Tail-anchored Research Articles

Regulation of the Get3 ATPase Cycle

The cytosolic ATPase Get3/TRC40 mediates targeting of tail‐anchored (TA) membrane proteins to the endoplasmic reticulum (ER). Get3 functions as a molecular chaperone by binding to transmembrane domains of newly synthesized TA proteins and delivering them to a membrane docking complex in the ER. Previous work have shown that ATP binding and hydrolysis can regulate Get3's function and are essential for the targeting of TA proteins. However, the precise mechanisms by which this is accomplished remain unclear. Using a combination of mechanistic enzymology and biophysical methods, we delineate Get3's ATPase cycle and show that it is required at multiple stages during TA protein targeting. Our data complements previous structural studies and contributes to our understanding of how Get3 harnesses the energy of ATP hydrolysis to spatially and temporally coordinate the delivery of TA proteins to the ER membrane.

The complex process of GETting tail-anchored membrane proteins to the ER

Biosynthesis of membrane proteins requires that hydrophobic transmembrane (TM) regions be shielded from the cytoplasm while being directed to the correct membrane. Tail-anchored (TA) membrane proteins, characterized by a single C-terminal TM, pose an additional level of complexity because they must be post-translationally targeted. In eukaryotes, the GET pathway shuttles TA-proteins to the endoplasmic reticulum. The key proteins required in yeast (Sgt2 and Get1-5) have been under extensive structural and biochemical investigation during recent years. The central protein Get3 utilizes nucleotide linked conformational changes to facilitate substrate loading and targeting. Here we analyze this complex process from a structural perspective, as understood in yeast, and further postulate on similar pathways in other domains of life.

The Nucleotide Dependent Mechanism of Get3 as Revealed by Molecular Dynamics Simulations

Tail-anchored (TA) proteins, which comprise approximately 5% of all transmembrane proteins, contain a single transmembrane domain (TMD) located near the C-terminus. This unique organization presents a topological problem that prevents the classical cotranslational translocation by the signal recognition particle, thus a distinct posttranslational targeting pathway is required for membrane insertion. In yeast, this is guided entry of TA-proteins (GET) pathway. At the center of this process is Get3, an enzyme that shuttles a TA protein from the cytosolic Get4/Get5/Sgt2 complex to the transmembrane Get1/Get2 site. Biochemical and structural studies have revealed that Get3 is a homodimer that leverages the power gained through ATP binding and hydrolysis to undergo large-scale conformational changes between an “open” and “closed” conformation. Through the use of conventional MD simulations, enhanced sampling, and rigorous free energy calculations in five possible symmetric and non-symmetric nucleotide states, we have mapped out the underlying potential of mean force for the protein as it progresses through the hydrolysis cycle. Results have lead to a mechanistic model that agrees well with experiments on the stabilities of the open and closed conformations in the symmetric states, and were able to not only predict the existence of the very recently discovered semi-open state, but also to closely match its structure. In addition, calculations predict the existence of a wide-open conformation, along with the probabilities of each of these states as nucleotides bind and unbind from Get3. This model improves our interpretation of experiments on Get3, suggests important details about the GET cycle, and provides a model system for understanding the coupling of nucleotide binding and hydrolysis to protein conformational changes.

Interaction of modified tail-anchored proteins with liposomes: Effect of extensions of hydrophilic segment at the COOH-terminus of holo-cytochromes b5

A group of membrane proteins having a single COOH-terminal hydrophobic domain capable of post-translational insertion into lipid bilayer is known as tail-anchored (TA) proteins. To clarify the insertion mechanism of the TA-domain of human cytochrome b(5) (Hcytb5) into ER membranes, we produced and purified various membrane-bound forms of Hcytb5 with their heme b-bound, in which various truncated forms of NH(2)-terminal bovine opsin sequence were appended at the COOH-terminus of the native form. We analyzed the integration of the TA-domains of these forms onto protein-free liposomes. The integration occurred efficiently even in the presence of a small amount of sodium cholate and, once incorporated, such proteoliposomes were very stable. The mode of the integration was further analyzed by treatment of the proteoliposomes with trypsin either on the extravesicular side or on the luminal side. LC-MS analyses of the trypsin digests obtained from the proteoliposomes indicated that most of the C-terminal hydrophilic segment of the native Hcytb5 were exposed towards the lumen of the vesicles and, further, a significant part of the population of the extended C-terminal hydrophilic segments of the modified Hcytb5 were exposed in the lumen as well, suggesting efficient translocation ability of the TA-domain without any assistance from other protein factors. Present results opened a route for the use of the C-terminal TA-domain as a convenient tool for the transport of proteins as well as short peptides into artificial liposomes.

