Tail-anchored Research Articles

The WRB Subunit of the Get3 Receptor is Required for the Correct Integration of its Partner CAML into the ER

Calcium-modulating cyclophilin ligand (CAML), together with Tryptophan rich basic protein (WRB, Get1 in yeast), constitutes the mammalian receptor for the Transmembrane Recognition Complex subunit of 40 kDa (TRC40, Get3 in yeast), a cytosolic ATPase with a central role in the post-translational targeting pathway of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) membrane. CAML has also been implicated in other cell-specific processes, notably in immune cell survival, and has been found in molar excess over WRB in different cell types. Notwithstanding the stoichiometric imbalance, WRB and CAML depend strictly on each other for expression. Here, we investigated the mechanism by which WRB impacts CAML levels. We demonstrate that CAML, generated in the presence of sufficient WRB levels, is inserted into the ER membrane with three transmembrane segments (TMs) in its C-terminal region. By contrast, without sufficient levels of WRB, CAML fails to adopt this topology, and is instead incompletely integrated to generate two aberrant topoforms; these congregate in ER-associated clusters and are degraded by the proteasome. Our results suggest that WRB, a member of the recently proposed Oxa1 superfamily, acts catalytically to assist the topogenesis of CAML and may have wider functions in membrane biogenesis than previously appreciated.

Cooperation of mitochondrial and ER factors in quality control of tail-anchored proteins.

Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation. Here we screened for factors involved in degradation of TA proteins mislocalized to mitochondria. We show that the ER-associated degradation (ERAD) E3 ubiquitin ligase Doa10 controls cytoplasmic level of Msp1 clients. Furthermore, we identified the uncharacterized OMM protein Fmp32 and the ectopically expressed subunit of the ER-mitochondria encounter structure (ERMES) complex Gem1 as native clients for Msp1 and Doa10. We propose that productive localization of TA proteins to the OMM is ensured by complex assembly, while orphan subunits are extracted by Msp1 and eventually degraded by Doa10.

Structural analysis of chloroplast tail-anchored membrane protein recognition by ArsA1.

In mammals and yeast, tail-anchored (TA) membrane proteins destined for the post-translational pathway are safely delivered to the endoplasmic reticulum (ER) membrane by a well-known targeting factor, TRC40/Get3. In contrast, the underlying mechanism for translocation of TA proteins in plants remains obscure. How this unique eukaryotic membrane-trafficking system correctly distinguishes different subsets of TA proteins destined for various organelles, including mitochondria, chloroplasts and the ER, is a key question of long standing. Here, we present crystal structures of algal ArsA1 (the Get3 homolog) in a distinct nucleotide-free open state and bound to adenylyl-imidodiphosphate. This approximately 80-kDa protein possesses a monomeric architecture, with two ATPase domains in a single polypeptide chain. It is capable of binding chloroplast (TOC34 and TOC159) and mitochondrial (TOM7) TA proteins based on features of its transmembrane domain as well as the regions immediately before and after the transmembrane domain. Several helices located above the TA-binding groove comprise the interlocking hook-like motif implicated by mutational analyses in TA substrate recognition. Our data provide insights into the molecular basis of the highly specific selectivity of interactions of algal ArsA1 with the correct sets of TA substrates before membrane targeting in plant cells.

Mitochondrial AAA-ATPase Msp1 detects mislocalized tail-anchored proteins through a dual-recognition mechanism.

The conserved AAA-ATPase Msp1 is embedded in the outer mitochondrial membrane and removes mislocalized tail-anchored (TA) proteins upon dysfunction of the guided entry of tail-anchored (GET) pathway. It remains unclear how Msp1 recognizes its substrates. Here, we extensively characterize Msp1 and its substrates, including the mitochondrially targeted Pex15Δ30, and full-length Pex15, which mislocalizes to mitochondria upon dysfunction of Pex19 but not the GET pathway. Moreover, we identify two new substrates, Frt1 and Ysy6. Our results suggest that mislocalized TA proteins expose hydrophobic surfaces in the cytoplasm and are recognized by Msp1 through conserved hydrophobic residues. Introducing a hydrophobic patch into mitochondrial TA proteins transforms them into Msp1 substrates. In addition, Pex15Δ30 and Frt1 contain basic inter-membrane space (IMS) residues critical for their mitochondrial mistargeting. Remarkably, Msp1 recognizes this feature through the acidic D12 residue in its IMS domain. This dual-recognition mechanism involving interactions at the cytoplasmic and IMS domains of Msp1 and substrates greatly facilitates substrate recognition and is required by Msp1 to safeguard mitochondrial functions.

