RESUMEN
Lipid droplets (LDs) and peroxisomes are ubiquitous organelles with central roles in eukaryotic cells. Although the mechanisms involved in biogenesis of these organelles remain elusive, both seem to require the endoplasmic reticulum (ER). Here we show that in yeast the ER budding of these structurally unrelated organelles has remarkably similar requirements and involves cooperation between Pex30 and the seipin complex. In the absence of these components, budding of both LDs and peroxisomes is inhibited, leading to the ER accumulation of their respective constituent molecules, such as triacylglycerols and peroxisomal membrane proteins, whereas COPII vesicle formation remains unaffected. This phenotype can be reversed by remodeling ER phospholipid composition highlighting a key function of these lipids in organelle biogenesis. We propose that seipin and Pex30 act in concert to organize membrane domains permissive for organelle budding, and that may have a lipid composition distinct from the bulk ER.
Asunto(s)
División Celular/fisiología , Retículo Endoplásmico/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Biogénesis de Organelos , Reproducción Asexuada/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Membranas Intracelulares/metabolismo , Gotas Lipídicas/metabolismo , Proteínas de la Membrana/genética , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Peroxisomas/metabolismo , Fosfolípidos/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Triglicéridos/metabolismoRESUMEN
Storage and consumption of neutral lipids in lipid droplets (LDs) are essential for energy homeostasis and tightly coupled to cellular metabolism. However, how metabolic cues are integrated in the life cycle of LDs is unclear. In this study, we characterize the function of Ldo16 and Ldo45, two splicing isoforms of the same protein in budding yeast. We show that Ldo proteins interact with the seipin complex, which regulates contacts between LDs and the endoplasmic reticulum (ER). Moreover, we show that the levels of Ldo16 and Ldo45 depend on the growth stage of cells and that deregulation of their relative abundance alters LD morphology, protein localization, and triglyceride content. Finally, we show that absence of Ldo proteins results in defects in LD morphology and consumption by lipophagy. Our findings support a model in which Ldo proteins modulate the activity of the seipin complex, thereby affecting LD properties. Moreover, we identify ER-LD contacts as regulatory targets coupling energy storage to cellular metabolism.
Asunto(s)
Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Gotas Lipídicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Metabolismo de los Lípidos/fisiología , Isoformas de Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Triglicéridos/metabolismoRESUMEN
Oxysterol binding protein-related proteins (ORPs) are conserved lipid binding polypeptides, enriched at ER contacts sites. ORPs promote non-vesicular lipid transport and work as lipid sensors in the context of many cellular tasks, but the determinants of their distinct localization and function are not understood. Here, we demonstrate that the yeast endocytic invaginations associate with the ER and that this association specifically requires the ORPs Osh2 and Osh3, which bridge the endocytic myosin-I Myo5 to the ER integral-membrane VAMP-associated protein (VAP) Scs2. Disruption of the ER contact with endocytic sites using ORP, VAP, myosin-I, or reticulon mutants delays and weakens actin polymerization and interferes with vesicle scission. Finally, we provide evidence suggesting that ORP-dependent sterol transfer facilitates actin polymerization at endocytic sites.
Asunto(s)
Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Metabolismo de los Lípidos/fisiología , Animales , Transporte Biológico , Miosina Tipo I/metabolismo , Receptores de Esteroides/metabolismo , Saccharomyces cerevisiae/metabolismo , Esteroles/metabolismoRESUMEN
Anaphase chromatin bridges can lead to chromosome breakage if not properly resolved before completion of cytokinesis. The NoCut checkpoint, which depends on Aurora B at the spindle midzone, delays abscission in response to chromosome segregation defects in yeast and animal cells. How chromatin bridges are detected, and whether abscission inhibition prevents their damage, remain key unresolved questions. We find that bridges induced by DNA replication stress and by condensation or decatenation defects, but not dicentric chromosomes, delay abscission in a NoCut-dependent manner. Decatenation and condensation defects lead to spindle stabilization during cytokinesis, allowing bridge detection by Aurora B. NoCut does not prevent DNA damage following condensin or topoisomerase II inactivation; however, it protects anaphase bridges and promotes cellular viability after replication stress. Therefore, the molecular origin of chromatin bridges is critical for activation of NoCut, which plays a key role in the maintenance of genome stability after replicative stress.
