RESUMO
Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.
Assuntos
Proteínas do Capsídeo/metabolismo , Peptídeos Penetradores de Células/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Endossomos/metabolismo , Complexo de Golgi/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Mutagênese , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Ligação Viral , Internalização do VírusRESUMO
The Golgi functions principally in the biogenesis and trafficking of glycoproteins and lipids. It is compartmentalized into multiple flattened adherent membrane sacs termed cisternae, which each contain a distinct repertoire of resident proteins, principally enzymes that modify newly synthesized proteins and lipids sequentially as they traffic through the stack of Golgi cisternae. Upon reaching the final compartments of the Golgi, the trans cisterna and trans-Golgi network (TGN), processed glycoproteins and lipids are packaged into coated and non-coated transport carriers derived from the trans Golgi and TGN. The cargoes of clathrin-coated vesicles are chiefly residents of endo-lysosomal organelles, while uncoated carriers ferry cargo to the cell surface. There are outstanding questions regarding the mechanisms of protein and lipid sorting within the Golgi for export to different organelles. Nonetheless, conceptual advances have begun to define the key molecular features of cargo clients and the mechanisms underlying their sorting into distinct export pathways, which we have collated in this Cell Science at a Glance article and the accompanying poster.
Assuntos
Complexo de Golgi , Rede trans-Golgi , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina , Humanos , Transporte Proteico , Rede trans-Golgi/metabolismoRESUMO
The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome-to-Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.
Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Membrana Celular/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Retromer orchestrates the selection and export of integral membrane proteins from the endosome via retrograde and plasma membrane recycling pathways. Long-standing hypotheses regarding the retromer sorting mechanism posit that oligomeric interactions between retromer and associated accessory factors on the endosome membrane drives clustering of retromer-bound integral membrane cargo prior to its packaging into a nascent transport carrier. To test this idea, we examined interactions between components of the sorting nexin 3 (SNX3)-retromer sorting pathway using quantitative single particle fluorescence microscopy in a reconstituted system. This system includes a supported lipid bilayer, fluorescently labeled retromer, SNX3, and two model cargo proteins, RAB7, and retromer-binding segments of the WASHC2C subunit of the WASH complex. We found that the distribution of membrane-associated retromer is predominantly comprised of monomer (â¼18%), dimer (â¼35%), trimer (â¼24%), and tetramer (â¼13%). Unexpectedly, neither the presence of membrane-associated cargo nor accessory factors substantially affected this distribution. The results indicate that retromer has an intrinsic propensity to form low order oligomers on a supported lipid bilayer and that neither membrane association nor accessory factors potentiate oligomerization. The results support a model whereby SNX3-retromer is a minimally concentrative coat protein complex adapted to bulk membrane trafficking from the endosomal system.
Assuntos
Bicamadas Lipídicas/química , Complexos Multiproteicos/química , Proteínas de Ligação a Fosfato/química , Nexinas de Classificação/química , Proteínas rab de Ligação ao GTP/química , Humanos , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Nexinas de Classificação/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
The large GTPase dynamin is the first protein shown to catalyze membrane fission. Dynamin and its related proteins are essential to many cell functions, from endocytosis to organelle division and fusion, and it plays a critical role in many physiological functions such as synaptic transmission and muscle contraction. Research of the past three decades has focused on understanding how dynamin works. In this review, we present the basis for an emerging consensus on how dynamin functions. Three properties of dynamin are strongly supported by experimental data: first, dynamin oligomerizes into a helical polymer; second, dynamin oligomer constricts in the presence of GTP; and third, dynamin catalyzes membrane fission upon GTP hydrolysis. We present the two current models for fission, essentially diverging in how GTP energy is spent. We further discuss how future research might solve the remaining open questions presently under discussion.
Assuntos
Membrana Celular/fisiologia , Dinaminas/fisiologia , Animais , Guanosina Trifosfato/fisiologia , HumanosRESUMO
The yeast SNX4 sub-family of sorting nexin containing a Bin-Amphiphysin-Rvs domain (SNX-BAR) proteins, Snx4/Atg24, Snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here, we show that Snx4 forms 2 functionally distinct heterodimers: Snx4-Atg20 and Snx4-Snx41. Each heterodimer coats an endosome-derived tubule that mediates retrograde sorting of distinct cargo; the v-SNARE, Snc1, is a cargo of the Snx4-Atg20 pathway, and Snx4-Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5-Vps17 retromer SNX-BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer-coated tubules, promotes fission of Snx4-Atg20 coated tubules. The results indicate that the yeast SNX-BAR proteins coat 3 distinct types of endosome-derived carriers that mediate endosome-to-Golgi retrograde trafficking.
