RESUMEN
Proper localization of membrane proteins is essential for the function of biological membranes and for the establishment of organelle identity within a cell. Molecular machineries that mediate membrane protein biogenesis need to not only achieve a high degree of efficiency and accuracy, but also prevent off-pathway aggregation events that can be detrimental to cells. The posttranslational targeting of tail-anchored proteins (TAs) provides tractable model systems to probe these fundamental issues. Recent advances in understanding TA-targeting pathways reveal sophisticated molecular machineries that drive and regulate these processes. These findings also suggest how an interconnected network of targeting factors, cochaperones, and quality control machineries together ensures robust membrane protein biogenesis.
Asunto(s)
Proteínas de la Membrana/metabolismo , Animales , Humanos , Proteínas de la Membrana/química , Modelos Biológicos , Señales de Clasificación de Proteína , Transporte de ProteínasRESUMEN
Approximately 40% of the mammalian proteome undergoes N-terminal methionine excision and acetylation, mediated sequentially by methionine aminopeptidase (MetAP) and N-acetyltransferase A (NatA), respectively1. Both modifications are strictly cotranslational and essential in higher eukaryotic organisms1. The interaction, activity and regulation of these enzymes on translating ribosomes are poorly understood. Here we perform biochemical, structural and in vivo studies to demonstrate that the nascent polypeptide-associated complex2,3 (NAC) orchestrates the action of these enzymes. NAC assembles a multienzyme complex with MetAP1 and NatA early during translation and pre-positions the active sites of both enzymes for timely sequential processing of the nascent protein. NAC further releases the inhibitory interactions from the NatA regulatory protein huntingtin yeast two-hybrid protein K4,5 (HYPK) to activate NatA on the ribosome, enforcing cotranslational N-terminal acetylation. Our results provide a mechanistic model for the cotranslational processing of proteins in eukaryotic cells.
RESUMEN
The signal recognition particle (SRP) and its receptor compose a universally conserved and essential cellular machinery that couples the synthesis of nascent proteins to their proper membrane localization. The past decade has witnessed an explosion in in-depth mechanistic investigations of this targeting machine at increasingly higher resolutions. In this review, we summarize recent work that elucidates how the SRP and SRP receptor interact with the cargo protein and the target membrane, respectively, and how these interactions are coupled to a novel GTPase cycle in the SRP·SRP receptor complex to provide the driving force and enhance the fidelity of this fundamental cellular pathway. We also discuss emerging frontiers in which important questions remain to be addressed.
Asunto(s)
Membrana Celular/metabolismo , GTP Fosfohidrolasas/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Animales , Proteínas Bacterianas/metabolismo , Humanos , Mamíferos , Unión Proteica , Transporte de Proteínas , Transducción de SeñalRESUMEN
The modular SCF (Skp1, cullin, and F box) ubiquitin ligases feature a large family of F box protein substrate receptors that enable recognition of diverse targets. However, how the repertoire of SCF complexes is sustained remains unclear. Real-time measurements of formation and disassembly indicate that SCF(Fbxw7) is extraordinarily stable, but, in the Nedd8-deconjugated state, the cullin-binding protein Cand1 augments its dissociation by one-million-fold. Binding and ubiquitylation assays show that Cand1 is a protein exchange factor that accelerates the rate at which Cul1-Rbx1 equilibrates with multiple F box protein-Skp1 modules. Depletion of Cand1 from cells impedes recruitment of new F box proteins to pre-existing Cul1 and profoundly alters the cellular landscape of SCF complexes. We suggest that catalyzed protein exchange may be a general feature of dynamic macromolecular machines and propose a hypothesis for how substrates, Nedd8, and Cand1 collaborate to regulate the cellular repertoire of SCF complexes.
