RESUMEN
Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.
RESUMEN
The lipid phosphatidylinositol-3-phosphate (PI3P) is a regulator of two fundamental but distinct cellular processes, endocytosis and autophagy, so its generation needs to be under precise temporal and spatial control. PI3P is generated by two complexes that both contain the lipid kinase VPS34: complex II on endosomes (VPS34/VPS15/Beclin 1/UVRAG), and complex I on autophagosomes (VPS34/VPS15/Beclin 1/ATG14L). The endosomal GTPase Rab5 binds complex II, but the mechanism of VPS34 activation by Rab5 has remained elusive, and no GTPase is known to bind complex I. Here we show that Rab5a-GTP recruits endocytic complex II to membranes and activates it by binding between the VPS34 C2 and VPS15 WD40 domains. Electron cryotomography of complex II on Rab5a-decorated vesicles shows that the VPS34 kinase domain is released from inhibition by VPS15 and hovers over the lipid bilayer, poised for catalysis. We also show that the GTPase Rab1a, which is known to be involved in autophagy, recruits and activates the autophagy-specific complex I, but not complex II. Both Rabs bind to the same VPS34 interface but in a manner unique for each. These findings reveal how VPS34 complexes are activated on membranes by specific Rab GTPases and how they are recruited to unique cellular locations.
Asunto(s)
Membrana Celular/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/química , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteínas de Unión al GTP rab1/química , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/metabolismo , Beclina-1/química , Beclina-1/genética , Beclina-1/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/genética , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Estructura Secundaria de Proteína , Tomografía , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína de Clasificación Vacuolar VPS15/química , Proteína de Clasificación Vacuolar VPS15/genética , Proteína de Clasificación Vacuolar VPS15/metabolismo , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
Retromer is a master regulator of cargo retrieval from endosomes, which is critical for many cellular processes including signaling, immunity, neuroprotection, and virus infection. The retromer core (VPS26/VPS29/VPS35) is present on cargo-transporting, tubular carriers along with a range of sorting nexins. Here, we elucidate the structural basis of membrane tubulation and coupled cargo recognition by metazoan and fungal retromer coats assembled with the non-Bin1/Amphiphysin/Rvs (BAR) sorting nexin SNX3 using cryo-electron tomography. The retromer core retains its arched, scaffolding structure but changes its mode of membrane recruitment when assembled with different SNX adaptors, allowing cargo recognition at subunit interfaces. Thus, membrane bending and cargo incorporation can be modulated to allow retromer to traffic cargoes along different cellular transport routes.
RESUMEN
Caveolae are spherically shaped nanodomains of the plasma membrane, generated by cooperative assembly of caveolin and cavin proteins. Cavins are cytosolic peripheral membrane proteins with negatively charged intrinsically disordered regions that flank positively charged α-helical regions. Here, we show that the three disordered domains of Cavin1 are essential for caveola formation and dynamic trafficking of caveolae. Electrostatic interactions between disordered regions and α-helical regions promote liquid-liquid phase separation behaviour of Cavin1 in vitro, assembly of Cavin1 oligomers in solution, generation of membrane curvature, association with caveolin-1, and Cavin1 recruitment to caveolae in cells. Removal of the first disordered region causes irreversible gel formation in vitro and results in aberrant caveola trafficking through the endosomal system. We propose a model for caveola assembly whereby fuzzy electrostatic interactions between Cavin1 and caveolin-1 proteins, combined with membrane lipid interactions, are required to generate membrane curvature and a metastable caveola coat.
Asunto(s)
Caveolas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Caveolas/química , Caveolina 1/genética , Caveolina 1/metabolismo , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de la Membrana/genética , Ratones , Dominios Proteicos , Proteínas de Unión al ARN/genética , Electricidad EstáticaRESUMEN
Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P 2 (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 ß2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.
RESUMEN
Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes1-3. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders4-7. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.
