3.A.21 The C-terminal Tail-Anchored Membrane Protein Biogenesis/Insertion Complex (TAMP-B) Family of the Guided Entry of Tail-anchored Protein (GET) Superfamily

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), and nuclear localization signals target tail-anchored membrane proteins to the inner nuclear envelope in plants (Groves et al. 2019).  Tail-anchored membrane protein (TA) targeting involves a network of chaperones, cochaperones, and targeting factors that together drive and regulate passage through the cytosol and insertion into the membrane (Shan 2019). Brito et al. 2019 have identified 859, 657 and 119 putative TAMPs in human (Homo sapiens), plant (Arabidopsis thaliana), and yeast (Saccharomyces cerevisiae), respectively, using the TAMPfinder program. Endoplasmic reticulum 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).

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. 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.

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). Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins (Culver and Mariappan 2021).

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). In plants, an RK/ST C-terminal motif is required for targeting of OEP7.2 and a subset of other Arabidopsis tail-anchored proteins to the plastid outer envelope membrane (Teresinski et al. 2018). Two cytochrome b5 forms, b5-ER and b5-RR in animals, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. Figueiredo Costa et al. 2018 demonstrated that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and revealed a novel, substrate-specific ER-targeting pathway. 

Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins, often characterized by a single TMS at their C-terminus. Consequently, the hydrophobic TMS, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation, precluding canonical co-translational targeting and membrane insertion. Peschke et al. 2018 identified DjlC (P77359) and Flk (P15286) as bona fide E. coli TAMPs and showed that their TMSs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, they showed that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and the insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMS of DjlC and Flk with both the Ffh subunit of SRP and YidC was confirmed.  The hydrophobicity of the TMS correlates with the dependency on SRP for efficient targeting (Peschke et al. 2018). 

Many tail-anchored (TA) membrane proteins are targeted to and inserted into the ER by the `guided entry of tail-anchored proteins' (GET) pathway. This post-translational pathway uses transmembrane-domain selective cytosolic chaperones for targeting, and a dedicated membrane protein complex for insertion. Mateja and Keenan 2018 reviewed the mechanisms underlying each step of the pathway, emphasizing available structural work.  The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER (Vitali et al. 2018). While the majority of integral membrane proteins are delivered to the ER membrane via the co-translational SRP-dependent route, tail-anchored proteins employ an alternative, post-translational route that relies on distinct factors such as a cytosolic protein quality control component, SGTA. Leznicki and High 2020 showed that SGTA is selectively recruited to ribosomes synthesising a diverse range of membrane proteins, suggesting that it can act on precursors of the co-translational ER delivery pathway. SGTA is recruited to nascent membrane proteins before their transmembrane domain emerges from the ribosome. Hence, SGTA can capture these aggregation prone regions shortly after their synthesis. SGTA thus complements SRP by reducing the co-translational ubiquitination of proteins with multiple hydrophobic signal sequences. It may act to mask specific transmembrane domains until they engage the ER translocon and become membrane inserted (Leznicki and High 2020).

The tail length of C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. Membrane proteins with C-TMS tails shorter than approximately 60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion (Sun and Mariappan 2020). These C-TMSs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMSs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD; TC# 3.A.25) pathway for removal. In contrast, C-TMSs with sufficient hydrophobicity or tails longer than approximately 80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins (Sun and Mariappan 2020). Sequence-based features determine tail-anchored membrane protein sorting in eukaryotes. The most inclusive predictor uses both hydrophobicity and C-terminal charge in tandem (Fry et al. 2021).

Tail-anchored (TA) proteins fulfill diverse cellular functions within different organellar membranes. Their characteristic C-terminal TMS renders TA proteins inherently prone to aggregation, necessitating posttranslational targeting. The guided entry of TA proteins (GET in yeast)/transmembrane recognition complex (TRC in humans) pathway represents a major route for TA proteins to the endoplasmic reticulum (ER). Farkas and Bohnsack 2021 reviewed the capture of nascent TA proteins at the ribosome by the GET pathway pretargeting complex and the mechanism of their delivery into the ER membrane by the GET receptor insertase. Several alternative routes by which TA proteins can be targeted to the ER have emerged, raising questions about how selectivity is achieved during TA protein capture. The quality control machineries in the ER and outer mitochondrial membrane for displacing mislocalized TA proteins have been reviewed (Farkas and Bohnsack 2021).

