RESUMEN
Retromer is a multi-protein complex that recycles transmembrane cargo from endosomes to the trans-Golgi network and the plasma membrane. Defects in retromer impair various cellular processes and underlie some forms of Alzheimer's disease and Parkinson's disease. Although retromer was discovered over 15 years ago, the mechanisms for cargo recognition and recruitment to endosomes have remained elusive. Here, we present an X-ray crystallographic analysis of a four-component complex comprising the VPS26 and VPS35 subunits of retromer, the sorting nexin SNX3, and a recycling signal from the divalent cation transporter DMT1-II. This analysis identifies a binding site for canonical recycling signals at the interface between VPS26 and SNX3. In addition, the structure highlights a network of cooperative interactions among the VPS subunits, SNX3, and cargo that couple signal-recognition to membrane recruitment.
Asunto(s)
Proteínas de Transporte de Catión/química , Complejos Multiproteicos/química , Nexinas de Clasificación/química , Proteínas de Transporte Vesicular/química , Secuencia de Aminoácidos , Proteínas de Transporte de Catión/metabolismo , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Dispersión del Ángulo Pequeño , Nexinas de Clasificación/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The activity of proteins is thought to be invariably determined by their amino acid sequence or composition, but we show that a long segment of a viral protein can support infection independent of its sequence or composition. During virus entry, the papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm to bind cellular factors such as retromer required for intracellular virus trafficking. Here, we show that an ~110 amino acid segment of L2 is predicted to be disordered and that large deletions in this segment abolish infectivity of HPV16 pseudoviruses by inhibiting cytoplasmic protrusion of L2, association with retromer, and proper virus trafficking. The activity of these mutants can be restored by insertion of protein segments with diverse sequences, compositions, and chemical properties, including scrambled amino acid sequences, a tandem array of a short sequence, and the intrinsically disordered region of an unrelated cellular protein. The infectivity of mutants with small in-frame deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or composition, determines its activity during HPV16 pseudovirus infection. We propose that a minimal length of L2 is required for it to protrude far enough into the cytoplasm to bind cytoplasmic trafficking factors, but the sequence of this segment is largely irrelevant. Thus, protein segments can carry out complex biological functions such as Human papillomavirus pseudovirus infection in a sequence-independent manner. This finding has important implications for protein function and evolution.
Asunto(s)
Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Humanos , Proteínas de la Cápside/química , Virus del Papiloma Humano , Internalización del Virus , Células HeLa , Cápside/metabolismo , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/químicaRESUMEN
AMPylation, the post-translational modification with adenosine monophosphate (AMP), is catalyzed by effector proteins from a variety of pathogens. Legionella pneumophila is thus far the only known pathogen that, in addition to encoding an AMPylase (SidM/DrrA), also encodes a deAMPylase, called SidD, that reverses SidM-mediated AMPylation of the vesicle transport GTPase Rab1. DeAMPylation is catalyzed by the N-terminal phosphatase-like domain of SidD. Here, we determined the crystal structure of full length SidD including the uncharacterized C-terminal domain (CTD). A flexible loop rich in aromatic residues within the CTD was required to target SidD to model membranes in vitro and to the Golgi apparatus within mammalian cells. Deletion of the loop (Δloop) or substitution of its aromatic phenylalanine residues rendered SidD cytosolic, showing that the hydrophobic loop is the primary membrane-targeting determinant of SidD. Notably, deletion of the two terminal alpha helices resulted in a CTD variant incapable of discriminating between membranes of different composition. Moreover, a L. pneumophila strain producing SidDΔloop phenocopied a L. pneumophila ΔsidD strain during growth in mouse macrophages and displayed prolonged co-localization of AMPylated Rab1 with LCVs, thus revealing that membrane targeting of SidD via its CTD is a critical prerequisite for its ability to catalyze Rab1 deAMPylation during L. pneumophila infection.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/microbiología , Legionella pneumophila/enzimología , Enfermedad de los Legionarios/microbiología , Adenosina Monofosfato/metabolismo , Animales , Proteínas Bacterianas/genética , Femenino , Aparato de Golgi/metabolismo , Humanos , Legionella pneumophila/química , Legionella pneumophila/genética , Ratones , Dominios ProteicosRESUMEN
