WRB and CAML Are Necessary and Sufficient to Mediate Tail-Anchored Protein Targeting to the ER Membrane

Tail-Anchored (TA) proteins are inserted into the endoplasmic reticulum (ER) membrane of yeast cells via the posttranslational Guided Entry of Tail-Anchored protein (GET) pathway. The key component of this targeting machinery is the ATPase Get3 that docks to the ER membrane by interacting with a receptor complex formed by the proteins Get1 and Get2. A conserved pathway is present in higher eukaryotes and is mediated by TRC40, homolog of Get3, and the recently identified membrane receptors WRB and CAML. Here, we used yeast lacking the GET1 and GET2 genes and substituted them with WRB and CAML. This rescued the growth phenotypes of the GET receptor mutant. We demonstrate that WRB and CAML efficiently recruit Get3 to the ER membrane and promote the targeting of the TA proteins in vivo. Our results show that the membrane spanning segments of CAML are essential to create a functional receptor with WRB and to ensure TA protein membrane insertion. Finally, we determined the binding parameters of TRC40 to the WRB/CAML receptor. We conclude that together, WRB and CAML are not only necessary but also sufficient to create a functional membrane receptor complex for TRC40. The yeast complementation assay can be used to further dissect the structure-function relationship of the WRB/CAML heteromultimer in the absence of endogenous receptor proteins.     

The Crystal Structures of Yeast Get3 Suggest a Mechanism for Tail-Anchored Protein Membrane Insertion

Tail-anchored (TA) proteins represent a unique class of membrane proteins that contain a single C-terminal transmembrane helix. The post-translational insertion of the yeast TA proteins into the ER membrane requires the Golgi ER trafficking (GET) complex which contains Get1, Get2 and Get3. Get3 is an ATPase that recognizes and binds the C-terminal transmembrane domain (TMD) of the TA proteins. We have determined the crystal structures of Get3 from two yeast species, S. cerevisiae and D. hansenii, respectively. These high resolution crystal structures show that Get3 contains a nucleotide-binding domain and a “finger” domain for binding the TA protein TMD. A large hydrophobic groove on the finger domain of S. cerevisiae Get3 structure might represent the binding site for TMD of TA proteins. A hydrophobic helix from a symmetry-related Get3 molecule sits in the TMD-binding groove and mimics the TA binding scenario. Interestingly, the crystal structures of the Get3 dimers from S. cerevisiae and D. hansenii exhibit distinct conformations. The S. cerevisiae Get3 dimer structure does not contain nucleotides and maintains an “open” conformation, while the D. hansenii Get3 dimer structure binds ADP and stays in a “closed” conformation. We propose that the conformational changes to switch the Get3 between the open and closed conformations may facilitate the membrane insertions for TA proteins.     

Analysis of tail-anchored protein translocation pathway in plants

Tail-anchored (TA) proteins are a special class of membrane proteins that carry out vital functions in all living cells. Targeting mechanisms of TA proteins are investigated as the best example for post-translational protein targeting in yeast. Of the several mechanisms, Guided Entry of Tail-anchored protein (GET) pathway plays a major role in TA protein targeting. Many in silico and in vivo analyses are geared to identify TA proteins and their targeting mechanisms in different systems including Arabidopsis thaliana. Yet, crop plants that grow in specific and/or different conditions are not investigated for the presence of TA proteins and GET pathway. This study majorly investigates GET pathway in two crop plants, Oryza sativa subsp. Indica and Solanum tuberosum, through detailed in silico analysis. 508 and 912 TA proteins are identified in Oryza sativa subsp. Indica and Solanum tuberosum respectively and their localization with respect to endoplasmic reticulum (ER), mitochondria, and chloroplast has been delineated. Similarly, the associated GET proteins are identified (Get1, Get3 and Get4) and their structural inferences are elucidated using homology modelling. Get3 models are based on yeast Get3. The cytoplasmic Get3 from O. sativa is identified to be very similar to yeast Get3 with conserved P-loop and TA binding groove. Three cytoplasmic Get3s are identified for S. tuberosum. Taken together, this is the first study to identify TA proteins and GET components in Oryza sativa subsp. Indica and Solanum tuberosum, forming the basis for any further experimental characterization of TA targeting and GET pathway mechanisms in crop plants.     

