Tail-anchored Proteins Research Articles

Msp1 cooperates with the proteasome for extraction of arrested mitochondrial import intermediates.

The mitochondrial AAA ATPase Msp1 is well known for extraction of mislocalized tail-anchored ER proteins from the mitochondrial outer membrane. Here, we analyzed the extraction of precursors blocking the import pore in the outer membrane. We demonstrate strong genetic interactions of Msp1 and the proteasome with components of the TOM complex, the main translocase in the outer membrane. Msp1 and the proteasome both contribute to the removal of arrested precursor proteins that specifically accumulate in these mutants. The proteasome activity is essential for the removal as proteasome inhibitors block extraction. Furthermore, the proteasomal subunit Rpn10 copurified with Msp1. The human Msp1 homologue has been implicated in neurodegenerative diseases, and we show that the lack of the Caenorhabditis elegans Msp1 homologue triggers an import stress response in the worm, which indicates a conserved role in metazoa. In summary, our results suggest a role of Msp1 as an adaptor for the proteasome that drives the extraction of arrested and mislocalized proteins at the mitochondrial outer membrane.

Morphologically Discrete ER Subdomains Support the Synthesis of Different Types of Protein

The endoplasmic reticulum (ER) has a complex morphology comprised of stacked sheets, tubules, and three-way junctions, which together function as a platform for protein synthesis of the membrane and secretory proteins. It is widely believed that specific ER subdomains are spatially organized for protein synthesis activities, but precisely where these domains occur in relation to the ER's overall architecture is unclear due to lack of functional-spatial resolution. Here, we use single-molecule tracking of ribosomes and mRNAs in combination with simultaneous imaging of ER to assess sites of membrane protein synthesis in the ER. We found that ribosomes were widely distributed throughout the different ER morphologies. However, synthesis of membrane proteins (including type I, II and multi-spanning) and an ER luminal protein occurred primarily at the three-way junctions. Unlike mRNAs coding for membrane proteins, mRNA coding for the translocon-independent, tail-anchored protein Sec61β was found primarily on ER tubules and was actively translated on the ER. Surprisingly, mRNA coding for the cytosolic protein β-actin was also primarily associated with ER tubules. These results support the idea that discrete ER subdomains exist to support specific types of protein synthesis activities, with ER morphological features playing an important role in this organization

Structure of the AAA protein Msp1 reveals mechanism of mislocalized membrane protein extraction.

The AAA protein Msp1 extracts mislocalized tail-anchored membrane proteins and targets them for degradation, thus maintaining proper cell organization. How Msp1 selects its substrates and firmly engages them during the energetically unfavorable extraction process remains a mystery. To address this question, we solved cryo-EM structures of Msp1-substrate complexes at near-atomic resolution. Akin to other AAA proteins, Msp1 forms hexameric spirals that translocate substrates through a central pore. A singular hydrophobic substrate recruitment site is exposed at the spiral's seam, which we propose positions the substrate for entry into the pore. There, a tight web of aromatic amino acids grips the substrate in a sequence-promiscuous, hydrophobic milieu. Elements at the intersubunit interfaces coordinate ATP hydrolysis with the subunits' positions in the spiral. We present a comprehensive model of Msp1's mechanism, which follows general architectural principles established for other AAA proteins yet specializes Msp1 for its unique role in membrane protein extraction.

A reference library for assigning protein subcellular localizations by image-based machine learning.

Confocal micrographs of EGFP fusion proteins localized at key cell organelles in murine and human cells were acquired for use as subcellular localization landmarks. For each of the respective 789,011 and 523,319 optically validated cell images, morphology and statistical features were measured. Machine learning algorithms using these features permit automated assignment of the localization of other proteins and dyes in both cell types with very high accuracy. Automated assignment of subcellular localizations for model tail-anchored proteins with randomly mutated C-terminal targeting sequences allowed the discovery of motifs responsible for targeting to mitochondria, endoplasmic reticulum, and the late secretory pathway. Analysis of directed mutants enabled refinement of these motifs and characterization of protein distributions in within cellular subcompartments.