Targeting of tail-anchored proteins to Trichomonas vaginalis hydrogenosomes.

Tail-anchored (TA) proteins are membrane proteins that are found in all domains of life. They consist of an N-terminal domain that performs various functions and a single transmembrane domain (TMD) near the C-terminus. In eukaryotes, TA proteins are targeted to the membranes of mitochondria, the endoplasmic reticulum (ER), peroxisomes and in plants, chloroplasts. The targeting of these proteins to their specific destinations correlates with the properties of the C-terminal domain, mainly the TMD hydrophobicity and the net charge of the flanking regions. Trichomonas vaginalis is a human parasite that has adapted to oxygen-poor environment. This adaptation is reflected by the presence of highly modified mitochondria (hydrogenosomes) and the absence of peroxisomes. The proteome of hydrogenosomes is considerably reduced; however, our bioinformatic analysis predicted 120 putative hydrogenosomal TA proteins. Seven proteins were selected to prove their localization. The elimination of the net positive charge in the C-tail of the hydrogenosomal TA4 protein resulted in its dual localization to hydrogenosomes and the ER, causing changes in ER morphology. Domain mutation and swap experiments with hydrogenosomal (TA4) and ER (TAPDI) proteins indicated that the general principles for specific targeting are conserved across eukaryotic lineages, including T. vaginalis; however, there are also significant lineage-specific differences.

Triplet-pore structure of a highly divergent TOM complex of hydrogenosomes in Trichomonas vaginalis.

Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.

Pex19 is involved in importing dually targeted tail-anchored proteins to both mitochondria and peroxisomes.

Tail-anchored (TA) proteins are embedded into their corresponding membrane via a single transmembrane segment at their C-terminus whereas the majority of the protein is facing the cytosol. So far, cellular factors that mediate the integration of such proteins into the mitochondrial outer membrane were not found. Using budding yeast as a model system, we identified the cytosolic Hsp70 chaperone Ssa1 and the peroxisome import factor Pex19 as import mediators for a subset of mitochondrial TA proteins. Accordingly, deletion of PEX19 results in: (1) growth defect under respiration conditions, (2) alteration in mitochondrial morphology, (3) reduced steady-state levels of the mitochondrial TA proteins Fis1 and Gem1, and (4) hampered in organello import of the TA proteins Fis1 and Gem1. Furthermore, recombinant Pex19 can bind directly the TA proteins Fis1 and Gem1. Collectively, this work identified the first factors that are involved in the biogenesis of mitochondrial TA proteins and uncovered an unexpected function of Pex19.

The GET pathway can increase the risk of mitochondrial outer membrane proteins to be mistargeted to the ER.

Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.

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 ER membrane insertase Get1/2 is required for efficient mitophagy in yeast

Mitophagy is an evolutionarily conserved autophagy pathway that selectively eliminates mitochondria to control mitochondrial quality and quantity. Although mitophagy is thought to be crucial for cellular homeostasis, how this catabolic process is regulated remains largely unknown. Here we demonstrate that mitophagy during prolonged respiratory growth is strongly impaired in yeast cells lacking Get1/2, a transmembrane complex mediating insertion of tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane. Under the same conditions, loss of Get1/2 caused only slight defects in other types of selective and bulk autophagy. In addition, mitophagy and other autophagy-related processes are mostly normal in cells lacking Get3, a cytosolic ATP-driven chaperone that promotes delivery of TA proteins to the Get1/2 complex. We also found that Get1/2-deficient cells exhibited wildtype-like induction and mitochondrial localization of Atg32, a protein essential for mitophagy. Notably, Get1/2 is important for Atg32-independent, ectopically promoted mitophagy. Together, we propose that Get1/2-dependent TA protein(s) and/or the Get1/2 complex itself may act specifically in mitophagy.

Reconstitution of a Tail‐anchored Mitochondrial Membrane Protein

Tail‐anchored (TA) proteins have a single transmembrane domain (TMD) located on the C‐terminus with the bulk of the protein facing the cytosol; in contrast with the conventional membrane protein that is translated directly into the membrane of interest. TA proteins are involved in organelle division, vesicle trafficking, and apoptosis. TA proteins lack an N‐terminal signal sequence and exhibit wide sequence variation in the TMD. Thus, TA proteins are likely inserted into the membrane post translationally. We asked if we can reconstitute Fis1, a mitochondrial outer membrane TA protein, in a detergent micelle to determine its structure. We were able to purify soluble recombinant Fis1 in the absence of detergent. Preliminary results suggest that in the absence of detergent, Fis1 exists in a soluble aggregate that can be disrupted by addition of detergent. This soluble aggregate is likely facilitated by the TMD of Fis1. We further show that in the detergent micelle Fis1 is oriented such that the cytoplasmic domain faces the solvent, reminiscent of the orientation on the mitochondrial surface. Using properly reconstituted Fis1 in detergent micelles, we collected CD, DLS, NMR, and EPR data with the goal of determining a high‐resolution structure of Fis1 in the membrane, which is an outstanding question in the mitochondrial fission field. More generally, this work has many implications for TA membrane protein reconstitution.Support or Funding InformationNIH R01‐GM067180This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail-anchored proteins.

Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.

The ATPase activity of Asna1/TRC40 is required for pancreatic progenitor cell survival.

Asna1, also known as TRC40, is implicated in the delivery of tail-anchored (TA) proteins into the endoplasmic reticulum (ER), in vesicle-mediated transport, and in chaperoning unfolded proteins during oxidative stress/ATP depletion. Here, we show that Asna1 inactivation in pancreatic progenitor cells leads to redistribution of the Golgi TA SNARE proteins syntaxin 5 and syntaxin 6, Golgi fragmentation, and accumulation of cytosolic p62+ puncta. Asna1−/− multipotent progenitor cells (MPCs) selectively activate integrated stress response signaling and undergo apoptosis, thereby disrupting endocrine and acinar cell differentiation, resulting in pancreatic agenesis. Rescue experiments implicate the Asna1 ATPase activity and a CXXC di-cysteine motif in ensuring Golgi integrity, syntaxin 5 localization and MPC survival. Ex vivo inhibition of retrograde transport reproduces the perturbed Golgi morphology, and syntaxin 5 and syntaxin 6 expression, whereas modulation of p53 activity, using PFT-α and Nutlin-3, prevents or reproduces apoptosis in Asna1-deficient and wild-type MPCs, respectively. These findings support a role for the Asna1 ATPase activity in ensuring the survival of pancreatic MPCs, possibly by counteracting p53-mediated apoptosis.

The AAA protein Msp1 mediates clearance of excess tail-anchored proteins from the peroxisomal membrane.

Msp1 is a conserved AAA ATPase in budding yeast localized to mitochondria where it prevents accumulation of mistargeted tail-anchored (TA) proteins, including the peroxisomal TA protein Pex15. Msp1 also resides on peroxisomes but it remains unknown how native TA proteins on mitochondria and peroxisomes evade Msp1 surveillance. We used live-cell quantitative cell microscopy tools and drug-inducible gene expression to dissect Msp1 function. We found that a small fraction of peroxisomal Pex15, exaggerated by overexpression, is turned over by Msp1. Kinetic measurements guided by theoretical modeling revealed that Pex15 molecules at mitochondria display age-independent Msp1 sensitivity. By contrast, Pex15 molecules at peroxisomes are rapidly converted from an initial Msp1-sensitive to an Msp1-resistant state. Lastly, we show that Pex15 interacts with the peroxisomal membrane protein Pex3, which shields Pex15 from Msp1-dependent turnover. In sum, our work argues that Msp1 selects its substrates on the basis of their solitary membrane existence.

Tail-Anchored Protein Insertion by a Single Get1/2 Heterodimer

The Get1/2 transmembrane complex drives the insertion of tail-anchored (TA) proteins from the cytosolic chaperone Get3 into the endoplasmic reticulum membrane. Mechanistic insight into how Get1/2 coordinates this process is confounded by a lack of understanding of the basic architecture of the complex. Here, we define the oligomeric state of full-length Get1/2 in reconstituted lipid bilayers by combining single-molecule and bulk fluorescence measurements with quantitative invitro insertion analysis. We show that a single Get1/2 heterodimer is sufficient for insertion and demonstrate that the conserved cytosolic regions of Get1 and Get2 bind asymmetrically to opposing subunits of the Get3 homodimer. Altogether, our results define a simplified model for how Get1/2 and Get3 coordinate TA protein insertion.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical forTA protein extraction invitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane.