The GET complex mediates insertion of tail-anchored proteins into the ER membrane

Tail-anchored (TA) proteins, defined by the presence of a single C-terminal transmembrane domain (TMD), play critical roles throughout the secretory pathway and in mitochondria, yet the machinery responsible for their proper membrane insertion remains poorly characterized. Here we show that Get3, the yeast homolog of the TA-interacting factor Asna1/Trc40, specifically recognizes TMDs of TA proteins destined for the secretory pathway. Get3 recognition represents a key decision step, whose loss can lead to misinsertion of TA proteins into mitochondria. Get3-TA protein complexes are recruited for endoplasmic reticulum (ER) membrane insertion by the Get1/Get2 receptor. In vivo, the absence of Get1/Get2 leads to cytosolic aggregation of Get3-TA complexes and broad defects in TA protein biogenesis. In vitro reconstitution demonstrates that the Get proteins directly mediate insertion of newly synthesized TA proteins into ER membranes. Thus, the GET complex represents a critical mechanism for ensuring efficient and accurate targeting of TA proteins.     

The structure of Get4 reveals an α-solenoid fold adapted for multiple interactions in tail-anchored protein biogenesis

Tail-anchored proteins play important roles in protein translocation, membrane fusion and apoptosis. They are targeted to the endoplasmic reticulum membrane via the guided-entry of tail-anchored proteins (Get) pathway. We present the 2 Å crystal structure of Get4 which participates in early steps of the Get pathway. The structure shows an α-solenoid fold with particular deviations from the regular pairwise arrangement of α-helices. A conserved hydrophobic groove accommodates the flexible C-terminal region in trans. The structural organization of the Get4 helical hairpin motifs provides a scaffold for protein-protein interactions in the Get pathway.     

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.     

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 et?al. and Mariappan et?al. now provide insights into TA protein insertion.     

Mechanism of Assembly of a Substrate Transfer Complex during Tail-anchored Protein Targeting

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C terminus, are post-translationally targeted to the endoplasmic reticulum membrane by the guided entry of TA proteins (GET) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 complex (Get4/5), which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3·Get4/5 complex. Here we report crystal structures of a Get3·Get4/5 complex from Saccharomyces cerevisiae at 2.8 and 6.0 Å that reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3·Get4/5 complex. Furthermore, we provide evidence that the Get3·Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limited electrostatically driven Get3·Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.     

Get1 stabilizes an open dimer conformation of get3 ATPase by binding two distinct interfaces

Tail-anchored (TA) proteins are integral membrane proteins that possess a single transmembrane domain near their carboxy terminus. TA proteins play critical roles in many important cellular processes such as membrane trafficking, protein translocation, and apoptosis. The GET complex mediates posttranslational insertion of newly synthesized TA proteins to the endoplasmic reticulum membrane. 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. We report 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. Our structures and biochemical data suggest that Get1 uses two interfaces to stabilize the open dimer conformation of Get3. We propose that one interface is sufficient for binding of Get1 by Get3, while the second interface stabilizes the open dimer conformation of Get3.     