Asunto(s)
Proteínas Ligasas SKP Cullina F-box/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Proteínas Cullin/metabolismo , Escherichia coli/genética , Proteínas F-Box/metabolismo , Humanos , Espectrometría de Masas , Proteínas Ligasas SKP Cullina F-box/químicaRESUMEN
The Hsp70 family of molecular chaperones acts as a central 'hub' in the cell that interacts with numerous newly synthesized proteins to assist in their biogenesis. Apart from its central and well-established role in facilitating protein folding, Hsp70s also act as key decision points in the cellular chaperone network that direct client proteins to distinct biogenesis and quality control pathways. In this paper, we review accumulating data that illustrate a new branch in the Hsp70 network: the post-translational targeting of nascent membrane and organellar proteins to diverse cellular organelles. Work in multiple pathways suggests that Hsp70, via its ability to interact with components of protein targeting and translocation machineries, can initiate elaborate substrate relays in a sophisticated cascade of chaperones, cochaperones, and receptor proteins, and thus provide a mechanism to safeguard and deliver nascent membrane proteins to the correct cellular membrane. We discuss the mechanistic principles gleaned from better-studied Hsp70-dependent targeting pathways and outline the observations and outstanding questions in less well-studied systems.
Asunto(s)
Proteínas HSP70 de Choque Térmico , Proteínas de la Membrana , Humanos , Proteínas de la Membrana/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Pliegue de Proteína , Proteínas Portadoras/metabolismoRESUMEN
J-domain proteins (JDPs) play essential roles in Hsp70 function by assisting Hsp70 in client trapping and regulating the Hsp70 ATPase cycle. Here, we report that JDPs can further enhance the targeting competence of Hsp70-bound client proteins during tail-anchored protein (TA) biogenesis. In the guided-entry-of-tail-anchored protein pathway in yeast, nascent TAs are captured by cytosolic Hsp70 and sequentially relayed to downstream chaperones, Sgt2 and Get3, for delivery to the ER. We found that two JDPs, Ydj1 and Sis1, function in parallel to support TA targeting to the ER in vivo. Biochemical analyses showed that, while Ydj1 and Sis1 differ in their ability to assist Hsp70 in TA trapping, both JDPs enhance the transfer of Hsp70-bound TAs to Sgt2. The ability of the JDPs to regulate the ATPase cycle of Hsp70 is essential for enhancing the transfer competence of Hsp70-bound TAs in vitro and for supporting TA insertion in vivo. These results demonstrate a role of JDPs in regulating the conformation of Hsp70-bound clients during membrane protein biogenesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/genética , Retículo Endoplásmico/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas HSP70 de Choque Térmico/genética , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Membrane proteins are aggregation-prone in aqueous environments, and their biogenesis poses acute challenges to cellular protein homeostasis. How the chaperone network effectively protects integral membrane proteins during their post-translational targeting is not well understood. Here, biochemical reconstitutions showed that the yeast cytosolic Hsp70 is responsible for capturing newly synthesized tail-anchored membrane proteins (TAs) in the soluble form. Moreover, direct interaction of Hsp70 with the cochaperone Sgt2 initiates a sequential series of TA relays to the dedicated TA targeting factor Get3. In contrast to direct loading of TAs to downstream chaperones, stepwise substrate loading via Hsp70 maintains the solubility and targeting competence of TAs, ensuring their efficient delivery to the endoplasmic reticulum (ER). Inactivation of cytosolic Hsp70 severely impairs TA translocation in vivo Our results demonstrate a new role of cytosolic Hsp70 in directly assisting the targeting of an essential class of integral membrane proteins and provide a paradigm for how "substrate funneling" through a chaperone cascade preserves the conformational quality of nascent membrane proteins during their biogenesis.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Portadoras/genética , Retículo Endoplásmico/genética , Factores de Intercambio de Guanina Nucleótido/genética , Proteínas HSP70 de Choque Térmico/genética , Proteínas de la Membrana/genética , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The nascent polypeptide exit site of the ribosome is a crowded environment where multiple ribosome-associated protein biogenesis factors (RPBs) compete for the nascent polypeptide to influence their localization, folding, or quality control. Here we address how N-terminal methionine excision (NME), a ubiquitous process crucial for the maturation of over 50% of the bacterial proteome, occurs in a timely and selective manner in this crowded environment. In bacteria, NME is mediated by 2 essential enzymes, peptide deformylase (PDF) and methionine aminopeptidase (MAP). We show that the reaction of MAP on ribosome-bound nascent chains approaches diffusion-limited rates, allowing immediate methionine excision of optimal substrates after deformylation. Specificity is achieved by kinetic competition of NME with translation elongation and by regulation from other RPBs, which selectively narrow the processing time window for suboptimal substrates. A mathematical model derived from the data accurately predicts cotranslational NME efficiency in the cytosol. Our results demonstrate how a fundamental enzymatic activity is reshaped by its associated macromolecular environment to optimize both efficiency and selectivity, and provides a platform to study other cotranslational protein biogenesis pathways.