Asunto(s)
Anafase , Aurora Quinasas/metabolismo , Puntos de Control del Ciclo Celular , Replicación del ADN , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Estrés Fisiológico , Actomiosina/metabolismo , Adenosina Trifosfatasas/metabolismo , Anafase/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN/efectos de los fármacos , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Histona Acetiltransferasas/metabolismo , Hidroxiurea/farmacología , Viabilidad Microbiana/efectos de los fármacos , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/ultraestructura , Huso Acromático/efectos de los fármacos , Huso Acromático/metabolismo , Estrés Fisiológico/efectos de los fármacosRESUMEN
Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER-LD contact sites.
Asunto(s)
Retículo Endoplásmico/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/fisiología , Gotas Lipídicas/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Mitocondriales/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Fosfolípidos/biosíntesis , Transporte de Proteínas , Saccharomyces cerevisiae/ultraestructuraRESUMEN
A transient burst of actin polymerization assists endocytic budding. How actin polymerization is controlled in this context is not understood. Here, we show that crosstalk between PI(4,5)P2and the CK2 catalytic subunit Cka2 controls actin polymerization at endocytic sites. We find that phosphorylation of the myosin-I Myo5 by Cka2 downregulates Myo5-induced Arp2/3-dependent actin polymerization, whereas PI(4,5)P2cooperatively relieves Myo5 autoinhibition and inhibits the catalytic activity of Cka2. Cka2 and the PI(4,5)P2-5-phosphatases Sjl1 and Sjl2, the yeast synaptojanins, exhibit genetic interactions indicating functional redundancy. The ultrastructural analysis of plasma membrane invaginations in CK2 and synaptojanin mutants demonstrates that both cooperate to initiate constriction of the invagination neck, a process coupled to the remodeling of the endocytic actin network. Our data demonstrate a holoenzyme-independent function of CK2 in endocytic budding and establish a robust genetic, functional, and molecular link between PI(4,5)P2and CK2, two masters of intracellular signaling.
Asunto(s)
Actinas/metabolismo , Quinasa de la Caseína II/metabolismo , Endocitosis , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Quinasa de la Caseína II/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Miosina Tipo I/genética , Miosina Tipo I/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eng2 is a glucanase required for spore release, although it is also expressed during vegetative growth, suggesting that it might play other cellular functions. Its homology to the Saccharomyces cerevisiae Acf2 protein, previously shown to promote actin polymerization at endocytic sites in vitro, prompted us to investigate its role in endocytosis. Interestingly, depletion of Eng2 caused profound defects in endocytic uptake, which were not due to the absence of its glucanase activity. Analysis of the dynamics of endocytic proteins by fluorescence microscopy in the eng2Δ strain unveiled a previously undescribed phenotype, in which assembly of the Arp2/3 complex appeared uncoupled from the internalization of the endocytic coat and resulted in a fission defect. Strikingly also, we found that Eng2-GFP dynamics did not match the pattern of other endocytic proteins. Eng2-GFP localized to bright cytosolic spots that moved around the cellular poles and occasionally contacted assembling endocytic patches just before recruitment of Wsp1, the Schizosaccharomyces pombe WASP. Interestingly, Csh3-YFP, a WASP-interacting protein, interacted with Eng2 by co-immunoprecipitation and was recruited to Eng2 in bright cytosolic spots. Altogether, our work defines a novel endocytic functional module, which probably couples the endocytic coat to the actin module.
Asunto(s)
Endocitosis , Glucano Endo-1,3-beta-D-Glucosidasa/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Glucano Endo-1,3-beta-D-Glucosidasa/genética , Unión Proteica , Transporte de Proteínas , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
Endocytic budding implies the remodeling of a plasma membrane portion from a flat sheet to a closed vesicle. Clathrin- and actin-mediated endocytosis in yeast has proven a very powerful model to study this process, with more than 60 evolutionarily conserved proteins involved in fashioning primary endocytic vesicles. Major progress in the field has been made during the last decades by defining the sequential recruitment of the endocytic machinery at the cell cortex using live-cell fluorescence microscopy. Higher spatial resolution has been recently achieved by developing time-resolved electron microscopy methods, allowing for the first time the visualization of changes in the plasma membrane shape, coupled to the dynamics of the endocytic machinery. Here, we highlight these advances and review recent findings from yeast and mammals that have increased our understanding of where and how endocytic proteins may apply force to remodel the plasma membrane during different stages of the process.