Expression and biophysical analysis of a triple-transmembrane domain-containing fragment from a yeast G protein-coupled receptor

Structural characterization of G protein-coupled receptors (GPCRs) is hindered by the inherent hydrophobicity, flexibility, and large size of these signaling proteins. Insights into conformational preferences and the three-dimensional (3D) structure of domains of these receptors can be obtained using polypeptide fragments of these proteins. Herein, we report the expression, purification, and biophysical characterization of a three-transmembrane domain-containing 131-residue fragment of the yeast α-factor receptor, Ste2p. Ste2p TM1–TM3 (G31–R161) was expressed as a TrpΔLE fusion protein in Escherichia coli. The expressed protein was subject to CNBr cleavage to remove the fusion tag and TM1–TM3 was purified by reverse-phased HPLC. The cleavage product was isolated in yields of up to 20 mg per liter of culture in both unlabeled and uniformly [15N]-labeled and [15N, 13C, 2H]-labeled forms. The secondary structure of TM1–TM3 was determined to be helical in a number of membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE):water and lysomyristoylphosphatidylglycerol (LMPG) detergent micelles by circular dichroism. Preliminary HSQC analysis in 50% TFE:water and LMPG micelles prepared in sodium phosphate and 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffers revealed that this fragment is suitable for structural analysis by nuclear magnetic resonance (NMR). Complete backbone assignments and a detailed localization of the secondary structural elements of TM1–TM3 in 50% TFE:water have been achieved.     

Searching for remote homologs of CAML among eukaryotes

The tryptophan rich basic protein/calcium signal‐modulating cyclophilin ligand (WRB/CAML) and Get1p/Get2p complexes, in vertebrates and yeast, respectively, mediate the final step of tail‐anchored protein insertion into the endoplasmic reticulum membrane via the Get pathway. While WRB appears to exist in all eukaryotes, CAML homologs were previously recognized only among chordates, raising the question as to how CAML's function is performed in other phyla. Furthermore, whereas WRB was recognized as the metazoan homolog of Get1, CAML and Get2, although functionally equivalent, were not considered to be homologous. CAML contains an N‐terminal basic, TRC40/Get3‐interacting, region, three transmembrane segments near the C‐terminus, and a poorly conserved region between these domains. Here, I searched the NCBI protein database for remote CAML homologs in all eukaryotes, using position‐specific iterated‐basic local alignment search tool, with the C‐terminal, the N‐terminal or the full‐length sequence of human CAML as query. The N‐terminal basic region and full‐length CAML retrieved homologs among metazoa, plants and fungi. In the latter group several hits were annotated as GET2. The C‐terminal query did not return entries outside of the animal kingdom, but did retrieve over one hundred invertebrate metazoan CAML‐like proteins, which all conserved the N‐terminal TRC40‐binding domain. The results indicate that CAML homologs exist throughout the eukaryotic domain of life, and suggest that metazoan CAML and yeast GET2 share a common evolutionary origin. They further reveal a tight link between the particular features of the metazoan membrane‐anchoring domain and the TRC40‐interacting region. The list of sequences presented here should provide a useful resource for future studies addressing structure‐function relationships in CAML proteins.     

Structural and Functional Characterization of Ybr137wp Implicates Its Involvement in the Targeting of Tail-Anchored Proteins to Membranes

Nearly 5% of membrane proteins are guided to nuclear, endoplasmic reticulum (ER), mitochondrial, Golgi, or peroxisome membranes by their C-terminal transmembrane domain and are classified as tail-anchored (TA) membrane proteins. In Saccharomyces cerevisiae, the guided entry of TA protein (GET) pathway has been shown to function in the delivery of TA proteins to the ER. The sorting complex for this pathway is comprised of Sgt2, Get4, and Get5 and facilitates the loading of nascent tail-anchored proteins onto the Get3 ATPase. Multiple pulldown assays also indicated that Ybr137wp associates with this complex in vivo. Here, we report a 2.8-Å-resolution crystal structure for Ybr137wp from Saccharomyces cerevisiae. The protein is a decamer in the crystal and also in solution, as observed by size exclusion chromatography and analytical ultracentrifugation. In addition, isothermal titration calorimetry indicated that the C-terminal acidic motif of Ybr137wp interacts with the tetratricopeptide repeat (TPR) domain of Sgt2. Moreover, an in vivo study demonstrated that Ybr137wp is induced in yeast exiting the log phase and ameliorates the defect of TA protein delivery and cell viability derived by the impaired GET system under starvation conditions. Therefore, this study suggests a possible role for Ybr137wp related to targeting of tail-anchored proteins.     

