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3.A.21 The C-terminal Tail-Anchored Membrane Protein Biogenesis/ Insertion Complex (TAMP-B) Family

Sophisticated mechanisms have evolved to target eukaryotic membrane proteins to the correct membrane-surrounded organelle and to protect them from aggregation. Of central importance are cytosolic factors that decode the targeting information of a signal sequence by transient association and escort membrane proteins to their designated location (Schuldiner et al., 2008). Tail-anchored membrane proteins (TA proteins) consist of an N-terminal soluble domain and a single C-terminal transmembrane segment (TMS) (Borgese et al., 2007). The C-terminal localization of their targeting signal requires release of the synthesized protein from the ribosome before interaction with an insertion machinery (Cross et al., 2009). This precludes TA proteins from cotranslational insertion mediated by the signal recognition particle (SRP)-Sec61 translocon system (TC# 3.A.5), which mainly targets membrane proteins with an N-terminal signal sequence.  The tail-anchored assemblly has been studied in unicellular eukaryotes such as Toxoplasma gondii (Padgett et al. 2016).

Although some studies suggest an unassisted insertion of TA proteins into the mitochondrial outer membrane (Meineke et al., 2008), most endoplasmic reticulum (ER)-destined TA proteins are thought to insert by an energy-dependent process, which involves several cytosolic factors. In yeast, it has been shown that the adenosine triphosphatase (ATPase) Get3 (guided-entry of TA proteins-3 pathway) is necessary for the biogenesis of TA proteins (Schuldiner et al., 2008). Get3 is a dimeric protein with each subunit comprising a nucleotide-binding domain (NBD) and a methionine-rich α-helical domain that has been implicated in TA protein binding (TA binding domain, TABD). Mateja et al. 2015 reconstituted the assembly pathway for a functional targeting complex and showed that it comprises a TA protein bound to a Get3 homodimer. Crystal structures of Get3 bound to different TA proteins showed an α-helical TMS occupying a hydrophobic groove that spans the Get3 homodimer.

Crystal structures of Get3 have shown that the protein switches between open and closed states, depending on its nucleotide load. Whereas in the apo and magnesium-free adenosine diphosphate (ADP) forms, the open state is favored (Bozkurt et al., 2009), ADP-Mg2+ and the nonhydrolyzable ATP analog 5′-adenylyl-β,γ-imidodiphosphate-Mg2+ (AMPPNP-Mg2+) induce the closed state, which is further tightened up in the transition state of adenosine triphosphate (ATP) hydrolysis (Mateja et al., 2009). The hydrophobic groove responsible for TA binding seems fully assembled only in the transition state. At the membrane, Get3 interacts with the two receptor proteins, Get1 and Get2, which are essential for TA protein insertion (Schuldiner et al., 2008). 

Tail-anchored (TA) proteins in yeast contain a C-terminal membrane anchor and are posttranslationally delivered to the endoplasmic reticulum (ER) membrane by the Get3 adenosine triphosphatase (an ArsA homologue) interacting with the hetero-oligomeric 3 TMS Get1/2 membrane receptor. Stefer et al. (2011) have determined crystal structures of Get3 in complex with the cytosolic domains of Get1 and Get2 in different functional states. The heterotetrameric Get1/Get2 complex stoichiometry is (Get1)2(Get2)2. The structural data, together with biochemical experiments, show that Get1 and Get2 use adjacent, partially overlapping binding sites and that both can bind simultaneously to Get3. Docking to the Get1/2 complex allows for conformational changes in Get3 that are required for TA protein insertion. A molecular mechanism for nucleotide-regulated delivery of TA proteins was proposed (Stefer et al., 2011).

GET3 cooperates with the HDEL receptor ERD2 to mediate ATP-dependent retrieval of ER proteins that contain a C-terminal HDEL sequence (Schuldiner et al., 2005). This sequence is the retention signal from the Golgi to the ER. It may also be involved in low-level resistance to oxyanions such as arsenite, and in heat tolerance (Shen et al., 2003). It interacts with the Gef1 Cl- transporter in a copper-dependent fashion (Metz et al., 2006). Metz et al. claimed that the arsenite binding site of the E. coli ArsA is not conserved in Arr4p. The homodimer binds to the C-terminus of Gef1p. Both Gef1p and GET3 are required for normal growth under iron-limiting conditions. gef1 mutants lose high-affinity iron uptake because the Fet3p multi-copper oxidase involved in iron uptake (TC #1.A.11.1.1) does not mature normally in a gef1 mutant. This is because copper loading of Fet3p in the lumen of the late secretory pathway requires Cl- which enters the compartment via Gef1p. Therefore, copper and iron are limiting for growth at alkaline pH. Arr4p antagonizes the function of Gef1p (Metz et al., 2006). Thus, Arr4p is a negative regulator of Gef1p which binds directly to the C-terminus of the latter. GET3 (Arr4) thus inhibits Cl- transport via Gef1p (TC #1.A.11.1.1).