Assuntos
Proteínas Fúngicas/metabolismo , Transporte Proteico/fisiologia , Proteínas R-SNARE/metabolismo , Nexinas de Classificação/metabolismo , Leveduras/metabolismo , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismoRESUMO
One of the principal functions of the trans Golgi network (TGN) is the sorting of proteins into distinct vesicular transport carriers that mediate secretion and interorganelle trafficking. Are lipids also sorted into distinct TGN-derived carriers? The Golgi is the principal site of the synthesis of sphingomyelin (SM), an abundant sphingolipid that is transported. To address the specificity of SM transport to the plasma membrane, we engineered a natural SM-binding pore-forming toxin, equinatoxin II (Eqt), into a nontoxic reporter termed Eqt-SM and used it to monitor intracellular trafficking of SM. Using quantitative live cell imaging, we found that Eqt-SM is enriched in a subset of TGN-derived secretory vesicles that are also enriched in a glycophosphatidylinositol-anchored protein. In contrast, an integral membrane secretory protein (CD8α) is not enriched in these carriers. Our results demonstrate the sorting of native SM at the TGN and its transport to the plasma membrane by specific carriers.
Assuntos
Antígenos CD8/metabolismo , Membrana Celular/metabolismo , Vesículas Secretórias/metabolismo , Esfingomielinas/metabolismo , Rede trans-Golgi/metabolismo , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/fisiologia , Antígenos CD8/genética , Membrana Celular/genética , Venenos de Cnidários/farmacologia , Células HeLa , Humanos , Vesículas Secretórias/genética , Esfingomielinas/genética , Rede trans-Golgi/genéticaRESUMO
Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.
Assuntos
Proteínas do Capsídeo/metabolismo , Núcleo Celular/virologia , Papillomavirus Humano 16/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Liberação de Vírus/fisiologia , Antígenos Virais/metabolismo , Sítios de Ligação , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Linhagem Celular , Endossomos/virologia , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Células HEK293 , Células HeLa , Humanos , Proteínas Oncogênicas Virais/genética , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais , Internalização do VírusRESUMO
Retromer is an evolutionarily conserved protein complex composed of the VPS26, VPS29, and VPS35 proteins that selects and packages cargo proteins into transport carriers that export cargo from the endosome. The mechanisms by which retromer is recruited to the endosome and captures cargo are unknown. We show that membrane recruitment of retromer is mediated by bivalent recognition of an effector of PI3K, SNX3, and the RAB7A GTPase, by the VPS35 retromer subunit. These bivalent interactions prime retromer to capture integral membrane cargo, which enhances membrane association of retromer and initiates cargo sorting. The role of RAB7A is severely impaired by a mutation, K157N, that causes Charcot-Marie-Tooth neuropathy 2B. The results elucidate minimal requirements for retromer assembly on the endosome membrane and reveal how PI3K and RAB signaling are coupled to initiate retromer-mediated cargo export.
Assuntos
Endossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/química , Transporte Biológico , Proteínas de Transporte/química , Reagentes de Ligações Cruzadas , Humanos , Lipossomos/química , Espectrometria de Massas , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais , Nexinas de Classificação/química , Proteínas rab de Ligação ao GTP/química , proteínas de unión al GTP Rab7RESUMO
Sorting of macromolecules within the endosomal system is vital for physiological control of nutrient homeostasis, cell motility, and proteostasis. Trafficking routes that export macromolecules from the endosome via vesicle and tubule transport carriers constitute plasma membrane recycling and retrograde endosome-to-Golgi pathways. Proteins of the sorting nexin family have been discovered to function at nearly every step of endosomal transport carrier biogenesis and it is becoming increasingly clear that they form the core machineries of cargo-specific transport pathways that are closely integrated with cellular physiology. Here, we summarize recent progress in elucidating the pathways that mediate the biogenesis of endosome-derived transport carriers.
