RESUMEN
In cells, microtubules (MTs) assemble from α/ß-tubulin subunits at nucleation sites containing the γ-tubulin ring complex (γ-TuRC). Within the γ-TuRC, exposed γ-tubulin molecules act as templates for MT assembly by interacting with α/ß-tubulin. The vertebrate γ-TuRC is scaffolded by γ-tubulin-interacting proteins GCP2-6 arranged in a specific order. Interestingly, the γ-tubulin molecules in the γ-TuRC deviate from the cylindrical geometry of MTs, raising the question of how the γ-TuRC structure changes during MT nucleation. Recent studies on the structure of the vertebrate γ-TuRC attached to the end of MTs came to varying conclusions. In vitro assembly of MTs, facilitated by an α-tubulin mutant, resulted in a closed, cylindrical γ-TuRC showing canonical interactions between all γ-tubulin molecules and α/ß-tubulin subunits. Conversely, native MTs formed in a frog extract were capped by a partially closed γ-TuRC, with some γ-tubulin molecules failing to align with α/ß-tubulin. This review discusses these outcomes, along with the broader implications.
RESUMEN
The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/ß-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Microtúbulos , Tubulina (Proteína) , Xenopus laevis , Proteína de Unión al GTP ran , Animales , Microscopía por Crioelectrón , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/metabolismo , Proteína de Unión al GTP ran/metabolismo , Proteína de Unión al GTP ran/genética , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genéticaRESUMEN
Cellular functionality relies on a well-balanced, but highly dynamic proteome. Dysfunction of mitochondrial protein import leads to the cytosolic accumulation of mitochondrial precursor proteins which compromise cellular proteostasis and trigger a mitoprotein-induced stress response. To dissect the effects of mitochondrial dysfunction on the cellular proteome as a whole, we developed pre-post thermal proteome profiling. This multiplexed time-resolved proteome-wide thermal stability profiling approach with isobaric peptide tags in combination with a pulsed SILAC labelling elucidated dynamic proteostasis changes in several dimensions: In addition to adaptations in protein abundance, we observed rapid modulations of the thermal stability of individual cellular proteins. Different functional groups of proteins showed characteristic response patterns and reacted with group-specific kinetics, allowing the identification of functional modules that are relevant for mitoprotein-induced stress. Thus, our new pre-post thermal proteome profiling approach uncovered a complex response network that orchestrates proteome homeostasis in eukaryotic cells by time-controlled adaptations of the abundance and the conformation of proteins.
Asunto(s)
Proteoma , Proteostasis , Proteoma/metabolismo , Péptidos , Proteínas Mitocondriales/metabolismo , Mitocondrias/metabolismoRESUMEN
In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is hampered by the lack of structural information on the augmin complex. Here, we elucidate the molecular architecture and conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.
Asunto(s)
Huso Acromático , Tubulina (Proteína) , Proteínas Asociadas a Microtúbulos/química , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Ribosome-associated quality-control (RQC) surveys incomplete nascent polypeptides produced by interrupted translation. Central players in RQC are the human ribosome- and tRNA-binding protein, NEMF, and its orthologs, yeast Rqc2 and bacterial RqcH, which sense large ribosomal subunits obstructed with nascent chains and then promote nascent-chain proteolysis. In canonical eukaryotic RQC, NEMF stabilizes the LTN1/Listerin E3 ligase binding to obstructed ribosomal subunits for nascent-chain ubiquitylation. Furthermore, NEMF orthologs across evolution modify nascent chains by mediating C-terminal, untemplated polypeptide elongation. In eukaryotes, this process exposes ribosome-buried nascent-chain lysines, the ubiquitin acceptor sites, to LTN1. Remarkably, in both bacteria and eukaryotes, C-terminal tails also have an extra-ribosomal function as degrons. Here, we discuss recent findings on RQC mechanisms and briefly review how ribosomal stalling is sensed upstream of RQC, including via ribosome collisions, from an evolutionary perspective. Because RQC defects impair cellular fitness and cause neurodegeneration, this knowledge provides a framework for pathway-related biology and disease studies.