The GET complex is composed of the homodimeric Get3 ATPase and its heterooligomeric receptor, Get1/2. During insertion, the Get3 dimer shuttles between open and closed conformational states, coupled with ATP hydrolysis and the binding/release of TA proteins. Kubota et al. (2012) reported crystal structures of ADP-bound Get3 in complex with the cytoplasmic domain of Get1 (Get1CD) in open and semi-open conformations at 3.0- and 4.5-Å resolutions, respectively. Their structures and biochemical data suggest that Get1 uses two interfaces to stabilize the open dimer conformation of Get3. They propose that one interface is sufficient for binding of Get1 by Get3, while the second interface stabilizes the open dimer conformation of Get3.  Evidence supports the conclusion that Get1/2 functions as the insertase directly (Kubota et al. 2012; Wang et al. 2014).

The generalized reaction for the C-terminal tail-anchored membrane insertion complex is:

TA protein (cytoplasm) + ATP Wang et al. 2014).

The generalized reaction for the C-terminal tail-anchored membrane insertion complex is:

TA protein (cytoplasm) + ATP  TA protein (membrane inserted) +ADP + Pi

References associated with 3.A.21 family:

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Bozkurt, G., G. Stjepanovic, F. Vilardi, S. Amlacher, K. Wild, G. Bange, V. Favaloro, K. Rippe, E. Hurt, B. Dobberstein, and I. Sinning. (2009). Structural insights into tail-anchored protein binding and membrane insertion by Get3. Proc. Natl. Acad. Sci. USA 106: 21131-21136. 19948960
Castillo, R. and M.H. Saier. (2010). Functional Promiscuity of Homologues of the Bacterial ArsA ATPases. Int J Microbiol 2010: 187373. 20981284
Cross, B.C., I. Sinning, J. Luirink, and S. High. (2009). Delivering proteins for export from the cytosol. Nat Rev Mol. Cell Biol. 10: 255-264. 19305415
Kubota, K., A. Yamagata, Y. Sato, S. Goto-Ito, and S. Fukai. (2012). Get1 stabilizes an open dimer conformation of get3 ATPase by binding two distinct interfaces. J. Mol. Biol. 422: 366-375. 22684149
Mateja, A., A. Szlachcic, M.E. Downing, M. Dobosz, M. Mariappan, R.S. Hegde, and R.J. Keenan. (2009). The structural basis of tail-anchored membrane protein recognition by Get3. Nature 461: 361-366. 19675567
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
Meineke, B., G. Engl, C. Kemper, A. Vasiljev-Neumeyer, H. Paulitschke, and D. Rapaport. (2008). The outer membrane form of the mitochondrial protein Mcr1 follows a TOM-independent membrane insertion pathway. FEBS Lett. 582: 855-860. 18279676
Metz, J., A. Wächter, B. Schmidt, J.M. Bujnicki, and B. Schwappach. (2006). The yeast Arr4p ATPase binds the chloride transporter Gef1p when copper is available in the cytosol. J. Biol. Chem. 281: 410-417. 16260785
Padgett, L.R., G. Arrizabalaga, and W.J. Sullivan, Jr. (2016). Targeting of tail-anchored membrane proteins to subcellular organelles in Toxoplasma gondii. Traffic. [Epub: Ahead of Print] 27991712
Schuldiner, M., J. Metz, V. Schmid, V. Denic, M. Rakwalska, H.D. Schmitt, B. Schwappach, and J.S. Weissman. (2008). The GET complex mediates insertion of tail-anchored proteins into the ER membrane. Cell 134: 634-645. 18724936
Schuldiner, M., S.R. Collins, N.J. Thompson, V. Denic, A. Bhamidipati, T. Punna, J. Ihmels, B. Andrews, C. Boone, J.F. Greenblatt, J.S. Weissman, and N.J. Krogan. (2005). Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123: 507-519. 16269340
Shen, J., C.M. Hsu, B.K. Kang, B.P. Rosen, and H. Bhattacharjee. (2003). The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance. Biometals 16: 369-378. 12680698
Stefer, S., S. Reitz, F. Wang, K. Wild, Y.Y. Pang, D. Schwarz, J. Bomke, C. Hein, F. Löhr, F. Bernhard, V. Denic, V. Dötsch, and I. Sinning. (2011). Structural basis for tail-anchored membrane protein biogenesis by the Get3-receptor complex. Science 333: 758-762. 21719644
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
Wang, F., C. Chan, N.R. Weir, and V. Denic. (2014). The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase. Nature 512: 441-444. 25043001