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

TAMP (cytoplasm) + ATP _{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. 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.}

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). Deubiquitinases USP20/33 promote the biogenesis of tail-anchored membrane proteins (Culver and Mariappan 2021).

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). In plants, an RK/ST C-terminal motif is required for targeting of OEP7.2 and a subset of other Arabidopsis tail-anchored proteins to the plastid outer envelope membrane (Teresinski et al. 2018). Two cytochrome b5 forms, b5-ER and b5-RR in animals, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. Figueiredo Costa et al. 2018 demonstrated that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and revealed a novel, substrate-specific ER-targeting pathway. 

Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins, often characterized by a single TMS at their C-terminus. Consequently, the hydrophobic TMS, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation, precluding canonical co-translational targeting and membrane insertion. Peschke et al. 2018 identified DjlC (P77359) and Flk (P15286) as bona fide E. coli TAMPs and showed that their TMSs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, they showed that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and the insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMS of DjlC and Flk with both the Ffh subunit of SRP and YidC was confirmed.  The hydrophobicity of the TMS correlates with the dependency on SRP for efficient targeting (Peschke et al. 2018). 

Many tail-anchored (TA) membrane proteins are targeted to and inserted into the ER by the `guided entry of tail-anchored proteins' (GET) pathway. This post-translational pathway uses transmembrane-domain selective cytosolic chaperones for targeting, and a dedicated membrane protein complex for insertion. Mateja and Keenan 2018 reviewed the mechanisms underlying each step of the pathway, emphasizing available structural work.  The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER (Vitali et al. 2018). While the majority of integral membrane proteins are delivered to the ER membrane via the co-translational SRP-dependent route, tail-anchored proteins employ an alternative, post-translational route that relies on distinct factors such as a cytosolic protein quality control component, SGTA. Leznicki and High 2020 showed that SGTA is selectively recruited to ribosomes synthesising a diverse range of membrane proteins, suggesting that it can act on precursors of the co-translational ER delivery pathway. SGTA is recruited to nascent membrane proteins before their transmembrane domain emerges from the ribosome. Hence, SGTA can capture these aggregation prone regions shortly after their synthesis. SGTA thus complements SRP by reducing the co-translational ubiquitination of proteins with multiple hydrophobic signal sequences. It may act to mask specific transmembrane domains until they engage the ER translocon and become membrane inserted (Leznicki and High 2020).

The tail length of C-terminal transmembrane domains (C-TMDs) determines efficient insertion and assembly of membrane proteins in the ER. Membrane proteins with C-TMS tails shorter than approximately 60 amino acids are poorly inserted into the ER membrane, which suggests that translation is terminated before they are recognized by the Sec61 translocon for insertion (Sun and Mariappan 2020). These C-TMSs with insufficient hydrophobicity are post-translationally recognized and retained by the Sec61 translocon complex, providing a time window for efficient assembly with TMDs from partner proteins. Retained TMSs that fail to assemble with their cognate TMDs are slowly translocated into the ER lumen and are recognized by the ER-associated degradation (ERAD; TC# 3.A.25) pathway for removal. In contrast, C-TMSs with sufficient hydrophobicity or tails longer than approximately 80 residues are quickly released from the Sec61 translocon into the membrane or the ER lumen, resulting in inefficient assembly with partner TMDs. Thus, C-terminal tails harbor crucial signals for both the insertion and assembly of membrane proteins (Sun and Mariappan 2020). Sequence-based features determine tail-anchored membrane protein sorting in eukaryotes. The most inclusive predictor uses both hydrophobicity and C-terminal charge in tandem (Fry et al. 2021).

Tail-anchored (TA) proteins fulfill diverse cellular functions within different organellar membranes. Their characteristic C-terminal TMS renders TA proteins inherently prone to aggregation, necessitating posttranslational targeting. The guided entry of TA proteins (GET in yeast)/transmembrane recognition complex (TRC in humans) pathway represents a major route for TA proteins to the endoplasmic reticulum (ER). Farkas and Bohnsack 2021 reviewed the capture of nascent TA proteins at the ribosome by the GET pathway pretargeting complex and the mechanism of their delivery into the ER membrane by the GET receptor insertase. Several alternative routes by which TA proteins can be targeted to the ER have emerged, raising questions about how selectivity is achieved during TA protein capture. The quality control machineries in the ER and outer mitochondrial membrane for displacing mislocalized TA proteins have been reviewed (Farkas and Bohnsack 2021).

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

TAMP (cytoplasm) + ATP   TAMP (membrane inserted) +ADP + P_i