The eukaryotic ubiquitylation machinery catalyzes the covalent attachment of the small protein modifier ubiquitin to cellular target proteins in order to alter their fate. Microbial pathogens exploit this post-translational modification process by encoding molecular mimics of E3 ubiquitin ligases, eukaryotic enzymes that catalyze the final step in the ubiquitylation cascade. Here, we show that the Legionella pneumophila effector protein RavN belongs to a growing class of bacterial proteins that mimic host cell E3 ligases to exploit the ubiquitylation pathway. The E3 ligase activity of RavN was located within its N-terminal region and was dependent upon interaction with a defined subset of E2 ubiquitin-conjugating enzymes. The crystal structure of the N-terminal region of RavN revealed a U-box-like motif that was only remotely similar to other U-box domains, indicating that RavN is an E3 ligase relic that has undergone significant evolutionary alteration. Substitution of residues within the predicted E2 binding interface rendered RavN inactive, indicating that, despite significant structural changes, the mode of E2 recognition has remained conserved. Using hidden Markov model-based secondary structure analyses, we identified and experimentally validated four additional L. pneumophila effectors that were not previously recognized to possess E3 ligase activity, including Lpg2452/SdcB, a new paralog of SidC. Our study provides strong evidence that L. pneumophila is dedicating a considerable fraction of its effector arsenal to the manipulation of the host ubiquitylation pathway.
Asunto(s)
Legionella pneumophila/enzimología , Ubiquitina-Proteína Ligasas/fisiología , Secuencia de Aminoácidos , Clonación Molecular , Células HEK293 , Humanos , Legionella pneumophila/genética , Enfermedad de los Legionarios/genética , Enfermedad de los Legionarios/microbiología , Modelos Moleculares , Conformación Proteica en Hélice alfa , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/aislamiento & purificación , Ubiquitinación/genéticaRESUMEN
Microbial pathogens employ sophisticated virulence strategies to cause infections in humans. The intracellular pathogen Legionella pneumophila encodes RidL to hijack the host scaffold protein VPS29, a component of retromer and retriever complexes critical for endosomal cargo recycling. Here, we determined the crystal structure of L. pneumophila RidL in complex with the human VPS29-VPS35 retromer subcomplex. A hairpin loop protruding from RidL inserts into a conserved pocket on VPS29 that is also used by cellular ligands, such as Tre-2/Bub2/Cdc16 domain family member 5 (TBC1D5) and VPS9-ankyrin repeat protein for VPS29 binding. Consistent with the idea of molecular mimicry in protein interactions, RidL outcompeted TBC1D5 for binding to VPS29. Furthermore, the interaction of RidL with retromer did not interfere with retromer dimerization but was essential for association of RidL with retromer-coated vacuolar and tubular endosomes. Our work thus provides structural and mechanistic evidence into how RidL is targeted to endosomal membranes.
Asunto(s)
Proteínas Bacterianas/química , Legionella pneumophila/química , Multimerización de Proteína , Factores de Virulencia/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Legionella pneumophila/genética , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidad , Dominios Proteicos , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The cell surface delivery of extracellular matrix (ECM) and integrins is fundamental for cell migration in wound healing and during cancer cell metastasis. This process is not only driven by several soluble NSF attachment protein (SNAP) receptor (SNARE) proteins, which are key players in vesicle transport at the cell surface and intracellular compartments, but is also tightly modulated by cholesterol. Cholesterol-sensitive SNAREs at the cell surface are relatively well characterized, but it is less well understood how altered cholesterol levels in intracellular compartments impact on SNARE localization and function. Recent insights from structural biology, protein chemistry and cell microscopy have suggested that a subset of the SNAREs engaged in exocytic and retrograde pathways dynamically 'sense' cholesterol levels in the Golgi and endosomal membranes. Hence, the transport routes that modulate cellular cholesterol distribution appear to trigger not only a change in the location and functioning of SNAREs at the cell surface but also in endomembranes. In this Commentary, we will discuss how disrupted cholesterol transport through the Golgi and endosomal compartments ultimately controls SNARE-mediated delivery of ECM and integrins to the cell surface and, consequently, cell migration.