Tail-anchor targeting by a Get3 tetramer: the structure of an archaeal homologue.

Efficient delivery of membrane proteins is a critical cellular process. The recently elucidated GET (Guided Entry of TA proteins) pathway is responsible for the targeted delivery of tail-anchored (TA) membrane proteins to the endoplasmic reticulum. The central player is the ATPase Get3, which in its free form exists as a dimer. Biochemical evidence suggests a role for a tetramer of Get3. Here, we present the first crystal structure of an archaeal Get3 homologue that exists as a tetramer and is capable of TA protein binding. The tetramer generates a hydrophobic chamber that we propose binds the TA protein. We use small-angle X-ray scattering to provide the first structural information of a fungal Get3/TA protein complex showing that the overall molecular envelope is consistent with the archaeal tetramer structure. Moreover, we show that this fungal tetramer complex is capable of TA insertion. This allows us to suggest a model where a tetramer of Get3 sequesters a TA protein during targeting to the membrane.

Ca2+-calmodulin inhibits tail-anchored protein insertion into the mammalian endoplasmic reticulum membrane

Cytosolic components and pathways have been identified that are involved in inserting tail-anchored (TA) membrane proteins into the yeast or mammalian endoplasmic reticulum (ER) membrane. Searching for regulatory mechanisms of TA protein biogenesis, we found that Ca2+-calmodulin (CaM) inhibits the insertion of TA proteins into mammalian ER membranes and that this inhibition is prevented by trifluoperazine, a CaM antagonist that interferes with substrate binding of Ca2+-CaM. The effects of Ca2+-CaM on cytochrome b5 and Synaptobrevin 2 suggest a direct interaction between Ca2+-CaM and TA proteins. Thus, CaM appears to regulate TA insertion into the ER membrane in a Ca2+ dependent manner.

It Takes Two to Get3

Tail-anchored (TA) membrane proteins perform essential cellular functions. They are posttranslationally inserted into the endoplasmic reticulum (ER) membrane by interaction of the Get3 chaperone with the Get1/2 receptor. Two independent structural and functional analyses of the Get3/receptor complex by Stefer etal. and Mariappan etal. now provide insights into TA protein insertion.

The mechanism of membrane-associated steps in tail-anchored protein insertion.

Tail-anchored (TA) membrane proteins destined for the endoplasmic reticulum are chaperoned by cytosolic targeting factors that deliver them to a membrane receptor for insertion. Although a basic framework for TA protein recognition is now emerging, the decisive targeting and membrane insertion steps are not understood. Here we reconstitute the TA protein insertion cycle with purified components, present crystal structures of key complexes between these components and perform mutational analyses based on the structures. We show that a committed targeting complex, formed by a TA protein bound to the chaperone ATPase Get3, is initially recruited to the membrane through an interaction with Get2. Once the targeting complex has been recruited, Get1 interacts with Get3 to drive TA protein release in an ATPase-dependent reaction. After releasing its TA protein cargo, the now-vacant Get3 recycles back to the cytosol concomitant with ATP binding. This work provides a detailed structural and mechanistic framework for the minimal TA protein insertion cycle.