Assuntos
Transporte Biológico/fisiologia , Endossomos/metabolismo , Endossomos/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Complexo de Golgi/metabolismo , Complexo de Golgi/fisiologia , HumanosRESUMO
Bridge-like lipid transfer protein family member 2 (BLTP2) is an evolutionary conserved protein with unknown function(s). The absence of BLTP2 in Drosophila melanogaster results in impaired cellular secretion and larval death, while in mice (Mus musculus), it causes preweaning lethality. Structural predictions propose that BLTP2 belongs to the repeating ß-groove domain-containing (also called the VPS13) protein family, forming a long tube with a hydrophobic core, suggesting that it operates as a lipid transfer protein (LTP). We establish BLTP2 as a negative regulator of ciliogenesis in RPE-1 cells based on a strong genetic interaction with WDR44, a gene that also suppresses ciliogenesis. Like WDR44, BLTP2 localizes to membrane contact sites involving the endoplasmic reticulum and the tubular endosome network in HeLa cells and that BLTP2 depletion enhanced ciliogenesis in RPE-1 cells grown in serum-containing medium, a condition where ciliogenesis is normally suppressed. This study establishes human BLTP2 as a putative LTP acting between tubular endosomes and ER that regulates primary cilium biogenesis.
Assuntos
Proteínas de Transporte , Drosophila melanogaster , Humanos , Animais , Camundongos , Células HeLa , Drosophila melanogaster/metabolismo , Proteínas de Transporte/metabolismo , Proteínas/metabolismo , FamíliaRESUMO
Bidirectional traffic between the Golgi apparatus and the endosomal system sustains the functions of the trans-Golgi network (TGN) in secretion and organelle biogenesis. Export of cargo from the TGN via anterograde trafficking pathways depletes the organelle of sorting receptors, processing proteases, SNARE molecules, and other factors, and these are subsequently retrieved from endosomes via the retrograde pathway. Recent studies indicate that retrograde trafficking is vital to early metazoan development, nutrient homeostasis, and for processes that protect against Alzheimer's and other neurological diseases.
Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Endossomos/patologia , Endossomos/fisiologia , Rede trans-Golgi/patologia , Rede trans-Golgi/fisiologia , Animais , Endossomos/metabolismo , Humanos , Transporte Proteico , Transdução de Sinais , Rede trans-Golgi/metabolismoRESUMO
The sorting and trafficking of lipids between organelles gives rise to a dichotomy of bulk membrane properties between organelles of the secretory and endolysosome networks, giving rise to two "membrane territories" based on differences in lipid-packing density, net membrane charge, and bilayer leaflet asymmetries. The cellular organelle membrane dichotomy emerges from ER-to-PM anterograde membrane trafficking and the synthesis of sphingolipids and cholesterol flux at the trans-Golgi network, which constitutes the interface between the two membrane territories. Organelle homeostasis is maintained by vesicle-mediated retrieval of bulk membrane from the distal organelles of each territory to the endoplasmic reticulum or plasma membrane and by soluble lipid transfer proteins that traffic particular lipids. The concept of cellular membrane territories emphasizes the contrasting features of organelle membranes of the secretory and endolysosome networks and the essential roles of lipid-sorting pathways that maintain organelle function.
Assuntos
Retículo Endoplasmático , Lipídeos , Retículo Endoplasmático/metabolismo , Membrana Celular/metabolismo , Transporte Proteico , Transporte BiológicoRESUMO
Sphingomyelin plays a key role in cellular cholesterol homeostasis by binding to and sequestering cholesterol in the plasma membrane. We discovered that synthesis of very long chain (VLC) sphingomyelins is inversely regulated by cellular cholesterol levels; acute cholesterol depletion elicited a rapid induction of VLC-sphingolipid synthesis, increased trafficking to the Golgi apparatus and plasma membrane, while cholesterol loading reduced VLC-sphingolipid synthesis. This sphingolipid-cholesterol metabolic axis is distinct from the sterol responsive element binding protein pathway as it requires ceramide synthase 2 (CerS2) activity, epidermal growth factor receptor signaling, and was unaffected by inhibition of protein translation. Depletion of VLC-ceramides reduced plasma membrane cholesterol content, reduced plasma membrane lipid packing, and unexpectedly resulted in the accumulation of cholesterol in the cytoplasmic leaflet of the lysosome membrane. This study establishes the existence of a cholesterol-sphingolipid regulatory axis that maintains plasma membrane lipid homeostasis via regulation of sphingomyelin synthesis and trafficking.