A structural model of the Sgt2 protein and its interactions with chaperones and the Get4/Get5 complex

The insertion of tail-anchored transmembrane (TA) proteins into the appropriate membrane is a post-translational event that requires stabilization of the transmembrane domain and targeting to the proper destination. Sgt2 is a heat-shock protein cognate (HSC) co-chaperone that preferentially binds endoplasmic reticulum-destined TA proteins and directs them to the GET pathway via Get4 and Get5. Here, we present the crystal structure from a fungal Sgt2 homolog of the tetratrico-repeat (TPR) domain and part of the linker that connects to the C-terminal domain. The linker extends into the two-carboxylate clamp of the TPR domain from a symmetry-related molecule mimicking the binding to HSCs. Based on this structure, we provide biochemical evidence that the Sgt2 TPR domain has the ability to directly bind multiple HSC family members. The structure allows us to propose features involved in this lower specificity relative to other TPR containing co-chaperones. We further show that a dimer of Sgt2 binds a single Get5 and use small angle x-ray scattering to characterize the domain arrangement of Sgt2 in solution. These results allow us to present a structural model of the Sgt2-Get4/Get5-HSC complex.     

Deciphering the molecular organization of GET pathway chaperones through native mass spectrometry

Get3/4/5 chaperone complex is responsible for targeting C-terminal tail-anchored membrane proteins to the endoplasmic reticulum. Despite the availability of several crystal structures of independent proteins and partial structures of subcomplexes, different models of oligomeric states and structural organization have been proposed for the protein complexes involved. Here, using native mass spectrometry (Native-MS), coupled with intact dissociation, we show that Get4/5 exclusively forms a tetramer using both Get5/5 and a novel Get4/4 dimerization interface. Addition of Get3 to this leads to a hexameric (Get3)₂-(Get4)₂-(Get5)₂ complex with closed-ring cyclic architecture. We further validate our claims through molecular modeling and mutational abrogation of the proposed interfaces. Native-MS has become a principal tool to determine the state of oligomeric organization of proteins. The work demonstrates that for multiprotein complexes, native-MS, coupled with molecular modeling and mutational perturbation, can provide an alternative route to render a detailed view of both the oligomeric states as well as the molecular interfaces involved. This is especially useful for large multiprotein complexes with large unstructured domains that make it recalcitrant to conventional structure determination approaches.     

Evidence for similar function of transmembrane segments in receptor and membrane-anchored proteins

Transmembrane (TM) regions of receptor proteins should have unique structural and/or chemical characteristics if these regions contain residues functional in TM signal transduction. However, in a survey of the membrane-occurring residues in 37 integral membrane proteins, we found that amino acid compositions of TM regions of receptor proteins (n = 11) could not be distinguished statistically from corresponding regions of membrane-anchored proteins (e.g., recognition molecules) with a functional external domain attached to a single hydrophobic membrane-spanning anchor segment (n = 16). TM regions in both categories of proteins differed from the compositions of TM regions in membrane-transport proteins (n = 10). The analysis implies that TM regions in receptor proteins may function mainly to anchor (and position) receptors in their cellular membranes, and therefore residues in receptors that participate in signal transduction need not be restricted to these regions. In addition to mechanisms involving receptor aggregation, ligand-activated conformational perturbation of a receptor external aqueous domain, resulting in membrane penetration of hydrophobic segment(s) of this domain to produce intramembranous contact with its cytoplasmic domain, is hypothesized as a further possible mode of signal transduction.     