The Mechanism of Tail-Anchored Protein Insertion into the ER Membrane

Tail-anchored (TA) proteins access the secretory pathway via posttranslational insertion of their C-terminal transmembrane domain into the endoplasmic reticulum (ER). Get3 is an ATPase that delivers TA proteins to the ER by interacting with the Get1-Get2 transmembrane complex, but how Get3's nucleotide cycle drives TA protein insertion remains unclear. Here, we establish that nucleotide binding to Get3 promotes Get3-TA protein complex formation by recruiting Get3 to a chaperone that hands over TA proteins to Get3. Biochemical reconstitution and mutagenesis reveal that the Get1-Get2 complex comprises the minimal TA protein insertion machinery with functionally critical cytosolic regions. By engineering a soluble heterodimer of Get1-Get2 cytosolic domains, we uncover the mechanism of TA protein release from Get3: Get2 tethers Get3-TA protein complexes into proximity with the ATPase-dependent, substrate-releasing activity of Get1. Lastly, we show that ATP enhances Get3 dissociation from the membrane, thus freeing Get1-Get2 for new rounds of substrate insertion.

A conserved archaeal pathway for tail-anchored membrane protein insertion.

Eukaryotic tail-anchored (TA) membrane proteins are inserted into the endoplasmic reticulum by a post-translational TRC40 pathway, but no comparable pathway is known in other domains of life. The crystal structure of an archaebacterial TRC40 sequence homolog bound to ADP•AlF(4) (-) reveals characteristic features of eukaryotic TRC40, including a zinc-mediated dimer and a large hydrophobic groove. Moreover, archaeal TRC40 interacts with the transmembrane domain of TA substrates and directs their membrane insertion. Thus, the TRC40 pathway is more broadly conserved than previously recognized.

Cooperative and independent activities of Sgt2 and Get5 in the targeting of tail-anchored proteins

Abstract TPR proteins modulate the activity of molecular chaperones. Here, we describe the S. cerevisiae TPR protein Sgt2 as interaction partner of Ssa1 and Hsp104 and as a component of the GET pathway by interacting with Get5. The GET pathway mediates the sorting of tail-anchored (TA) proteins, harboring a C-terminal trans-membrane segment, to the ER membrane. S. cerevisiae sgt2Δ cells show partial defects in TA protein sorting. Sgt2 activity in vivo relies on its N- and C-terminal domains, whereas the central TPR domain and thus chaperone interactions are dispensable. We show that TA protein sorting defects are more severe in sgt2Δ get5Δ mutants compared to single knockouts. Furthermore, overproduction of Sgt2 becomes toxic to get3Δ but not to get5Δ cells. Together, these findings indicate an additional, Get5-independent role of Sgt2 in TA protein sorting, pointing to parallel pathways of substrate delivery to Get3.

A biochemical analysis of the constraints of tail-anchored protein biogenesis

TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61β and Cytb5 (cytochrome b5) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61β, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein α) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61β polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process.

WRB is the receptor for TRC40/Asna1-mediated insertion of tail-anchored proteins into the ER membrane

Tail-anchored (TA) proteins are post-translationally targeted to and inserted into the endoplasmic reticulum (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. We have identified 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. Using biochemical and cell imaging approaches, we demonstrate that WRB is an ER-resident membrane protein that interacts with TRC40/Asna1 and recruits it to the ER membrane. We identify the coiled-coil domain of WRB as the binding site for TRC40/Asna1 and show that a soluble form of the coiled-coil domain interferes with TRC40/Asna1-mediated membrane insertion of TA proteins. The identification of WRB as a component of the TRC (Get) pathway for membrane insertion of TA proteins raises new questions concerning the proposed roles of WRB (CHD5) in congenital heart disease, and heart and eye development.

EBV Protein BNLF2a Exploits Host Tail-Anchored Protein Integration Machinery To Inhibit TAP