Asunto(s)
Ribosomas , Proteínas de Saccharomyces cerevisiae , Bacterias/genética , Bacterias/metabolismo , Humanos , Péptidos/metabolismo , Biosíntesis de Proteínas , Ribosomas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Ribosome stalling during translation is detrimental to cellular fitness, but how this is sensed and elicits recycling of ribosomal subunits and quality control of associated mRNA and incomplete nascent chains is poorly understood1,2. Here we uncover Bacillus subtilis MutS2, a member of the conserved MutS family of ATPases that function in DNA mismatch repair3, as an unexpected ribosome-binding protein with an essential function in translational quality control. Cryo-electron microscopy analysis of affinity-purified native complexes shows that MutS2 functions in sensing collisions between stalled and translating ribosomes and suggests how ribosome collisions can serve as platforms to deploy downstream processes: MutS2 has an RNA endonuclease small MutS-related (SMR) domain, as well as an ATPase/clamp domain that is properly positioned to promote ribosomal subunit dissociation, which is a requirement both for ribosome recycling and for initiation of ribosome-associated protein quality control (RQC). Accordingly, MutS2 promotes nascent chain modification with alanine-tail degrons-an early step in RQC-in an ATPase domain-dependent manner. The relevance of these observations is underscored by evidence of strong co-occurrence of MutS2 and RQC genes across bacterial phyla. Overall, the findings demonstrate a deeply conserved role for ribosome collisions in mounting a complex response to the interruption of translation within open reading frames.
Asunto(s)
Adenosina Trifosfatasas , Ribosomas , Adenosina Trifosfatasas/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Microscopía por Crioelectrón , Reparación del ADN , Biosíntesis de Proteínas , Proteínas/metabolismo , Ribosomas/metabolismoRESUMEN
The gamma-tubulin ring complex (γ-TuRC) is the principal microtubule nucleation template in vertebrates. Recent cryo-EM reconstructions visualized the intricate quaternary structure of the γ-TuRC, containing more than thirty subunits, raising fundamental questions about γ-TuRC assembly and the role of actin as an integral part of the complex. Here, we reveal the structural mechanism underlying modular γ-TuRC assembly and identify a functional role of actin in microtubule nucleation. During γ-TuRC assembly, a GCP6-stabilized core comprising GCP2-3-4-5-4-6 is expanded by stepwise recruitment, selective stabilization and conformational locking of four pre-formed GCP2-GCP3 units. Formation of the lumenal bridge specifies incorporation of the terminal GCP2-GCP3 unit and thereby leads to closure of the γ-TuRC ring in a left-handed spiral configuration. Actin incorporation into the complex is not relevant for γ-TuRC assembly and structural integrity, but determines γ-TuRC geometry and is required for efficient microtubule nucleation and mitotic chromosome alignment in vivo.
Asunto(s)
Actinas/química , Microscopía por Crioelectrón/métodos , Proteínas Asociadas a Microtúbulos/química , Centro Organizador de los Microtúbulos/química , Microtúbulos/química , Tubulina (Proteína)/química , Actinas/metabolismo , Línea Celular , Humanos , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Proteínas Asociadas a Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Mitochondria are the powerhouse of eukaryotic cells. They possess their own gene expression machineries where highly divergent and specialized ribosomes, named hereafter mitoribosomes, translate the few essential messenger RNAs still encoded by mitochondrial genomes. Here, we present a biochemical and structural characterization of the mitoribosome in the model green alga Chlamydomonas reinhardtii, as well as a functional study of some of its specific components. Single particle cryo-electron microscopy resolves how the Chlamydomonas mitoribosome is assembled from 13 rRNA fragments encoded by separate non-contiguous gene pieces. Additional proteins, mainly OPR, PPR and mTERF helical repeat proteins, are found in Chlamydomonas mitoribosome, revealing the structure of an OPR protein in complex with its RNA binding partner. Targeted amiRNA silencing indicates that these ribosomal proteins are required for mitoribosome integrity. Finally, we use cryo-electron tomography to show that Chlamydomonas mitoribosomes are attached to the inner mitochondrial membrane via two contact points mediated by Chlamydomonas-specific proteins. Our study expands our understanding of mitoribosome diversity and the various strategies these specialized molecular machines adopt for membrane tethering.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Mitocondrias/metabolismo , ARN/metabolismo , Ribosomas/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/ultraestructura , Microscopía por Crioelectrón , Mitocondrias/química , Mitocondrias/genética , Mitocondrias/ultraestructura , Proteínas Mitocondriales , Ribosomas Mitocondriales/química , Ribosomas Mitocondriales/metabolismo , Ribosomas Mitocondriales/ultraestructura , ARN/química , ARN/genética , ARN/ultraestructura , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Proteínas Ribosómicas/ultraestructura , Ribosomas/química , Ribosomas/genética , Ribosomas/ultraestructuraRESUMEN
Cryogenic electron tomography (cryo-ET) visualizes the 3D spatial distribution of macromolecules at nanometer resolution inside native cells. However, automated identification of macromolecules inside cellular tomograms is challenged by noise and reconstruction artifacts, as well as the presence of many molecular species in the crowded volumes. Here, we present DeepFinder, a computational procedure that uses artificial neural networks to simultaneously localize multiple classes of macromolecules. Once trained, the inference stage of DeepFinder is faster than template matching and performs better than other competitive deep learning methods at identifying macromolecules of various sizes in both synthetic and experimental datasets. On cellular cryo-ET data, DeepFinder localized membrane-bound and cytosolic ribosomes (roughly 3.2 MDa), ribulose 1,5-bisphosphate carboxylase-oxygenase (roughly 560 kDa soluble complex) and photosystem II (roughly 550 kDa membrane complex) with an accuracy comparable to expert-supervised ground truth annotations. DeepFinder is therefore a promising algorithm for the semiautomated analysis of a wide range of molecular targets in cellular tomograms.