Asunto(s)
Colesterol/metabolismo , Membranas Intracelulares/metabolismo , Proteínas SNARE/metabolismo , Animales , Humanos , Transporte de ProteínasRESUMEN
A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD-Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Legionella pneumophila/enzimología , Fosfolipasas A1/química , Fosfolipasas A1/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab5/química , Proteínas de Unión al GTP rab5/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Unión Competitiva , Dominio Catalítico , Cristalografía por Rayos X , Endosomas/metabolismo , Interacciones Huésped-Patógeno , Humanos , Legionella pneumophila/genética , Legionella pneumophila/patogenicidad , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Mutagénesis Sitio-Dirigida , Fosfolipasas A1/genética , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab5/genéticaRESUMEN
The covalent attachment of adenosine monophosphate (AMP) to proteins, a process called AMPylation (adenylylation), has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.
Asunto(s)
Adenosina Monofosfato/química , Proteínas Bacterianas/química , Legionella pneumophila/enzimología , Simulación del Acoplamiento Molecular , Proteínas de Unión al GTP rab1/química , Adenosina Monofosfato/genética , Adenosina Monofosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Humanos , Legionella pneumophila/genética , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/genética , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2C , Estructura Cuaternaria de Proteína , Relación Estructura-Actividad , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
Endosomal trafficking ensures the proper distribution of lipids and proteins to various cellular compartments, facilitating intracellular communication, nutrient transport, waste disposal, and the maintenance of cell structure. Retromer, a peripheral membrane protein complex, plays an important role in this process by recruiting the associated actin-polymerizing WASH complex to establish distinct sorting domains. The WASH complex is recruited through the interaction of the VPS35 subunit of retromer with the WASH complex subunit FAM21. Here, we report the identification of two separate fragments of FAM21 that interact with VPS35, along with a third fragment that binds to the VPS29 subunit of retromer. The crystal structure of VPS29 bound to a peptide derived from FAM21 shows a distinctive sharp bend that inserts into a conserved hydrophobic pocket with a binding mode similar to that adopted by other VPS29 effectors. Interestingly, despite the network of interactions between FAM21 and retromer occurring near the Parkinson's disease-linked mutation (D620N) in VPS35, this mutation does not significantly impair the direct association with FAM21 in vitro.
Asunto(s)
Endosomas , Enfermedad de Parkinson , Humanos , Mutación , Transporte de Proteínas , Proteínas de Transporte Vesicular/genéticaRESUMEN
The retromer complex is required for the sorting of acid hydrolases to lysosomes, transcytosis of the polymeric immunoglobulin receptor, Wnt gradient formation, iron transporter recycling and processing of the amyloid precursor protein. Human retromer consists of two smaller complexes: the cargo recognition VPS26-VPS29-VPS35 heterotrimer and a membrane-targeting heterodimer or homodimer of SNX1 and/or SNX2 (ref. 13). Here we report the crystal structure of a VPS29-VPS35 subcomplex showing how the metallophosphoesterase-fold subunit VPS29 (refs 14, 15) acts as a scaffold for the carboxy-terminal half of VPS35. VPS35 forms a horseshoe-shaped, right-handed, alpha-helical solenoid, the concave face of which completely covers the metal-binding site of VPS29, whereas the convex face exposes a series of hydrophobic interhelical grooves. Electron microscopy shows that the intact VPS26-VPS29-VPS35 complex is a stick-shaped, flexible structure, approximately 21 nm long. A hybrid structural model derived from crystal structures, electron microscopy, interaction studies and bioinformatics shows that the alpha-solenoid fold extends the full length of VPS35, and that VPS26 is bound at the opposite end from VPS29. This extended structure presents multiple binding sites for the SNX complex and receptor cargo, and appears capable of flexing to conform to curved vesicular membranes.