EBV, the prototypic human γ(1)-herpesvirus, persists for life in infected individuals, despite the presence of vigorous antiviral immunity. CTLs play an important role in the protection against viral infections, which they detect through recognition of virus-encoded peptides presented in the context of HLA class I molecules at the cell surface. The viral peptides are generated in the cytosol and are transported into the endoplasmic reticulum (ER) by TAP. The EBV-encoded lytic-phase protein BNLF2a acts as a powerful inhibitor of TAP. Consequently, loading of antigenic peptides onto HLA class I molecules is hampered, and recognition of BNLF2a-expressing cells by cytotoxic T cells is avoided. In this study, we characterize BNLF2a as a tail-anchored (TA) protein and elucidate its mode of action. Its hydrophilic N-terminal domain is located in the cytosol, whereas its hydrophobic C-terminal domain is inserted into membranes posttranslationally. TAP has no role in membrane insertion of BNLF2a. Instead, Asna1 (also named TRC40), a cellular protein involved in posttranslational membrane insertion of TA proteins, is responsible for integration of BNLF2a into the ER membrane. Asna1 is thereby required for efficient BNLF2a-mediated HLA class I downregulation. To optimally accomplish immune evasion, BNLF2a is composed of two specialized domains: its C-terminal tail anchor ensures membrane integration and ER retention, whereas its cytosolic N terminus accomplishes inhibition of TAP function. These results illustrate how EBV exploits a cellular pathway for TA protein biogenesis to achieve immune evasion, and they highlight the exquisite adaptation of this virus to its host.

Tail-anchored membrane proteins: exploring the complex diversity of tail-anchored-protein targeting in plant cells

Tail-anchored (TA) proteins are special class of integral membrane proteins that in recent years have received a considerable amount of attention due to their diverse cellular functions and unique targeting and insertion mechanisms. Defined by the presence of a single, hydrophobic membrane-spanning domain at or near their C terminus, TA proteins must be inserted into membranes post-translationally and are orientated such that their larger N-terminal domain (most often the functional domain) faces the cytosol, while their shorter C-terminal domain faces the interior of the organelle. The C-terminal domain of TA proteins also usually contains the information responsible for their selective targeting to the proper subcellular membrane, a process that, based primarily on studies with yeasts and mammals, appears to be highly complex due to the presence of multiple pathways. Within this context, we discuss here the biogenesis of plant TA proteins and the potential for hundreds of new TA proteins identified via bioinformatics screens to contribute to the already remarkable number of roles that this class of membrane proteins participates in throughout plant growth and development.

A Chaperone Cascade Sorts Proteins for Posttranslational Membrane Insertion into the Endoplasmic Reticulum

Tail-anchored (TA) proteins are posttranslationally inserted into either the endoplasmic reticulum (ER)or the mitochondrial outer membrane. The C-terminal transmembrane domains (TMDs) of TA proteins enable their many essential cellular functions by specifying the membrane target, but how cells process these targeting signals is poorly understood. Here, we reveal the composition ofa conserved multiprotein TMD recognition complex (TRC) and show that distinct TRC subunits recognize the two types of TMD signals. By engineering mutations in a mitochondrial TMD, we switch over its TRC subunit recognition, thus leading to its misinsertion into the ER. Biochemical reconstitution with purified components demonstrates that TRC tethers and enzymatically activates Get3 to selectively hand off ER-bound TA proteins to Get3. Thus, ER-bound TA proteins are sorted at the top of a TMD chaperone cascade that culminates with the formation of Get3-TA protein complexes, which are recruited to the ER membrane for insertion.

Mitochondrial Redox Metabolism in Trypanosomatids Is Independent of Tryparedoxin Activity

Tryparedoxins (TXNs) are oxidoreductases unique to trypanosomatids (including Leishmania and Trypanosoma parasites) that transfer reducing equivalents from trypanothione, the major thiol in these organisms, to sulfur-dependent peroxidases and other dithiol proteins. The existence of a TXN within the mitochondrion of trypanosomatids, capable of driving crucial redox pathways, is considered a requisite for normal parasite metabolism. Here this concept is shown not to apply to Leishmania. First, removal of the Leishmania infantum mitochondrial TXN (LiTXN2) by gene-targeting, had no significant effect on parasite survival, even in the context of an animal infection. Second, evidence is presented that no other TXN is capable of replacing LiTXN2. In fact, although a candidate substitute for LiTXN2 (LiTXN3) was found in the genome of L. infantum, this was shown in biochemical assays to be poorly reduced by trypanothione and to be unable to reduce sulfur-containing peroxidases. Definitive conclusion that LiTXN3 cannot directly reduce proteins located within inner mitochondrial compartments was provided by analysis of its subcellular localization and membrane topology, which revealed that LiTXN3 is a tail-anchored (TA) mitochondrial outer membrane protein presenting, as characteristic of TA proteins, its N-terminal end (containing the redox-active domain) exposed to the cytosol. This manuscript further proposes the separation of trypanosomatid TXN sequences into two classes and this is supported by phylogenetic analysis: i) class I, encoding active TXNs, and ii) class II, coding for TA proteins unlikely to function as TXNs. Trypanosoma possess only two TXNs, one belonging to class I (which is cytosolic) and the other to class II. Thus, as demonstrated for Leishmania, the mitochondrial redox metabolism in Trypanosoma may also be independent of TXN activity. The major implication of these findings is that mitochondrial functions previously thought to depend on the provision of electrons by a TXN enzyme must proceed differently.