Asunto(s)
Algoritmos , Microscopía por Crioelectrón/métodos , Aprendizaje Profundo , Tomografía con Microscopio Electrónico/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Sustancias Macromoleculares/química , Redes Neurales de la Computación , Chlamydomonas reinhardtii/metabolismo , Complejo de Proteína del Fotosistema II/química , Ribosomas/química , Ribulosa-Bifosfato Carboxilasa/químicaRESUMEN
Microtubules are protein cylinders with functions in cell motility, signal sensing, cell organization, intracellular transport, and chromosome segregation. One of the key properties of microtubules is their dynamic architecture, allowing them to grow and shrink in length by adding or removing copies of their basic subunit, the heterodimer αß-tubulin. In higher eukaryotes, de novo assembly of microtubules from αß-tubulin is initiated by a 2 MDa multi-subunit complex, the gamma-tubulin ring complex (γ-TuRC). For many years, the structure of the γ-TuRC and the function of its subunits remained enigmatic, although structural data from the much simpler yeast counterpart, the γ-tubulin small complex (γ-TuSC), were available. Two recent breakthroughs in the field, high-resolution structural analysis and recombinant reconstitution of the complex, have revolutionized our knowledge about the architecture and function of the γ-TuRC and will form the basis for addressing outstanding questions about biogenesis and regulation of this essential microtubule organizer.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Tubulina (Proteína) , Animales , Centro Organizador de los Microtúbulos , Microtúbulos , Tubulina (Proteína)/genética , VertebradosRESUMEN
Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.
Asunto(s)
Tubulina (Proteína)/química , Animales , Humanos , Microtúbulos/química , Microtúbulos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Células Sf9 , Imagen Individual de Molécula , Spodoptera , Porcinos , Tubulina (Proteína)/metabolismo , XenopusRESUMEN
The nucleation of microtubules from αß-tubulin dimers is an essential cellular process dependent on γ-tubulin complexes. Mechanistic understanding of the nucleation reaction was hampered by the lack of γ-tubulin complex structures at sufficiently high resolution. The recent technical developments in cryo-electron microscopy have allowed resolving the vertebrate γ-tubulin ring complex (γ-TuRC) structure at near-atomic resolution. These studies clarified the arrangement and stoichiometry of gamma-tubulin complex proteins in the γ-TuRC, characterized the surprisingly versatile integration of the small proteins MZT1/2 into the complex, and identified actin as an integral component of the γ-TuRC. In this review, we summarize the structural insights into the molecular architecture, the assembly pathway, and the regulation of the microtubule nucleation reaction.
Asunto(s)
Proteínas Asociadas a Microtúbulos , Tubulina (Proteína) , Microscopía por Crioelectrón , Centro Organizador de los Microtúbulos , MicrotúbulosRESUMEN
Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Extensión de la Cadena Peptídica de Translación , ARN Bacteriano/metabolismo , ARN de Transferencia de Alanina/metabolismo , Bacillus subtilis/ultraestructura , Proteínas Bacterianas/genética , Microscopía por Crioelectrón , ARN Bacteriano/genética , ARN de Transferencia de Alanina/genéticaRESUMEN
Microtubules are essential cytoskeletal elements assembled from αß-tubulin dimers. In high eukaryotes, microtubule nucleation, the de novo assembly of a microtubule from its minus end, is initiated by the γ-tubulin ring complex (γ-TuRC). Despite many years of research, the structural and mechanistic principles of the microtubule nucleation machinery remained poorly understood. Only recently, cryoelectron microscopy studies uncovered the molecular organization and potential activation mechanisms of γ-TuRC. In vitro assays further deciphered the spatial and temporal cooperation between γ-TuRC and additional factors, for example, the augmin complex, the phase separation protein TPX2, and the microtubule polymerase XMAP215. These breakthroughs deepen our understanding of microtubule nucleation mechanisms and will link the assembly of individual microtubules to the organization of cellular microtubule networks.