Asunto(s)
Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/metabolismo , Secuencia de Aminoácidos , Biología Computacional , Cristalografía por Rayos X , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación Proteica , Transporte de Proteínas , Relación Estructura-Actividad , Especificidad por Sustrato , Proteínas de Transporte Vesicular/ultraestructuraRESUMEN
The multisubunit Golgi-associated retrograde protein (GARP) complex is required for tethering and fusion of endosome-derived transport vesicles to the trans-Golgi network. Mutation of leucine-967 to glutamine in the Vps54 subunit of GARP is responsible for spinal muscular atrophy in the wobbler mouse, an animal model of amyotrophic lateral sclerosis. The crystal structure at 1.7 A resolution of the mouse Vps54 C-terminal fragment harboring leucine-967, in conjunction with comparative sequence analysis, reveals that Vps54 has a continuous alpha-helical bundle organization similar to that of other multisubunit tethering complexes. The structure shows that leucine-967 is buried within the alpha-helical bundle through predominantly hydrophobic interactions that are critical for domain stability and folding in vitro. Mutation of this residue to glutamine does not prevent integration of Vps54 into the GARP complex but greatly reduces the half-life and levels of the protein in vivo. Severely reduced levels of mutant Vps54 and, consequently, of the whole GARP complex underlie the phenotype of the wobbler mouse.
Asunto(s)
Complejos Multiproteicos/metabolismo , Mutación/genética , Enfermedades Neurodegenerativas/genética , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Células HeLa , Humanos , Leucina/metabolismo , Ratones , Ratones Mutantes Neurológicos , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Especificidad de Órganos , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Relación Estructura-Actividad , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The papillomavirus L2 capsid protein protrudes through the endosome membrane into the cytoplasm during virus entry to bind cellular factors required for intracellular virus trafficking. Cytoplasmic protrusion of HPV16 L2, virus trafficking, and infectivity are inhibited by large deletions in an âË»110 amino acid segment of L2 that is predicted to be disordered. The activity of these mutants can be restored by inserting protein segments with diverse compositions and chemical properties into this region, including scrambled sequences, a tandem array of a short sequence, and the intrinsically disordered region of a cellular protein. The infectivity of mutants with small in-frame insertions and deletions in this segment directly correlates with the size of the segment. These results indicate that the length of the disordered segment, not its sequence or its composition, determines its activity during virus entry. Sequence independent but length dependent activity has important implications for protein function and evolution.
RESUMEN
Wnt proteins are secreted hydrophobic glycoproteins that act over long distances through poorly understood mechanisms. We discovered that Wnt7a is secreted on extracellular vesicles (EVs) following muscle injury. Structural analysis identified the motif responsible for Wnt7a secretion on EVs that we term the Exosome Binding Peptide (EBP). Addition of the EBP to an unrelated protein directed secretion on EVs. Disruption of palmitoylation, knockdown of WLS, or deletion of the N-terminal signal peptide did not affect Wnt7a secretion on purified EVs. Bio-ID analysis identified Coatomer proteins as candidates responsible for loading Wnt7a onto EVs. The crystal structure of EBP bound to the COPB2 coatomer subunit, the binding thermodynamics, and mutagenesis experiments, together demonstrate that a dilysine motif in the EBP mediates binding to COPB2. Other Wnts contain functionally analogous structural motifs. Mutation of the EBP results in a significant impairment in the ability of Wnt7a to stimulate regeneration, indicating that secretion of Wnt7a on exosomes is critical for normal regeneration in vivo . Our studies have defined the structural mechanism that mediates binding of Wnt7a to exosomes and elucidated the singularity of long-range Wnt signalling.
RESUMEN
Recycling of membrane proteins enables the reuse of receptors, ion channels and transporters. A key component of the recycling machinery is the endosomal sorting complex for promoting exit 1 (ESCPE-1), which rescues transmembrane proteins from the endolysosomal pathway for transport to the trans-Golgi network and the plasma membrane. This rescue entails the formation of recycling tubules through ESCPE-1 recruitment, cargo capture, coat assembly and membrane sculpting by mechanisms that remain largely unknown. Herein, we show that ESCPE-1 has a single-layer coat organization and suggest how synergistic interactions between ESCPE-1 protomers, phosphoinositides and cargo molecules result in a global arrangement of amphipathic helices to drive tubule formation. Our results thus define a key process of tubule-based endosomal sorting.