J-Domain Proteins Promote Client Relay from Hsp70 During Tail-Anchored Membrane Protein Targeting

J-domain proteins (JDPs) play essential roles in assisting Hsp70 function in diverse aspects of protein biogenesis. Previous work showed that JDPs act by recruiting Hsp70 to client proteins, assisting Hsp70 in client trapping, and regulating the ATPase cycle of Hsp70. Here we report that JDPs can further enhance the conformational quality of Hsp70-bound client proteins during tail-anchored membrane protein (TA) targeting. In the guided-entry-of-tail-anchored protein (GET) pathway in yeast, newly synthesized TAs are captured and solubilized by the cytosolic Hsp70 Ssa1, which further initiates sequential TA handovers to downstream chaperones, Sgt2 and Get3, to enable their delivery to the endoplasmic reticulum. We found that two J-domain proteins (JDPs), Ydj1 and Sis1, function in parallel to support the ER targeting of TAs in vivo . Biochemical analyses showed that, while Ydj1 and Sis1 differ in their ability to assist Ssa1 in TA trapping, both JDPs enhance the transfer of Ssa1-bound TA substrate to Sgt2. The ability of the JDPs to remodel the conformation and ATPase activity of Ssa1, rather than their client binding activity, is essential for enhancing the transfer competence of Ssa1-bound TAs and for supporting TA insertion in vivo. Our results define the Hsp70 cochaperone requirement for the GET pathway and demonstrate a role of JDPs in improving the conformational quality of Hsp70-bound membrane protein clients.

The Biogenesis of Mitochondrial Outer Membrane Proteins Show Variable Dependence on Import Factors

SummaryBiogenesis of mitochondrial outer membrane proteins involves their integration into the lipid bilayer. Among these proteins are those that form a single-span topology, but our understanding of their biogenesis is scarce. In this study, we found that the MIM complex is required for the membrane insertion of some single-span proteins. However, other such proteins integrate into the membrane in a MIM-independent manner. Moreover, the biogenesis of the studied proteins was dependent to a variable degree on the TOM receptors Tom20 and Tom70. We found that Atg32 C-terminal domain mediates dependency on Tom20, whereas the cytosolic domains of Atg32 and Gem1 facilitate MIM involvement. Collectively, our findings (1) enlarge the repertoire of MIM substrates to include also tail-anchored proteins, (2) provide new mechanistic insights to the functions of the MIM complex and TOM import receptors, and (3) demonstrate that the biogenesis of MOM single-span proteins shows variable dependence on import factors.

Quantifying the inhibitory effect of Bcl-xl on the action of Mff using live-cell fluorescence imaging.

Mitochondrial fission regulates mitochondrial function and morphology, and has been linked to apoptosis. The mitochondrial fission factor (Mff), a tail‐anchored membrane protein, induces excessive mitochondrial fission, contributing to mitochondrial dysfunction and apoptosis. Here, we evaluated the inhibitory effect of Bcl‐xl, an antiapoptotic protein, on the action of Mff by using live‐cell fluorescence imaging. Microscopic imaging analysis showed that overexpression of Mff induced mitochondrial fragmentation and apoptosis, which were reversed by coexpression of Bcl‐xl. Microscopic imaging and live‐cell fluorescence resonance energy transfer analysis demonstrated that Bcl‐xl reconstructs the Mff network from punctate distribution of higher‐order oligomers to filamentous distribution of lower‐order oligomers. Live‐cell fluorescence resonance energy transfer two‐hybrid assay showed that Bcl‐xl interacted with Mff to form heterogenous oligomers with 1 : 2 stoichiometry in cytoplasm and 1 : 1 stoichiometry on mitochondria, indicating that two Bcl‐xl molecules primarily interact with four Mff molecules in cytoplasm, but with two Mff molecules on mitochondria.

Genome-wide analysis of Homo sapiens, Arabidopsis thaliana, and Saccharomyces cerevisiae reveals novel attributes of tail-anchored membrane proteins

BackgroundTail-anchored membrane proteins (TAMPs) differ from other integral membrane proteins, because they contain a single transmembrane domain at the extreme carboxyl-terminus and are therefore obliged to target to membranes post-translationally. Although 3–5% of all transmembrane proteins are predicted to be TAMPs only a small number are well characterized.ResultsTo identify novel putative TAMPs across different species, we used TAMPfinder software to identify 859, 657 and 119 putative TAMPs in human (Homo sapiens), plant (Arabidopsis thaliana), and yeast (Saccharomyces cerevisiae), respectively. Bioinformatics analyses of these putative TAMP sequences suggest that the list is highly enriched for authentic TAMPs. To experimentally validate the software predictions several human and plant proteins identified by TAMPfinder that were previously uncharacterized were expressed in cells and visualized at subcellular membranes by fluorescence microscopy and further analyzed by carbonate extraction or by bimolecular fluorescence complementation. With the exception of the pro-apoptotic protein harakiri, which is, peripherally bound to the membrane this subset of novel proteins behave like genuine TAMPs. Comprehensive bioinformatics analysis of the generated TAMP datasets revealed previously unappreciated common and species-specific features such as the unusual size distribution of and the propensity of TAMP proteins to be part of larger complexes. Additionally, novel features of the amino acid sequences that anchor TAMPs to membranes were also revealed.ConclusionsThe findings in this study more than double the number of predicted annotated TAMPs and provide new insights into the common and species-specific features of TAMPs. Furthermore, the list of TAMPs and annotations provide a resource for further investigation.