Asunto(s)
Chaetomium/química , Chaetomium/ultraestructura , Microscopía por Crioelectrón , Tomografía con Microscopio Electrónico , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/ultraestructura , Chaetomium/metabolismo , Chlamydomonas reinhardtii/citología , Chlamydomonas reinhardtii/ultraestructura , Humanos , Modelos Moleculares , Unión Proteica , Transporte de Proteínas , Nexinas de Clasificación/química , Nexinas de Clasificación/metabolismo , Nexinas de Clasificación/ultraestructura , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Caveolae are plasma membrane invaginations involved in transport, signalling and mechanical membrane sensing in metazoans. Their formation depends upon multiple interactions between membrane-embedded caveolins, lipids and cytosolic cavin proteins. Of the four cavin family members, only cavin1 is strictly required for caveola formation. Here, we demonstrate that an eleven residue (undecad) repeat sequence (UC1) exclusive to cavin1 is essential for caveolar localization and promotes membrane remodelling through binding to phosphatidylserine. In the notochord of mechanically stimulated zebrafish embryos, the UC1 domain is required for caveolar stability and resistance to membrane stress. The number of undecad repeats in the cavin1 UC1 domain varies throughout evolution, and we find that an increased number also correlates with increased caveolar stability. Lastly, we show that the cavin1 UC1 domain induces dramatic remodelling of the plasma membrane when grafted into cavin2 suggesting an important role in membrane sculpting. Overall, our work defines a novel conserved cavin1 modular domain that controls caveolar assembly and stability.
Asunto(s)
Caveolas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Análisis Mutacional de ADN , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Notocorda/metabolismo , Células PC-3 , Proteínas de Unión a Fosfato , Proteínas de Unión al ARN/química , Estrés Mecánico , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Caveolae are abundant surface organelles implicated in a range of cellular processes. Two classes of proteins work together to generate caveolae: integral membrane proteins termed caveolins and cytoplasmic coat proteins called cavins. Caveolae respond to membrane stress by releasing cavins into the cytosol. A crucial aspect of this model is tight regulation of cytosolic pools of cavin under resting conditions. We now show that a recently identified region of cavin1 that can bind phosphoinositide (PI) lipids is also a major site of ubiquitylation. Ubiquitylation of lysines within this site leads to rapid proteasomal degradation. In cells that lack caveolins and caveolae, cavin1 is cytosolic and rapidly degraded as compared with cells in which cavin1 is associated with caveolae. Membrane stretching causes caveolar disassembly, release of cavin complexes into the cytosol, and increased proteasomal degradation of wild-type cavin1 but not mutant cavin1 lacking the major ubiquitylation site. Release of cavin1 from caveolae thus leads to exposure of key lysine residues in the PI-binding region, acting as a trigger for cavin1 ubiquitylation and down-regulation. This mutually exclusive PI-binding/ubiquitylation mechanism may help maintain low levels of cytosolic cavin1 in resting cells, a prerequisite for cavins acting as signaling modules following release from caveolae.
Asunto(s)
Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Sitios de Unión , Caveolas/metabolismo , Caveolina 1/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Perros , Humanos , Células MCF-7 , Células de Riñón Canino Madin Darby , Proteínas de la Membrana/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/genética , Transducción de Señal , UbiquitinaciónRESUMEN
Caveolae are an abundant feature of the plasma membrane in many cells. Until recently, they were generally considered to be membrane invaginations whose formation primarily driven by integral membrane proteins called caveolins. However, the past decade has seen the emergence of the cavin family of peripheral membrane proteins as essential coat components and regulators of caveola biogenesis. In this Commentary, we summarise recent data on the role of cavins in caveola formation, highlighting structural studies that provide new insights into cavin coat assembly. In mammals, there are four cavin family members that associate through homo- and hetero-oligomerisation to form distinct subcomplexes on caveolae, which can be released into the cell in response to stimuli. Studies from several labs have provided a better understanding of cavin stoichiometry and the molecular basis for their oligomerisation, as well as identifying interactions with membrane phospholipids that may be important for caveola function. We propose a model in which coincident, low-affinity electrostatically controlled protein-protein and protein-lipid interactions allow the formation of caveolae, generating a meta-stable structure that can respond to plasma membrane stress by release of cavins.
Asunto(s)
Caveolas/metabolismo , Familia de Multigenes , Proteínas de Unión al ARN/metabolismo , Animales , Caveolas/química , Humanos , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genéticaRESUMEN
Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo.