Structures of Get3, Get4, and Get5 Provide New Models for TA Membrane Protein Targeting

The GET pathway, using several proteins (Gets 1-5 and probably Sgt2), posttranslationally conducts tail-anchored (TA) proteins to the endoplasmic reticulum (ER). At the ER, TA proteins are inserted into the lipid bilayer and then sorted and directed to their respective destinations in the secretory pathway. Until last year, there was no structural information on any of the GET components but now there are ten crystal structures of Get3 in a variety of nucleotide-bound states and conformations. The structures of Get4 and a portion of Get5 also emerged in 2010. This minireview provides a detailed comparison of the GET structures and discusses their mechanistic relevance to TA protein insertion. It also addresses the outstanding gaps in detailed molecular information on this system, including the structures of Get5, Sgt2, and the transmembrane complex comprising Get1 and Get2.

Structural characterization of the Get4/Get5 complex and its interaction with Get3

The recently elucidated Get proteins are responsible for the targeted delivery of the majority of tail-anchored (TA) proteins to the endoplasmic reticulum. Get4 and Get5 have been identified in the early steps of the pathway mediating TA substrate delivery to the cytoplasmic targeting factor Get3. Here we report a crystal structure of Get4 and an N-terminal fragment of Get5 from Saccharomyces cerevisae. We show Get4 and Get5 (Get4/5) form an intimate complex that exists as a dimer (two copies of Get4/5) mediated by the C-terminus of Get5. We further demonstrate that Get3 specifically binds to a conserved surface on Get4 in a nucleotide dependent manner. This work provides further evidence for a model in which Get4/5 operates upstream of Get3 and mediates the specific delivery of a TA substrate.

Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

BackgroundTail-anchored (TA) proteins are a distinct class of membrane proteins that are sorted post-translationally to various organelles and function in a number of important cellular processes, including redox reactions, vesicular trafficking and protein translocation. While the molecular targeting signals and pathways responsible for sorting TA proteins to their correct intracellular destinations in yeasts and mammals have begun to be characterized, relatively little is known about TA protein biogenesis in plant cells, especially for those sorted to the plastid outer envelope.Methodology/Principal FindingsHere we investigated the biogenesis of three plastid TA proteins, including the 33-kDa and 34-kDa GTPases of the translocon at the outer envelope of chloroplasts (Toc33 and Toc34) and a novel 9-kDa protein of unknown function that we define here as an outer envelope TA protein (OEP9). Using a combination of in vivo and in vitro assays we show that OEP9 utilizes a different sorting pathway than that used by Toc33 and Toc34. For instance, while all three TA proteins interact with the cytosolic OEP chaperone/receptor, AKR2A, the plastid targeting information within OEP9 is distinct from that within Toc33 and Toc34. Toc33 and Toc34 also appear to differ from OEP9 in that their insertion is dependent on themselves and the unique lipid composition of the plastid outer envelope. By contrast, the insertion of OEP9 into the plastid outer envelope occurs in a proteinaceous-dependent, but Toc33/34-independent manner and membrane lipids appear to serve primarily to facilitate normal thermodynamic integration of this TA protein.Conclusions/SignificanceCollectively, the results provide evidence in support of at least two sorting pathways for plastid TA outer envelope proteins and shed light on not only the complex diversity of pathways involved in the targeting and insertion of proteins into plastids, but also the molecular mechanisms that underlie the delivery of TA proteins to their proper intracellular locations in general.