Membrane-Specific Targeting of Tail-Anchored Proteins SECE1 and SECE2 Within Chloroplasts.

Membrane proteins that are imported into chloroplasts must be accurately targeted in order to maintain the identity and function of the highly differentiated internal membranes. Relatively little is known about the targeting information or pathways that direct proteins with transmembrane domains to either the inner envelope or thylakoids. In this study, we focused on a structurally simple class of membrane proteins, the tail-anchored proteins, which have stroma-exposed amino-terminal domains and a single transmembrane domain within 30 amino acids of the carboxy-terminus. SECE1 and SECE2 are essential tail-anchored proteins that function as components of the dual SEC translocases in chloroplasts. SECE1 localizes to the thylakoids, while SECE2 localizes to the inner envelope. We have used transient expression in Arabidopsis leaf protoplasts and confocal microscopy in combination with a domain-swapping strategy to identify regions that contain important targeting determinants. We show that membrane-specific targeting depends on features of the transmembrane domains and the short C-terminal tails. We probed the contributions of these regions to targeting processes further through site-directed mutagenesis. We show that thylakoid targeting still occurs when changes are made to the tail of SECE1, but changing residues in the tail of SECE2 abolishes inner envelope targeting. Finally, we discuss possible parallels between sorting of tail-anchored proteins in the stroma and in the cytosol.

Proteomic mapping by rapamycin-dependent targeting of APEX2 identifies binding partners of VAPB at the inner nuclear membrane

Vesicle-associated membrane protein-associated protein B (VAPB) is a tail-anchored protein that is present at several contact sites of the endoplasmic reticulum (ER). We now show by immunoelectron microscopy that VAPB also localizes to the inner nuclear membrane (INM). Using a modified enhanced ascorbate peroxidase 2 (APEX2) approach with rapamycin-dependent targeting of the peroxidase to a protein of interest, we searched for proteins that are in close proximity to VAPB, particularly at the INM. In combination with stable isotope labeling with amino acids in cell culture (SILAC), we confirmed many well-known interaction partners at the level of the ER with a clear distinction between specific and nonspecific hits. Furthermore, we identified emerin, TMEM43, and ELYS as potential interaction partners of VAPB at the INM and the nuclear pore complex, respectively.

Retro-2 protects cells from ricin toxicity by inhibiting ASNA1-mediated ER targeting and insertion of tail-anchored proteins.

The small molecule Retro-2 prevents ricin toxicity through a poorly-defined mechanism of action (MOA), which involves halting retrograde vesicle transport to the endoplasmic reticulum (ER). CRISPRi genetic interaction analysis revealed Retro-2 activity resembles disruption of the transmembrane domain recognition complex (TRC) pathway, which mediates post-translational ER-targeting and insertion of tail-anchored (TA) proteins, including SNAREs required for retrograde transport. Cell-based and in vitro assays show that Retro-2 blocks delivery of newly-synthesized TA-proteins to the ER-targeting factor ASNA1 (TRC40). An ASNA1 point mutant identified using CRISPR-mediated mutagenesis abolishes both the cytoprotective effect of Retro-2 against ricin and its inhibitory effect on ASNA1-mediated ER-targeting. Together, our work explains how Retro-2 prevents retrograde trafficking of toxins by inhibiting TA-protein targeting, describes a general CRISPR strategy for predicting the MOA of small molecules, and paves the way for drugging the TRC pathway to treat broad classes of viruses known to be inhibited by Retro-2.

Distinct Requirements for Tail-Anchored Membrane Protein Biogenesis in Escherichia coli.

Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented.IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus "regular" membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.

Molecular pathways of mitochondrial outer membrane protein degradation.