Functional analysis of skunk cabbage SfUCPB, a unique uncoupling protein lacking the fifth transmembrane domain, in yeast cells

Skunk cabbage, Symplocarpus foetidus, expresses two uncoupling proteins (UCPs), termed SfUCPA and SfUCPB, in the thermogenic organ spadix. SfUCPB exhibits unique structural features characterized by the absence of the putative fifth transmembrane domain (TM5) observed in SfUCPA, which is structurally similar to UCP1, and is abundantly expressed in the thermogenic spadix. Here, we conducted a series of comparative analyses of UCPs with six transmembrane domains, SfUCPA and rat UCP1, and TM5-deficient SfUCPB, using a heterologous yeast expression system. All UCPs were successfully expressed and targeted to the mitochondria, although the expression level of SfUCPB protein was approximately 10% of rat UCP1. The growth rate, mitochondrial membrane potential, and ATP content were significantly lower in cells expressing SfUCPB than in those expressing rat UCP1 and SfUCPA. These results suggest that SfUCPB, a novel TM5-deficient UCP, acts as an uncoupling protein in yeast cells.     

Isotopic Determination and Optimum Reaction Conditions of Pantothenic Acid Synthetase

When yeast protoplasts that were producing repressible acid phosphatase (r-APase) were treated with tunicamycin (TM), three specific proteins of 59k, 57k, and 55k daltons were accumulated in the membrane fraction in addition to the usual membrane proteins and these proteins were not detected in the secreted fraction. These proteins were immunoprecipitated with anti r-APase antiserum. Their molecular sizes were almost the same as those endo-H treated r-APase. Therefore these proteins were considered to be nonglycosylated forms of r-APase proteins. These results proved that nonglycosylated forms of r-APase produced by TM-treatment were not secreted by yeast protoplasts.     

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.     

Membrane integration of the second transmembrane segment of band 3 requires a closely apposed preceding signal-anchor sequence

We have investigated the topogenic rules of multispanning membrane proteins using erythrocyte band 3. Here, the fine structural requirements for the correct disposition of its second transmembrane segment (TM2) were assessed. We made fusion proteins where TM1 and the loop sequence preceding TM2 were changed and fused to prolactin. They were expressed in a cell-free system supplemented with rough microsomal membrane, and their topologies on the membrane were assessed by protease sensitivity and N-glycosylation. TM1 was demonstrated to be a signal-anchor sequence that mediates translocation of the downstream portion, and thus TM2 should be responsible to halt the translocation to acquire TM topology. When the loop between TM1 and TM2 was elongated, however, TM2 was readily translocated through the membrane and not integrated. For the membrane integration of TM2, TM2 must be in close proximity to TM1. The TM1 can be replaced with another signal-anchor sequence with a long hydrophobic segment but not with a signal sequence with shorter hydrophobic stretch. The length of the hydrophobic segment affected final topology of TM2. We concluded that the two TM segments work synergistically within the translocon to acquire the correct topology and that the length of the preceding signal sequence is critical for stable transmembrane assembly of TM2. We propose that direct interaction among the TM segments is one of the critical factors for the transmembrane topogenesis of multispanning membrane proteins.     

Membrane profile-based probabilistic method for predicting transmembrane segments via multiple protein sequence alignment

Prediction of transmembrane (TM) segments of amino acid sequences of membrane proteins is a well-known and very important problem. The accuracy of its solution can be improved for approaches that do not use a homology search in an additional data bank. There is a lack of tested data in this area of research, because information on the structure of membrane proteins is scarce. In this work we created a test sample of structural alignments for membrane proteins. The TM segments of these proteins were mapped according to aligned 3D structures resolved for these proteins. A method for predicting TM segments in an alignment was developed on the basis of the forward-backward algorithm from the HMM theory. This method allows a user not only to predict TM segments, but also to create a probabilistic membrane profile, which can be employed in multiple alignment procedures taking the secondary structure of proteins into account. The method was implemented in a computer program available at http://bioinf.fbb.msu.ru/fwdbck/. It provides better results than the MEMSAT method, which is nearly the only tool predicting TM segments in multiple alignments, without a homology search.