Assuntos
Membrana Celular , Membranas Intracelulares , Esfingomielinas , Esfingosina N-Aciltransferase , Citoplasma , Homeostase , Esfingomielinas/biossíntese , Esfingosina N-Aciltransferase/metabolismo , Colesterol , Receptores ErbB/metabolismoRESUMO
Amajor function of the endocytic system is the sorting of cargo to various organelles. Endocytic sorting of the yeast reductive iron transporter, which is composed of the Fet3 and Ftr1 proteins, is regulated by available iron. When iron is provided to iron-starved cells, Fet3p-Ftr1p is targeted to the lysosome-like vacuole and degraded. In contrast, when iron is not available, Fet3p-Ftr1p is maintained on the plasma membrane via an endocytic recycling pathway requiring the sorting nexin Grd19/Snx3p, the pentameric retromer complex, and the Ypt6p Golgi Rab GTPase module. A recycling signal in Ftr1p was identified and found to bind directly to Grd19/Snx3p. Retromer and Grd19/Snx3p partially colocalize to tubular endosomes, where they are physically associated. After export from the endosome, Fet3p-Ftr1p transits through the Golgi apparatus for resecretion. Thus, Grd19/Snx3p, functions as a cargo-specific adapter for the retromer complex, establishing a precedent for a mechanism by which sorting nexins expand the repertoire of retromer-dependent cargos.
Assuntos
Proteínas de Transporte/fisiologia , Endocitose , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Membrana Celular/metabolismo , Ceruloplasmina/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde/análise , Ferro/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
The trans-Golgi network must coordinate sorting and secretion of proteins and lipids to intracellular organelles and the plasma membrane. During polarization of epithelial cells, changes in the lipidome and the expression and distribution of proteins contribute to the formation of apical and basolateral plasma membrane domains. Previous studies using HeLa cells show that the syndecan-1 transmembrane domain confers sorting within sphingomyelin-rich vesicles in a sphingomyelin secretion pathway. In polarized Madin-Darby canine kidney cells, we reveal differences in the sorting of syndecan-1, whereupon the correct trafficking of the protein is not dependent on its transmembrane domain and changes in sphingomyelin content of cells during polarization. Instead, we reveal that correct basolateral targeting of syndecan-1 requires a full-length PDZ motif in syndecan-1 and the PDZ domain golgin protein GOPC. Moreover, we reveal changes in Golgi morphology elicited by GOPC overexpression. These results suggest that the role of GOPC in sorting syndecan-1 is indirect and likely due to GOPC effects on Golgi organization.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Sindecana-1 , Animais , Membrana Celular/metabolismo , Polaridade Celular , Cães , Células Epiteliais/metabolismo , Células HeLa , Humanos , Células Madin Darby de Rim Canino , Transporte Proteico , Esfingomielinas/metabolismo , Sindecana-1/metabolismoRESUMO
Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.
Assuntos
Cálcio , Esfingomielinas , Cálcio/metabolismo , Citosol/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lisossomos/metabolismo , Esfingomielinas/metabolismoRESUMO
Sphingomyelin is a dominant sphingolipid in mammalian cells. Its production in the trans-Golgi traps cholesterol synthesized in the ER to promote formation of a sphingomyelin/sterol gradient along the secretory pathway. This gradient marks a fundamental transition in physical membrane properties that help specify organelle identify and function. We previously identified mutations in sphingomyelin synthase SMS2 that cause osteoporosis and skeletal dysplasia. Here, we show that SMS2 variants linked to the most severe bone phenotypes retain full enzymatic activity but fail to leave the ER owing to a defective autonomous ER export signal. Cells harboring pathogenic SMS2 variants accumulate sphingomyelin in the ER and display a disrupted transbilayer sphingomyelin asymmetry. These aberrant sphingomyelin distributions also occur in patient-derived fibroblasts and are accompanied by imbalances in cholesterol organization, glycerophospholipid profiles, and lipid order in the secretory pathway. We postulate that pathogenic SMS2 variants undermine the capacity of osteogenic cells to uphold nonrandom lipid distributions that are critical for their bone forming activity.
Assuntos
Via Secretória , Esfingomielinas , Animais , Colesterol , Glicerofosfolipídeos , Mamíferos/metabolismo , Camundongos , Camundongos Knockout , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)RESUMO
Myristoylation of ARF family GTPases is required for their association with Golgi and endosomal membranes, where they regulate protein sorting and the lipid composition of these organelles. The Golgi-localized ARF-like GTPase Arl3p/ARP lacks a myristoylation signal, indicating that its targeting mechanism is distinct from myristoylated ARFs. We demonstrate that acetylation of the N-terminal methionine of Arl3p requires the NatC N(alpha)-acetyltransferase and that this modification is required for its Golgi localization. Chemical crosslinking and fluorescence microscopy experiments demonstrate that localization of Arl3p also requires Sys1p, a Golgi-localized integral membrane protein, which may serve as a receptor for acetylated Arl3p.