CAML mediates survival of Myc-induced lymphoma cells independent of tail-anchored protein insertion

Calcium-modulating cyclophilin ligand (CAML) is an endoplasmic reticulum (ER) protein that functions, along with WRB and TRC40, to mediate tail-anchored (TA) protein insertion into the ER membrane. Physiologic roles for CAML include endocytic trafficking, intracellular calcium signaling, and the survival and proliferation of specialized immune cells, recently attributed to its requirement for TA protein insertion. To identify a possible role for CAML in cancer cells, we generated Eμ-Myc transgenic mice that carry a tamoxifen-inducible deletion allele of Caml. In multiple B-cell lymphoma cell lines derived from these mice, homozygous loss of Caml activated apoptosis. Cell death was blocked by Bcl-2/Bcl-xL overexpression; however, rescue from apoptosis was insufficient to restore proliferation. Tumors established from an Eμ-Myc lymphoma cell line completely regressed after tamoxifen administration, suggesting that CAML is also required for these cancer cells to survive and grow in vivo. Cell cycle analyses of Caml-deleted lymphoma cells revealed an arrest in G2/M, accompanied by low expression of the mitotic marker, phospho-histone H3 (Ser10). Surprisingly, lymphoma cell viability did not depend on the domain of CAML required for its interaction with TRC40. Furthermore, a small protein fragment consisting of the C-terminal 111 amino acid residues of CAML, encompassing the WRB-binding domain, was sufficient to rescue growth and survival of Caml-deleted lymphoma cells. Critically, this minimal region of CAML did not restore TA protein insertion in knockout cells. Taken together, these data reveal an essential role for CAML in supporting survival and mitotic progression in Myc-driven lymphomas that is independent of its TA protein insertion function.

In search of tail-anchored protein machinery in plants: reevaluating the role of arsenite transporters

Although the mechanisms underlying selective targeting of tail-anchored (TA) membrane proteins are well established in mammalian and yeast cells, little is known about their role in mediating intracellular membrane trafficking in plant cells. However, a recent study suggested that, in green algae, arsenite transporters located in the cytosol (ArsA1 and ArsA2) control the insertion of TA proteins into the membrane-bound organelles. In the present work, we overproduced and purified these hydrophilic proteins to near homogeneity. The analysis of their catalytic properties clearly demonstrates that C. reinhardtii ArsA proteins exhibit oxyanion-independent ATPase activity, as neither arsenite nor antimonite showed strong effects. Co-expression of ArsA proteins with TA-transmembrane regions showed not only that the former interact with the latter, but that ArsA1 does not share the same ligand specificity as ArsA2. Together with a structural model and molecular dynamics simulations, we propose that C. reinhadtii ArsA proteins are not arsenite transporters, but a TA-protein targeting factor. Further, we propose that ArsA targeting specificity is achieved at the ligand level, with ArsA1 mainly carrying TA-proteins to the chloroplast, while ArsA2 to the endoplasmic reticulum.

Mechanistic basis for a molecular triage reaction.

Newly synthesized proteins are triaged between biosynthesis and degradation to maintain cellular homeostasis, but the decision-making mechanisms are unclear. We reconstituted the core reactions for membrane targeting and ubiquitination of nascent tail-anchored membrane proteins to understand how their fate is determined. The central six-component triage system is divided into an uncommitted client-SGTA complex, a self-sufficient targeting module, and an embedded but self-sufficient quality control module. Client-SGTA engagement of the targeting module induces rapid, private, and committed client transfer to TRC40 for successful biosynthesis. Commitment to ubiquitination is dictated primarily by comparatively slower client dissociation from SGTA and nonprivate capture by the BAG6 subunit of the quality control module. Our results provide a paradigm for how priority and time are encoded within a multichaperone triage system.

Targeting of tail-anchored membrane proteins to subcellular organelles in Toxoplasma gondii.

Proper protein localization is essential for critical cellular processes, including vesicle-mediated transport and protein translocation. Tail-anchored (TA) proteins are integrated into organellar membranes via the C-terminus, orienting the N-terminus towards the cytosol. Localization of TA proteins occurs posttranslationally and is governed by the C-terminus, which contains the integral transmembrane domain (TMD) and targeting sequence. Targeting of TA proteins is dependent on the hydrophobicity of the TMD as well as the length and composition of flanking amino acid sequences. We previously identified an unusual homologue of elongator protein, Elp3, in the apicomplexan parasite Toxoplasma gondii as a TA protein targeting the outer mitochondrial membrane. We sought to gain further insight into TA proteins and their targeting mechanisms using this early-branching eukaryote as a model. Our bioinformatics analysis uncovered 59 predicted TA proteins in Toxoplasma, 9 of which were selected for follow-up analyses based on representative features. We identified novel TA proteins that traffic to specific organelles in Toxoplasma, including the parasite endoplasmic reticulum, mitochondrion, and Golgi apparatus. Domain swap experiments elucidated that targeting of TA proteins to these specific organelles was strongly influenced by the TMD sequence, including charge of the flanking C-terminal sequence.