Asunto(s)
Bacterias/genética , Proteínas Bacterianas/genética , Biosíntesis de Proteínas , Bacterias/química , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cinética , Modelos Teóricos , Péptidos/genética , Péptidos/metabolismo , Modificación Traduccional de las Proteínas , Ribosomas/química , Ribosomas/genética , Ribosomas/metabolismo , Factores de TiempoRESUMEN
The universally conserved signal recognition particle (SRP) system mediates the targeting of membrane proteins to the translocon in a multistep process controlled by GTP hydrolysis. Here we present the 2.6 Å crystal structure of the GTPase domains of the E. coli SRP protein (Ffh) and its receptor (FtsY) in complex with the tetraloop and the distal region of SRP-RNA, trapped in the activated state in presence of GDP:AlF4. The structure reveals the atomic details of FtsY recruitment and, together with biochemical experiments, pinpoints G83 as the key RNA residue that stimulates GTP hydrolysis. Insertion of G83 into the FtsY active site orients a single glutamate residue provided by Ffh (E277), triggering GTP hydrolysis and complex disassembly at the end of the targeting cycle. The complete conservation of the key residues of the SRP-RNA and the SRP protein implies that the suggested chemical mechanism of GTPase activation is applicable across all kingdoms.
Asunto(s)
Proteínas Bacterianas/genética , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , ARN/genética , Receptores Citoplasmáticos y Nucleares/genética , Partícula de Reconocimiento de Señal/genética , Partícula de Reconocimiento de Señal/metabolismo , Compuestos de Aluminio/farmacología , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Dominio Catalítico/efectos de los fármacos , Dominio Catalítico/genética , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fluoruros/farmacología , Activadores de GTP Fosfohidrolasa/farmacología , Guanosina Difosfato/genética , Guanosina Difosfato/metabolismo , Hidrólisis/efectos de los fármacos , Datos de Secuencia Molecular , ARN/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Análisis de Secuencia de ADNRESUMEN
Assembly of light-harvesting complexes requires synchronization of chlorophyll (Chl) biosynthesis with biogenesis of light-harvesting Chl a/b-binding proteins (LHCPs). The chloroplast signal recognition particle (cpSRP) pathway is responsible for transport of nucleus-encoded LHCPs in the stroma of the plastid and their integration into the thylakoid membranes. Correct folding and assembly of LHCPs require the incorporation of Chls, whose biosynthesis must therefore be precisely coordinated with membrane insertion of LHCPs. How the spatiotemporal coordination between the cpSRP machinery and Chl biosynthesis is achieved is poorly understood. In this work, we demonstrate a direct interaction between cpSRP43, the chaperone that mediates LHCP targeting and insertion, and glutamyl-tRNA reductase (GluTR), a rate-limiting enzyme in tetrapyrrole biosynthesis. Concurrent deficiency for cpSRP43 and the GluTR-binding protein (GBP) additively reduces GluTR levels, indicating that cpSRP43 and GBP act nonredundantly to stabilize GluTR. The substrate-binding domain of cpSRP43 binds to the N-terminal region of GluTR, which harbors aggregation-prone motifs, and the chaperone activity of cpSRP43 efficiently prevents aggregation of these regions. Our work thus reveals a function of cpSRP43 in Chl biosynthesis and suggests a striking mechanism for posttranslational coordination of LHCP insertion with Chl biosynthesis.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Clorofila/metabolismo , Proteínas de Cloroplastos/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Chaperonas Moleculares/metabolismo , Unión Proteica , Pliegue de Proteína , Transporte de Proteínas , Tetrapirroles/biosíntesisRESUMEN
Signal recognition particle (SRP) is a universally conserved targeting machine that mediates the targeted delivery of â¼30% of the proteome. The molecular mechanism by which eukaryotic SRP achieves efficient and selective protein targeting remains elusive. Here, we describe quantitative analyses of completely reconstituted human SRP (hSRP) and SRP receptor (SR). Enzymatic and fluorescence analyses showed that the ribosome, together with a functional signal sequence on the nascent polypeptide, are required to activate SRP for rapid recruitment of the SR, thereby delivering translating ribosomes to the endoplasmic reticulum. Single-molecule fluorescence spectroscopy combined with cross-complementation analyses reveal a sequential mechanism of activation whereby the ribosome unlocks the hSRP from an autoinhibited state and primes SRP to sample a variety of conformations. The signal sequence further preorganizes the mammalian SRP into the optimal conformation for efficient recruitment of the SR. Finally, the use of a signal sequence to activate SRP for receptor recruitment is a universally conserved feature to enable efficient and selective protein targeting, and the eukaryote-specific components confer upon the mammalian SRP the ability to sense and respond to ribosomes.