Asunto(s)
Caveolas/química , Caveolas/metabolismo , Caveolinas/química , Caveolinas/metabolismo , Citoplasma/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Caveolas/ultraestructura , Cristalografía por Rayos X , Citoplasma/química , Citoplasma/ultraestructura , Proteínas de la Membrana/metabolismo , Microscopía Electrónica , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Transducción de Señal/fisiología , Pez Cebra/metabolismoRESUMEN
Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Fosfatos de Fosfatidilinositol/química , Proteínas Recombinantes de Fusión/química , Nexinas de Clasificación/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Movimiento Celular , Secuencia Conservada , Cristalografía por Rayos X , Endosomas/química , Endosomas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Unión al GTP/genética , Expresión Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transducción de Señal , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
We isolated a novel, atypical long-chain three-finger toxin (TFT), α-elapitoxin-Dpp2d (α-EPTX-Dpp2d), from black mamba (Dendroaspis polylepis polylepis) venom. Proteolytic digestion with trypsin and V8 protease, together with MS/MS de novo sequencing, indicated that the mature toxin has an amidated C-terminal arginine, a posttranslational modification rarely observed for snake TFTs. α-EPTX-Dpp2d was found to potently inhibit α7 neuronal nicotinic acetylcholine receptors (nAChR; IC50, 58 ± 24 nM) and muscle-type nAChR (IC50, 114 ± 37 nM) but did not affect α3ß2 and α3ß4 nAChR isoforms at 1 µM concentrations. Competitive radioligand binding assays demonstrated that α-EPTX-Dpp2d competes with epibatidine binding to the Lymnea stagnalis acetylcholine-binding protein (Ls-AChBP; IC50, 4.9 ± 2.3 nM). The activity profile and binding data are reminiscent of classical long-chain TFTs with a free carboxyl termini, suggesting that amidation does not significantly affect toxin selectivity. The crystal structure of α-EPTX-Dpp2d was determined at 1.7 Å resolution and displayed a dimeric toxin assembly with each monomer positioned in an antiparallel orientation. The dimeric structure is stabilized by extensive intermolecular hydrogen bonds and electrostatic interactions, which raised the possibility that the toxin may exist as a noncovalent homodimer in solution. However, chemical cross-linking and size-exclusion chromatography coupled with multiangle laser light scattering (MALLS) data indicated that the toxin is predominantly monomeric under physiological conditions. Because of its high potency and selectivity, we expect this toxin to be a valuable pharmacological tool for studying the structure and function of nAChRs.
Asunto(s)
Venenos Elapídicos/química , Elapidae/metabolismo , Neurotoxinas/farmacología , Antagonistas Nicotínicos/farmacología , Procesamiento Proteico-Postraduccional , Proteínas de Reptiles/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Unión Competitiva , Señalización del Calcio/efectos de los fármacos , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Venenos Elapídicos/aislamiento & purificación , Venenos Elapídicos/metabolismo , Venenos Elapídicos/farmacología , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/química , Neurotoxinas/aislamiento & purificación , Neurotoxinas/metabolismo , Agonistas Nicotínicos/química , Agonistas Nicotínicos/farmacología , Antagonistas Nicotínicos/química , Antagonistas Nicotínicos/aislamiento & purificación , Antagonistas Nicotínicos/metabolismo , Conformación Proteica , Estabilidad Proteica , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Reptiles/química , Proteínas de Reptiles/aislamiento & purificación , Proteínas de Reptiles/metabolismo , Alineación de Secuencia , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
The retromer is a trimeric cargo-recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinson's disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromer's subcellular localization and its capacity to sort cargo. We show that expression of the PD-linked Vps35 D620N mutant redistributes retromer-positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild-type Vps35. While PD-linked point mutant Vps35 D620N interacts with the cation-independent mannose-6-phosphate receptor (CI-M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI-M6PR ligand and protease responsible for degradation of α-synuclein, a causative agent of PD. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.
Asunto(s)
Mutación Missense , Enfermedad de Parkinson/genética , Proteínas de Transporte Vesicular/metabolismo , Anciano , Catepsina D/metabolismo , Línea Celular Tumoral , Células Cultivadas , Endosomas/metabolismo , Células HEK293 , Humanos , Masculino , Enfermedad de Parkinson/metabolismo , Unión Proteica , Transporte de Proteínas , Receptor IGF Tipo 2/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Biochemical and structural analysis of macromolecular protein assemblies remains challenging due to technical difficulties in recombinant expression, engineering and reconstitution of multisubunit complexes. Here we use a recently developed cell-free protein expression system based on the protozoan Leishmania tarentolae to produce in vitro all six subunits of the 600 kDa HOPS and CORVET membrane tethering complexes. We demonstrate that both subcomplexes and the entire HOPS complex can be reconstituted in vitro resulting in a comprehensive subunit interaction map. To our knowledge this is the largest eukaryotic protein complex in vitro reconstituted to date. Using the truncation and interaction analysis, we demonstrate that the complex is assembled through short hydrophobic sequences located in the C-terminus of the individual Vps subunits. Based on this data we propose a model of the HOPS and CORVET complex assembly that reconciles the available biochemical and structural data.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Secuencia de Aminoácidos , Leishmania/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae. DOI: http://dx.doi.org/10.7554/eLife.01434.001.