Asunto(s)
Proteínas Portadoras , Endosomas , Endosomas/metabolismo , Transporte de Proteínas , Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Membrana Celular/metabolismoRESUMEN
Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.
Asunto(s)
Endosomas/metabolismo , Proteínas Fúngicas/metabolismo , Transporte de Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animales , Cristalografía por Rayos X/métodos , Citoplasma/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Técnica del Anticuerpo Fluorescente/métodos , Proteínas Fúngicas/química , Regulación Fúngica de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes/metabolismo , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis/fisiología , Unión Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Proteínas de Saccharomyces cerevisiae/química , Homología Estructural de Proteína , Proteínas de Transporte Vesicular/químicaRESUMEN
The multivesicular-body (MVB) pathway delivers transmembrane proteins and lipids to the lumen of the endosome. The multivesicular-body sorting pathway has crucial roles in growth-factor-receptor downregulation, developmental signalling, regulation of the immune response and the budding of certain enveloped viruses such as human immunodeficiency virus. Ubiquitination is a signal for sorting into the MVB pathway, which also requires the functions of three protein complexes, termed ESCRT-I, -II and -III (endosomal sorting complex required for transport). Here we report the crystal structure of the core of the yeast ESCRT-II complex, which contains one molecule of the Vps protein Vps22, the carboxy-terminal domain of Vps36 and two molecules of Vps25, and has the shape of a capital letter 'Y'. The amino-terminal coiled coil of Vps22 and the flexible linker leading to the ubiquitin-binding NZF domain of Vps36 both protrude from the tip of one branch of the 'Y'. Vps22 and Vps36 form nearly equivalent interactions with the two Vps25 molecules at the centre of the 'Y'. The structure suggests how ubiquitinated cargo could be passed between ESCRT components of the MVB pathway through the sequential transfer of ubiquitinated cargo from one complex to the next.
Asunto(s)
Proteínas Portadoras/química , Endosomas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sitios de Unión , Transporte Biológico , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Mutación/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte VesicularRESUMEN
The endolysosomal system is a highly dynamic network of membranes for degradation and recycling. During endosomal maturation, cargo molecules destined for lysosomal degradation are progressively concentrated through continuous rounds of fusion and fission reactions concomitant with inbound and outbound membrane fluxes. Of the cargo molecules delivered to endosomes, about two-thirds are rescued from degradation and recycled for reuse. This balance between degradation and recycling is essential to preserve the proteostatic plasticity of the cell under variable physiological demands. Cargo retrieval from endosomes involves several sorting complexes with stable core compositions that associate with multidomain regulatory proteins, consequently displaying complex interaction networks. The vacuolar protein sorting 29 (VPS29) has emerged as a central scaffold that coordinates the physical assembly of retrieval complexes with regulatory components in what appears to be an elegant solution for regulating distinct retrieval stations. This review summarizes the VPS29-binding partners and its integration into retrieval complexes for endosomal sorting and trafficking.
Asunto(s)
Endosomas/metabolismo , Transporte de Proteínas/genética , Proteínas de Transporte Vesicular/genética , HumanosRESUMEN
Lucas and Hierro introduce the retromer and its role in endosomal protein sorting and trafficking.
Asunto(s)
Endosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Transporte de ProteínasRESUMEN
Eukaryotic cells use sophisticated mechanisms to direct protein traffic between subcellular compartments. In eukaryotic cells, transmembrane proteins are delivered for degradation in the lysosome or yeast vacuole via multivesicular bodies. The sorting of proteins into lumenal vesicles within multivesicular bodies is directed by the three ESCRT protein complexes. Here we describe the expression and purification of the ESCRT-II complex using the polycistronic expression vector pST39 developed by Tan. In a modification of Tan's procedure, Pfu polymerase amplification with overlapping oligonucleotides was used to generate the translation cassettes for subcloning into pST39 expression vector in a single step. This approach reduces the number of restriction sites and subcloning steps required to express a heterooligomeric protein complex, facilitating rapid screening of multiple complexes and complex variants for crystallization or biochemical characterization.