Asunto(s)
Señales de Clasificación de Proteína/fisiología , Transporte de Proteínas/fisiología , Ribosomas/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Retículo Endoplásmico/metabolismo , Escherichia coli/metabolismo , Humanos , Péptidos/metabolismo , Unión Proteica/fisiología , Biosíntesis de Proteínas/fisiología , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismoRESUMEN
Fidelity of protein targeting is essential for the proper biogenesis and functioning of organelles. Unlike replication, transcription and translation processes, in which multiple mechanisms to recognize and reject noncognate substrates are established in energetic and molecular detail, the mechanisms by which cells achieve a high fidelity in protein localization remain incompletely understood. Signal recognition particle (SRP), a conserved pathway to mediate the localization of membrane and secretory proteins to the appropriate cellular membrane, provides a paradigm to understand the molecular basis of protein localization in the cell. In this chapter, we review recent progress in deciphering the molecular mechanisms and substrate selection of the mammalian SRP pathway, with an emphasis on the key role of the cotranslational chaperone NAC in preventing protein mistargeting to the ER and in ensuring the organelle specificity of protein localization.
Asunto(s)
Retículo Endoplásmico/metabolismo , Biosíntesis de Proteínas , Animales , Humanos , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Transporte de Proteínas , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
The GTPase superfamily of proteins provides molecular switches to regulate numerous cellular processes. The 'GTPase switch' paradigm, in which external regulatory factors control the switch of a GTPase between 'on' and 'off' states, has been used to interpret the regulatory mechanism of many GTPases. However, recent work unveiled a class of nucleotide hydrolases that do not adhere to this classical paradigm. Instead, they use nucleotide-dependent dimerization cycles to regulate key cellular processes. In this review article, recent studies of dimeric GTPases and ATPases involved in intracellular protein targeting are summarized. It is suggested that these proteins can use the conformational plasticity at their dimer interface to generate multiple points of regulation, thereby providing the driving force and spatiotemporal coordination of complex cellular pathways.