Asunto(s)
Proteínas Portadoras/análisis , Caveolas/química , Péptidos y Proteínas de Señalización Intracelular/análisis , Multimerización de Proteína , Proteínas de Unión al ARN/análisis , Animales , Línea Celular , Microscopía Inmunoelectrónica , Imagen Molecular , Imagen Óptica , Proteínas de Unión a Fosfato , Unión ProteicaRESUMEN
Cell-free protein expression is an important tool for a rapid production, engineering and labeling of recombinant proteins. However the complex protocols for preparation of eukaryotic cell-free protein expression systems result in high manufacturing costs and limit their utility. Recently we reported a novel cell-free expression system based on the lysate of a fermentable protozoan Leishmania tarentolae. Herein we describe a protocol for high throughput protein expression using Leishmania cell-free lysate. The protocol combines PCR-based synthesis and engineering of translation templates with a combined transcription-translation system. The protocol is adapted to multiwell plate format and allows translation of large protein libraries. In the presented example we translate in vitro and isolate a nearly complete complement of mammalian Rab GTPases. Further applications and developments of the system are discussed.
Asunto(s)
Sistema Libre de Células/metabolismo , Ensayos Analíticos de Alto Rendimiento , Leishmania/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas de Unión al GTP rab/biosíntesis , Animales , Sistema Libre de Células/química , Expresión Génica , Humanos , Leishmania/química , Leishmania/genética , Mamíferos , Biblioteca de Péptidos , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Proteómica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/aislamiento & purificaciónRESUMEN
The green fluorescent protein (GFP)-nanobody is a single-chain VHH antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X-ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP-nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP-nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP-nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP-nanobody.
Asunto(s)
Anticuerpos/química , Anticuerpos/metabolismo , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteínas Luminiscentes/química , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , TermodinámicaRESUMEN
The adaptation of organisms to a parasitic life style is often accompanied by the emergence of novel biochemical pathways absent in free-living organisms. As a result, the genomes of specialized parasitic organisms often code for a large number (>50%) of proteins with no detectable homology or predictable function. Although understanding the biochemical properties of these proteins and their roles in parasite biogenesis is the next challenge of molecular parasitology, analysis tools developed for free-living organisms are often inadequate for this purpose. Here we attempt to solve some of these problems by developing a methodology for the rapid production of expressed proteomes in cell-free systems based on parasitic organisms. To do so we take advantage of Species Independent Translational Sequences (SITS), which can efficiently mediate translation initiation in any organism. Using these sequences we developed a single-tube in vitro translation system based on the parasitic protozoan Leishmania tarentolae. We demonstrate that the system can be primed directly with SITS containing templates constructed by overlap extension PCR. To test the systems we simultaneously amplified 31 of L. tarentolae's putative translation initiation factors and phosphatases directly from the genomic DNA and subjected them to expression, purification and activity analysis. All of the amplified products produced soluble recombinant proteins, and putative phosphatases could be purified to at least 50% purity in one step. We further compared the ability of L. tarentolae and E. coli based cell-free systems to express a set of mammalian, L. tarentolae and Plasmodium falciparum Rab GTPases in functional form. We demonstrate that the L. tarentolae cell-free system consistently produced higher quality proteins than E. coli-based system. The differences were particularly pronounced in the case of open reading frames derived from P. falciparum. The implications of these developments are discussed.
Asunto(s)
Leishmania/metabolismo , Biología Molecular/métodos , Proteoma , Proteómica/métodos , Proteínas Protozoarias/genética , Sistema Libre de Células/metabolismo , Clonación Molecular , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Modelos Genéticos , Plasmodium falciparum/enzimología , Reacción en Cadena de la Polimerasa/métodos , Pliegue de Proteína , Proteínas Protozoarias/química , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rab/metabolismoRESUMEN
Cell-free protein synthesis enables the rapid production and engineering of recombinant proteins. Existing cell-free systems differ substantially from each other with respect to efficiency, scalability and the ability to produce functional eukaryotic proteins. Here we describe species-independent translational sequences (SITS) that mediate efficient cell-free protein synthesis in multiple prokaryotic and eukaryotic systems, presumably through bypassing the early translation initiation factors. We use these leaders in combination with targeted suppression of the endogenous Leishmania tarentolae mRNAs to create a cell-free system based on this protozoan. The system can be directly programmed with unpurified PCR products, enabling rapid generation of large protein libraries and protein variants. L. tarentolae extract can produce up to 300 microg/ml of recombinant protein in 2 h. We further demonstrate that protein-protein and protein-small molecule interactions can be quantitatively analyzed directly in the translation mixtures using fluorescent (cross-) correlation spectroscopy.