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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).

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 which 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 reaction mediated by the TRC40/GET pathway is:

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

This family belongs to the: ArsA ATPase (ArsA) Superfamily.

References associated with 3.A.19 family:

Beilharz, T., B. Egan, P.A. Silver, K. Hofmann, and T. Lithgow. (2003). Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in Saccharomyces cerevisiae. J. Biol. Chem. 278: 8219-8223. 12514182
Colombo, S.F., S. Cardani, A. Maroli, A. Vitiello, P. Soffientini, A. Crespi, R.F. Bram, R. Benfante, and N. Borgese. (2016). Tail-anchored Protein Insertion in Mammals: FUNCTION AND RECIPROCAL INTERACTIONS OF THE TWO SUBUNITS OF THE TRC40 RECEPTOR. J. Biol. Chem. 291: 15292-15306. 27226539
Denic, V. (2012). A portrait of the GET pathway as a surprisingly complicated young man. Trends. Biochem. Sci. 37: 411-417. 22951232
Favaloro, V., F. Vilardi, R. Schlecht, M.P. Mayer, and B. Dobberstein. (2010). Asna1/TRC40-mediated membrane insertion of tail-anchored proteins. J Cell Sci 123: 1522-1530. 20375064
Gristick, H.B., M.E. Rome, J.W. Chartron, M. Rao, S. Hess, S.O. Shan, and W.M. Clemons, Jr. (2015). Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting. J. Biol. Chem. 290: 30006-30017. 26451041
Kutay, U., G. Ahnert-Hilger, E. Hartmann, B. Wiedenmann, and T.A. Rapoport. (1995). Transport route for synaptobrevin via a novel pathway of insertion into the endoplasmic reticulum membrane. EMBO. J. 14: 217-223. 7835332
Mandon, E.C., and R. Gilmore. (2007). The tail end of membrane insertion. Cell. 128: 1031-1032. 17382875
Mateja, A., M. Paduch, H.Y. Chang, A. Szydlowska, A.A. Kossiakoff, R.S. Hegde, and R.J. Keenan. (2015). Protein targeting. Structure of the Get3 targeting factor in complex with its membrane protein cargo. Science 347: 1152-1155. 25745174
Ott, M., D. Marques, C. Funk, and S.M. Bailer. (2016). Asna1/TRC40 that mediates membrane insertion of tail-anchored proteins is required for efficient release of Herpes simplex virus 1 virions. Virol J 13: 175. 27765046
Simon, A.C., P.J. Simpson, R.M. Goldstone, E.M. Krysztofinska, J.W. Murray, S. High, and R.L. Isaacson. (2013). Structure of the Sgt2/Get5 complex provides insights into GET-mediated targeting of tail-anchored membrane proteins. Proc. Natl. Acad. Sci. USA 110: 1327-1332. 23297211
Stefanovic, S., and R.S. Hegde. (2007). Identification of a targeting factor for posttranslational membrane protein insertion into the ER. Cell. 128: 1147-1159. 17382883
Suloway, C.J., M.E. Rome, and W.M. Clemons, Jr. (2012). Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue. EMBO. J. 31: 707-719. 22124326
Vilardi, F., H. Lorenz, and B. Dobberstein. (2011). WRB is the receptor for TRC40/Asna1-mediated insertion of tail-anchored proteins into the ER membrane. J Cell Sci 124: 1301-1307. 21444755
Xing, S., D.G. Mehlhorn, N. Wallmeroth, L.Y. Asseck, R. Kar, A. Voss, P. Denninger, V.A. Schmidt, M. Schwarzländer, Y.D. Stierhof, G. Grossmann, and C. Grefen. (2017). Loss of GET pathway orthologs in Arabidopsis thaliana causes root hair growth defects and affects SNARE abundance. Proc. Natl. Acad. Sci. USA 114: E1544-E1553. 28096354