Mitochondrial outer membrane (MOM) encloses inner compartments of mitochondria and integrates cytoplasmic signals to regulate essential mitochondrial processes, such as protein import, dynamics, metabolism, cell death, etc. A substantial understanding of MOM associated proteostatic stresses and quality control pathways has been obtained in recent years. Six MOM associated protein degradation (MAD) pathways center on three AAA ATPases: Cdc48 in the cytoplasm, Msp1 integral to MOM, and Yme1 integral to the inner membrane. These pathways survey MOM proteome from the cytoplasmic and the inter-membrane space (IMS) sides. They detect and degrade MOM proteins with misfolded cytoplasmic and IMS domains, remove mistargeted tail-anchored proteins, and clear mitochondrial precursor proteins clogged in the TOM import complex. These MOM associated protein quality control pathways collaboratively maintain mitochondrial proteostasis and cell viability.

P1230Impact of regulated junctophilin-2 clustering at axial tubule junctions on atrial excitation-contraction coupling and therapeutic implications

Abstract Objectives Atrial dysfunction is highly prevalent and known to significantly aggravate heart failure. While rapid excitation-contraction (EC) coupling depends on axial tubule junctions in atrial myocytes (AMs), the mechanisms leading to atrial loss-of-function remain unclear. Junctophilin-2 (JP2), a tail-anchored protein of the sarcoplasmic reticulum, stabilizes the integrity of ventricular Ca2+ release units, which is disrupted in ventricular myocytes by reduced JP2 expression or proteolysis. Here we aim to characterize the abundance and subcellular localisation of JP2 in AMs, to assess the impact of decreased JP2 expression on atrial remodelling, and to investigate the potential to correct JP2 expression and atrial dysfunction. Results We identified 5-fold lower JP2 levels in atrial compared to ventricular tissue in mouse and human hearts by SDS-PAGE. Surprisingly, in AMs, this resulted in subcellular expression of large JP2 clusters at axial tubule junctions together with highly phosphorylated ryanodine receptor (RyR2) channels visualized by STED superresolution microscopy. Importantly, left atrial hypertrophy induced by aortic pressure overload led to an additional strong decrease in JP2 expression compared to sham control, disrupted junctional RyR2 clustering and EC-coupling. This loss-of-function mechanism was confirmed by conditional shRNA-mediated JP2 knockdown. Quantitative image analysis after atrial JP2 knockdown showed a 50% decrease in area overlap between RyR2 and JP2 in AMs (JP2 knockdown 0.03±0.003 μm2 vs. control 0.06±0.004 μm2, p<0.001), and a ∼2-fold increased Ca2+ spark frequency, consistent with decreased left atrial fractional shortening (JP2 knockdown 12.9±0.8% vs. control 16.5±0.9%, p<0.01). Whereas atrial-ventricular dysfunction due to aortic pressure overload resulted in 40% mortality, additional JP2 knockdown exacerbated mortality to 100% (n: 10 control vs. 9 JP2 knockdown mice). In contrast, transgenic JP2 overexpressor mice showed greatly improved atrial contractility without mortality after induced aortic pressure overload (n: 21 control vs. 16 JP2 overexpressor mice). JP2-OE not only augmented atrial RyR2-clustering, but induced the de-novo biogenesis of large poly-adic junctional membrane complexes, which were resolved by STED microscopy via high-resolution cholesterol-based membrane staining in live AMs and electron tomography. Conclusions Nanoscale imaging identifies a new subcellular mechanism of significantly limited atrial JP2 protein expression in large clusters at axial tubule junctions. In atrial hypertrophy, JP2 is further decreased with junctional RyR2 cluster disruption leading to impaired Ca2+ release and decreased contractility. Importantly, JP2 overexpression effectively protected from atrial dysfunction, providing a novel therapeutic rationale for atrial cardiomyopathies.