Asunto(s)
Centro Organizador de los Microtúbulos/química , Microtúbulos/química , Tubulina (Proteína)/química , Animales , Microscopía por Crioelectrón , Humanos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructura , Centro Organizador de los Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Polimerizacion , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestructuraRESUMEN
The nucleation of microtubules from αß-tubulin subunits is mediated by γ-tubulin complexes, which vary in composition across organisms. Aiming to understand how de novo microtubule formation is achieved and regulated by a minimal microtubule nucleation system, we here determined the cryo-electron microscopy structure of the heterotetrameric γ-tubulin small complex (γ-TuSC) from C. albicans at near-atomic resolution. Compared to the vertebrate γ-tubulin ring complex (γ-TuRC), we observed a vastly remodeled interface between the SPC/GCP-γ-tubulin spokes, which stabilizes the complex and defines the γ-tubulin arrangement. The relative positioning of γ-tubulin subunits indicates that a conformational rearrangement of the complex is required for microtubule nucleation activity, which follows opposing directionality as predicted for the vertebrate γ-TuRC. Collectively, our data suggest that the assembly and regulation mechanisms of γ-tubulin complexes fundamentally differ between the microtubule nucleation systems in lower and higher eukaryotes.
Asunto(s)
Candida albicans/metabolismo , Microtúbulos/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Tubulina (Proteína)/química , Candida albicans/química , Microscopía por Crioelectrón , Evolución Molecular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Guanosina Difosfato/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Modelos Moleculares , Complejos Multiproteicos/genética , Mutación , Conformación ProteicaRESUMEN
Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.
Asunto(s)
Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Complejos Multiproteicos/metabolismo , Línea Celular , Secuencia Conservada , Evolución Molecular , Humanos , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Fosfatidilinositoles/metabolismo , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.
Asunto(s)
Estrés Oxidativo , Ribosomas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Microscopía por Crioelectrón , Regulación Fúngica de la Expresión Génica , Modelos Moleculares , MutaciónRESUMEN
In mammalian cells, one-third of all polypeptides is transported into or through the ER-membrane via the Sec61-channel. While the Sec61-complex facilitates the transport of all polypeptides with amino-terminal signal peptides (SP) or SP-equivalent transmembrane helices (TMH), the translocating chain-associated membrane protein (now termed TRAM1) was proposed to support transport of a subset of precursors. To identify possible determinants of TRAM1 substrate specificity, we systematically identified TRAM1-dependent precursors by analyzing cellular protein abundance changes upon TRAM1 depletion in HeLa cells using quantitative label-free proteomics. In contrast to previous analysis after TRAP depletion, SP and TMH analysis of TRAM1 clients did not reveal any distinguishing features that could explain its putative substrate specificity. To further address the TRAM1 mechanism, live-cell calcium imaging was carried out after TRAM1 depletion in HeLa cells. In additional contrast to previous analysis after TRAP depletion, TRAM1 depletion did not affect calcium leakage from the ER. Thus, TRAM1 does not appear to act as SP- or TMH-receptor on the ER-membrane's cytosolic face and does not appear to affect the open probability of the Sec61-channel. It may rather play a supportive role in protein transport, such as making the phospholipid bilayer conducive for accepting SP and TMH in the vicinity of the lateral gate of the Sec61-channel.Abbreviations: ER, endoplasmic reticulum; OST, oligosaccharyltransferase; RAMP, ribosome-associated membrane protein; SP, signal peptide; SR, SRP-receptor; SRP, signal recognition particle; TMH, signal peptide-equivalent transmembrane helix; TRAM, translocating chain-associated membrane protein; TRAP, translocon-associated protein.
Asunto(s)
Retículo Endoplásmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Fosfolípidos/metabolismo , Canales de Translocación SEC/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Membrana Dobles de Lípidos/química , Glicoproteínas de Membrana/genética , Proteínas de Transporte de Membrana/genética , Transporte de Proteínas/fisiología , ProteómicaRESUMEN
Human Ebp1 is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp1 shares the methionine aminopeptidase (MetAP) fold and binds to mature 80S ribosomes for translational control. Here, we present a cryo-EM single particle analysis reconstruction of Ebp1 bound to non-translating human 80S ribosomes at a resolution range from 3.3 to ~8 Å. Ebp1 blocks the tunnel exit with major interactions to the general uL23/uL29 docking site for nascent chain-associated factors complemented by eukaryote-specific eL19 and rRNA helix H59. H59 is defined as dynamic adaptor undergoing significant remodeling upon Ebp1 binding. Ebp1 recruits rRNA expansion segment ES27L to the tunnel exit via specific interactions with rRNA consensus sequences. The Ebp1-ribosome complex serves as a template for MetAP binding and provides insights into the structural principles for spatial coordination of co-translational events and molecular triage at the ribosomal tunnel exit.