArsA1 mainly carries TA-proteins to the chloroplast, while ArsA2 carries them to the endoplasmic reticulum (Maestre-Reyna et al. 2017).
ArsA1 of Chlamydomonas reinhardtii (Chlamydomonas smithii)
ArsA2 of 362 aas (see description for ArsA1 (TC# 3.A.19.1.4) which has a similar function, but instead of targetting the chloroplast, ArsA2 targets the ER. It recognizes and selectively binds the transmembrane domain of TA proteins in the cytosol. This complex then targets to the endoplasmic reticulum by membrane-bound receptors, where the tail-anchored protein is released for insertion. This process is regulated by ATP binding and hydrolysis (Maestre-Reyna et al. 2017). ATP binding drives the homodimer towards the closed dimer state, facilitating recognition of newly synthesized TA membrane proteins. ATP hydrolysis is required for insertion. Subsequently, the homodimer reverts towards the open dimer state, lowering its affinity for the membrane-bound receptor, and returning it to the cytosol to initiate a new round of targeting.
ArsA2 of Chlamydomonas reinhardtii (Chlamydomonas smithii)
GET1 homologue of Glycine max (I1L4Q8)
GET homologue of Arabdiopsis thaliana (Q1H5D2)