Biallelic Variants in ASNA1 , Encoding a Cytosolic Targeting Factor of Tail-Anchored Proteins, Cause Rapidly Progressive Pediatric Cardiomyopathy

Pediatric cardiomyopathies are a clinically and genetically heterogeneous group of heart muscle disorders associated with high morbidity and mortality. Although knowledge of the genetic basis of pediatric cardiomyopathy has improved considerably, the underlying cause remains elusive in a substantial proportion of cases. Exome sequencing was used to screen for the causative genetic defect in a pair of siblings with rapidly progressive dilated cardiomyopathy and death in early infancy. Protein expression was assessed in patient samples, followed by an in vitro tail-anchored protein insertion assay and functional analyses in zebrafish. We identified compound heterozygous variants in the highly conserved ASNA1 gene (arsA arsenite transporter, ATP-binding, homolog), which encodes an ATPase required for post-translational membrane insertion of tail-anchored proteins. The c.913C>T variant on the paternal allele is predicted to result in a premature stop codon p.(Gln305*), and likely explains the decreased protein expression observed in myocardial tissue and skin fibroblasts. The c.488T>C variant on the maternal allele results in a valine to alanine substitution at residue 163 (p.Val163Ala). Functional studies showed that this variant leads to protein misfolding as well as less effective tail-anchored protein insertion. Loss of asna1 in zebrafish resulted in reduced cardiac contractility and early lethality. In contrast to wild-type mRNA, injection of either mutant mRNA failed to rescue this phenotype. Biallelic variants in ASNA1 cause severe pediatric cardiomyopathy and early death. Our findings point toward a critical role of the tail-anchored membrane protein insertion pathway in vertebrate cardiac function and disease.

Msp1 Clears Mistargeted Proteins by Facilitating Their Transfer from Mitochondria to the ER.

Normal mitochondrial functions rely on optimized composition of their resident proteins, and proteins mistargeted to mitochondria need to be efficiently removed. Msp1, an AAA-ATPase in the mitochondrial outer membrane (OM), facilitates degradation of tail-anchored (TA) proteins mistargeted to the OM, yet how Msp1 cooperates with other factors to conduct this process was unclear. Here, we show that Msp1 recognizes substrate TA proteins and facilitates their transfer to the endoplasmic reticulum (ER). Doa10 in the ER membrane then ubiquitinates them with Ubc6 and Ubc7. Ubiquitinated substrates are extracted from the ER membrane by another AAA-ATPase in the cytosol, Cdc48, with Ufd1 and Npl4 for proteasomal degradation in the cytosol. Thus, Msp1 functions as an extractase that mediates clearance of mistargeted TA proteins by facilitating their transfer to the ER for protein quality control.

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

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

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.

ER import of small human presecretory proteins: components and mechanisms.

Protein transport into the mammalian endoplasmic reticulum (ER) used to be seen as strictly cotranslational, that is temporarily and mechanistically coupled to protein synthesis. In the course of the last decades, however, several classes of precursors of soluble and membrane proteins were found to be post-translationally imported into the ER, without any involvement of the ribosome. The first such class to be identified were the small presecretory proteins; tail-anchored membrane proteins followed next. In both classes, the inherent address tag is released from the translating ribosome before the initiation of ER import, as part of the fully synthesized precursor. In small presecretory proteins, the information for ER targeting and -translocation via the polypeptide-conducting Sec61-channel is encoded by a classical N-terminal signal peptide, which is released from the ribsosome before targeting due to the small size of the full-length precursor. Here, we discuss the current state of research on targeting and translocation of small presecretory proteins into the mammalian ER. In closing, we present a unifying hypothesis for ER protein translocation in terms of an energy diagram for Sec61-channel gating.

A trap mutant reveals the physiological client spectrum of TRC40.

ABSTRACTThe transmembrane recognition complex (TRC) pathway targets tail-anchored (TA) proteins to the membrane of the endoplasmic reticulum (ER). While many TA proteins are known to be able to use this pathway, it is essential for the targeting of only a few. Here, we uncover a large number of TA proteins that engage with TRC40 when other targeting machineries are fully operational. We use a dominant-negative ATPase-impaired mutant of TRC40 in which aspartate 74 was replaced by a glutamate residue to trap TA proteins in the cytoplasm. Manipulation of the hydrophobic TA-binding groove in TRC40 (also known as ASNA1) reduces interaction with most, but not all, substrates suggesting that co-purification may also reflect interactions unrelated to precursor protein targeting. We confirm known TRC40 substrates and identify many additional TA proteins interacting with TRC40. By using the trap approach in combination with quantitative mass spectrometry, we show that Golgi-resident TA proteins such as the golgins golgin-84, CASP and giantin as well as the vesicle-associated membrane-protein-associated proteins VAPA and VAPB interact with TRC40. Thus, our results provide new avenues to assess the essential role of TRC40 in metazoan organisms.This article has an associated First Person interview with the first author of the paper.