Asunto(s)
Adenosina Trifosfatasas/química , Evolución Molecular , GTP Fosfohidrolasas/química , Factores de Intercambio de Guanina Nucleótido/química , Nucleotidasas/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfatasas/clasificación , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Archaea/clasificación , Archaea/genética , Archaea/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , GTP Fosfohidrolasas/clasificación , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Nucleotidasas/clasificación , Nucleotidasas/genética , Nucleotidasas/metabolismo , Filogenia , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/clasificación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Newly synthesized integral membrane proteins must traverse the aqueous cytosolic environment before arrival at their membrane destination and are prone to aggregation, misfolding, and mislocalization during this process. The biogenesis of integral membrane proteins therefore poses acute challenges to protein homeostasis within a cell and requires the action of effective molecular chaperones. Chaperones that mediate membrane protein targeting not only need to protect the nascent transmembrane domains from improper exposure in the cytosol, but also need to accurately select client proteins and actively guide their clients to the appropriate target membrane. The mechanisms by which cellular chaperones work together to coordinate this complex process are only beginning to be delineated. Here, we summarize recent advances in studies of the tail-anchored membrane protein targeting pathway, which revealed a network of chaperones, cochaperones, and targeting factors that together drive and regulate this essential process. This pathway is emerging as an excellent model system to decipher the mechanism by which molecular chaperones overcome the multiple challenges during post-translational membrane protein biogenesis and to gain insights into the functional organization of multicomponent chaperone networks.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Proteínas de la Membrana/química , Chaperonas Moleculares/química , Biosíntesis de Proteínas , Dominios Proteicos , Ribosomas/metabolismoRESUMEN
Proper localization of proteins to target membranes is a fundamental cellular process. How the nature and dynamics of the targeting complex help guide substrate proteins to the target membrane is not understood for most pathways. Here, we address this question for the conserved ATPase guided entry of tail-anchored protein 3 (Get3), which targets the essential class of tail-anchored proteins (TAs) to the endoplasmic reticulum (ER). Single-molecule fluorescence spectroscopy showed that, contrary to previous models of a static closed Get3â¢TA complex, Get3 samples open conformations on the submillisecond timescale upon TA binding, generating a fluctuating "protean clamp" that stably traps the substrate. Point mutations at the ATPase site bias Get3 toward closed conformations, uncouple TA binding from induced Get3â¢Get4/5 disassembly, and inhibit the ER targeting of the Get3â¢TA complex. These results demonstrate an essential role of substrate-induced Get3 dynamics in driving TA targeting to the membrane, and reveal a tightly coupled channel of communication between the TA-binding site, ATPase site, and effector interaction surfaces of Get3. Our results provide a precedent for large-scale dynamics in a substrate-bound chaperone, which provides an effective mechanism to retain substrate proteins with high affinity while also generating functional switches to drive vectorial cellular processes.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Factores de Intercambio de Guanina Nucleótido/química , Modelos Moleculares , Chaperonas Moleculares , Conformación Molecular , Unión Proteica , Transporte de Proteínas , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Integral membrane proteins are prone to aggregation and misfolding in aqueous environments and therefore require binding by molecular chaperones during their biogenesis. Chloroplast signal recognition particle 43 (cpSRP43) is an ATP-independent chaperone required for the biogenesis of the most abundant class of membrane proteins, the light-harvesting chlorophyll a/b-binding proteins (LHCPs). Previous work has shown that cpSRP43 specifically recognizes an L18 loop sequence conserved among LHCP paralogs. However, how cpSRP43 protects the transmembrane domains (TMDs) of LHCP from aggregation was unclear. In this work, alkylation-protection and site-specific cross-linking experiments found that cpSRP43 makes extensive contacts with all the TMDs in LHCP. Site-directed mutagenesis identified a class of cpSRP43 mutants that bind tightly to the L18 sequence but are defective in chaperoning full-length LHCP. These mutations mapped to hydrophobic surfaces on or near the bridging helix and the ß-hairpins lining the ankyrin repeat motifs of cpSRP43, suggesting that these regions are potential sites for interaction with the client TMDs. Our results suggest a working model for client protein interactions in this membrane protein chaperone.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión a Clorofila/metabolismo , Complejo de Proteína del Fotosistema II/metabolismo , Mapas de Interacción de Proteínas , Partícula de Reconocimiento de Señal/metabolismo , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Unión a Clorofila/química , Modelos Moleculares , Complejo de Proteína del Fotosistema II/química , Mutación Puntual , Agregado de Proteínas , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Partícula de Reconocimiento de Señal/química , Partícula de Reconocimiento de Señal/genéticaRESUMEN
Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP's transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Cloroplastos/química , Complejos de Proteína Captadores de Luz/metabolismo , Chaperonas Moleculares/metabolismo , Partícula de Reconocimiento de Señal/química , Secuencia de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Proteínas de Cloroplastos/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Partícula de Reconocimiento de Señal/metabolismo , Solubilidad , Relación Estructura-Actividad , Proteínas de las Membranas de los Tilacoides/metabolismo , Tilacoides/metabolismoRESUMEN
The universally conserved signal recognition particle (SRP) co-translationally delivers newly synthesized membrane and secretory proteins to the target cellular membrane. The only exception is found in the chloroplast of green plants, where the chloroplast SRP (cpSRP) post-translationally targets light-harvesting chlorophyll a/b-binding proteins (LHCP) to the thylakoid membrane. The mechanism and regulation of this post-translational mode of targeting by cpSRP remain unclear. Using biochemical and biophysical methods, here we show that anionic phospholipids activate the cpSRP receptor cpFtsY to promote rapid and stable cpSRP54·cpFtsY complex assembly. Furthermore, the stromal domain of the Alb3 translocase binds with high affinity to and regulates GTP hydrolysis in the cpSRP54·cpFtsY complex, suggesting that cpFtsY is primarily responsible for initial recruitment of the targeting complex to Alb3. These results suggest a new model for the sequential recruitment, remodeling, and unloading of the targeting complex at membrane translocase sites in the post-translational cpSRP pathway.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Unión a Clorofila/metabolismo , Complejos de Proteína Captadores de Luz/metabolismo , Fosfolípidos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Péptidos/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Aniones , Arabidopsis/crecimiento & desarrollo , Membrana Celular/metabolismo , Cloroplastos/metabolismo , Evolución Molecular , GTP Fosfohidrolasas/metabolismo , Filogenia , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
The signal recognition particle (SRP) is an essential ribonucleoprotein particle that mediates the co-translational targeting of newly synthesized proteins to cellular membranes. The SRP RNA is a universally conserved component of SRP that mediates key interactions between two GTPases in SRP and its receptor, thus enabling rapid delivery of cargo to the target membrane. Notably, this essential RNA is bypassed in the chloroplast (cp) SRP of green plants. Previously, we showed that the cpSRP and cpSRP receptor GTPases (cpSRP54 and cpFtsY, respectively) interact efficiently by themselves without the SRP RNA. Here, we explore the molecular mechanism by which this is accomplished. Fluorescence analyses showed that, in the absence of SRP RNA, the M-domain of cpSRP54 both accelerates and stabilizes complex assembly between cpSRP54 and cpFtsY. Cross-linking coupled with mass spectrometry and mutational analyses identified a new interaction between complementarily charged residues on the cpFtsY G-domain and the vicinity of the cpSRP54 M-domain. These residues are specifically conserved in plastids, and their evolution coincides with the loss of SRP RNA in green plants. These results provide an example of how proteins replace the functions of RNA during evolution.
Asunto(s)
Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Plastidios/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Secuencia de Aminoácidos , Proteínas de Cloroplastos/química , Cristalografía por Rayos X , Evolución Molecular , GTP Fosfohidrolasas/química , Filogenia , Unión Proteica , Conformación Proteica , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Partícula de Reconocimiento de Señal/químicaRESUMEN
Approximately one-third of the proteome is initially destined for the eukaryotic endoplasmic reticulum or the bacterial plasma membrane. The proper localization of these proteins is mediated by a universally conserved protein-targeting machinery, the signal recognition particle (SRP), which recognizes ribosomes carrying signal sequences and, through interactions with the SRP receptor, delivers them to the protein-translocation machinery on the target membrane. The SRP is an ancient ribonucleoprotein particle containing an essential, elongated SRP RNA for which precise functions have remained elusive. Here we used single-molecule fluorescence microscopy to show that the Escherichia coli SRP-SRP receptor GTPase complex, after initial assembly at the tetraloop end of SRP RNA, travels over 100 Å to the distal end of this RNA, where rapid GTP hydrolysis occurs. This movement is negatively regulated by the translating ribosome and, at a later stage, positively regulated by the SecYEG translocon, providing an attractive mechanism for ensuring the productive exchange of the targeting and translocation machineries at the ribosome exit site with high spatial and temporal accuracy. Our results show that large RNAs can act as molecular scaffolds that enable the easy exchange of distinct factors and precise timing of molecular events in a complex cellular process; this concept may be extended to similar phenomena in other ribonucleoprotein complexes.