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The ATP hydrolysis-dependent TRC receptor TRC40 (Asna-1) which functions as a chaparone protein, feeding into the WRB/CAML transporter complex (McDowell et al. 2020; 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). It is 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). Retro-2 (Morgens et al. 2019) protects cells from ricin and Shiga-like toxin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins (Morgens et al. 2019). Get3 (TRC40) binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. McDowell et al. 2020 identified a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40, important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) suggests an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove (McDowell et al. 2020).  The WRB subunit of the Get3 receptor is required for the correct integration of its partner CAML into the ER (Carvalho et al. 2019). Close coordination between chaperones is essential for the delivery of tail-anchored (TA) proteins containing a single C-terminal TMS to the ER by the 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. 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 (Keszei et al. 2021). 

The TRC complex of Homo sapiens
TRC40 (O43681)
WRB (CHO5) (O00258)
CAML (P49069)

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 is required for the post-translational delivery of tail-anchored (TA) proteins to the chloroplast. It is also 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 interacts 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). Lin et al. 2019 presented crystal structures of algal ArsA1 (a Get3 homolog) in a distinct nucleotide-free open state and bound to adenylyl-imidodiphosphate. This approximately 80-kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA-binding groove comprise the interlocking hook-like motif implicated by mutational analyses in TA substrate recognition. The data of Lin et al. 2019 provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.

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)

CHD5 homologue

GET1 homologue of Glycine max (I1L4Q8)

CHD5 homologue

GET homologue of Arabdiopsis thaliana (Q1H5D2)