Asunto(s)
Endocitosis , Microscopía Electrónica/métodos , Actinas/metabolismo , Animales , Humanos , Microscopía Electrónica/instrumentación , Miosina Tipo I/metabolismo , Levaduras/citología , Levaduras/metabolismoRESUMEN
Fluorescence live-cell imaging has temporally resolved the conserved choreography of more than 30 proteins involved in clathrin and actin-mediated endocytic budding from the plasma membrane. However, the resolution of these studies is insufficient to unveil how the endocytic machinery actually drives membrane deformation in vivo. In this study, we use quantitative immuno-EM to introduce the temporal dimension to the ultrastructural analysis of membrane budding and define changes in the topography of the lipid bilayer coupled to the dynamics of endocytic proteins with unprecedented spatiotemporal resolution. Using this approach, we frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and show that constriction of the invaginations correlates with translocation of membrane-sculpting proteins. Furthermore, we show that initial bending of the plasma membrane is independent of actin and clathrin polymerization and precedes building of an actin cap branched by the Arp2/3 complex. Finally, our data indicate that constriction and additional elongation of the endocytic profiles require the mechanochemical activity of the myosins-I. Altogether, this work provides major insights into the molecular mechanisms driving membrane deformation in a cellular context.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Membrana Celular/metabolismo , Endocitosis/fisiología , Miosina Tipo I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/ultraestructura , Membrana Celular/genética , Membrana Celular/ultraestructura , Miosina Tipo I/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Myosins-I are widely expressed actin-dependent motors which bear a phospholipid-binding domain. In addition, some members of the family can trigger Arp2/3 complex (actin-related protein 2/3 complex)-dependent actin polymerization. In the early 1990s, the development of powerful genetic tools in protozoa and mammals and discovery of these motors in yeast allowed the demonstration of their roles in membrane traffic along the endocytic and secretory pathways, in vacuole contraction, in cell motility and in mechanosensing. The powerful yeast genetics has contributed towards dissecting in detail the function and regulation of Saccharomyces cerevisiae myosins-I Myo3 and Myo5 in endocytic budding from the plasma membrane. In the present review, we summarize the evidence, dissecting their exact role in membrane budding and the molecular mechanisms controlling their recruitment and biochemical activities at the endocytic sites.
Asunto(s)
Endocitosis/fisiología , Miosina Tipo I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Modelos Biológicos , Miosina Tipo I/genética , Saccharomyces cerevisiae/citología , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C(ext). The TH1 domain prevented binding of the Myo5 C(ext) to the yeast WIP homologue Vrp1, Myo5 C(ext)-induced actin polymerization and recruitment of the Myo5 C(ext) to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C(ext). Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.
Asunto(s)
Calmodulina/metabolismo , Miosina Tipo I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Endocitosis , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Genetic analysis of endocytosis in yeast early pointed to the essential role of actin in the uptake step. Efforts to identify the machinery involved demonstrated the important contribution of Arp2/3 and the myosins-I. Analysis of the process using live-cell fluorescence microscopy and electron microscopy have recently contributed to refine molecular models explaining clathrin and actin-dependent endocytic uptake. Increasing evidence now also indicates that actin plays important roles in post-internalization events along the endocytic pathway in yeast, including transport of vesicles, motility of endosomes and vacuole fusion. This review describes the present knowledge state on the roles of actin in endocytosis in yeast and points to similarities and differences with analogous processes in mammals.
Asunto(s)
Actinas/fisiología , Endocitosis/fisiología , Evolución Molecular , Saccharomyces cerevisiae/fisiología , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Animales , Endocitosis/genética , Miosina Tipo I/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475-487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission.
Asunto(s)
Actinas/metabolismo , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Miosina Tipo I/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Membrana Celular/ultraestructura , Endocitosis/fisiología , Microscopía Inmunoelectrónica , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/ultraestructura , Vesículas Transportadoras/ultraestructuraRESUMEN
Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy chains (HC) bound by a light chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p. Here, using in vivo analysis in yeast, we provide novel insight into the role of this interaction. We find that overexpression of LC partially restores endocytosis to cells lacking clathrin HC. This suppression is dependent on the Sla2p binding region of LC. Using live cell imaging techniques to visualize endocytic vesicle formation, we find that the N-terminal Sla2p binding region of LC promotes the progression of arrested Sla2p patches that form in the absence of HC. We propose that LC binding to Sla2p positively regulates Sla2p for efficient endocytic vesicle formation.
Asunto(s)
Proteínas Portadoras/metabolismo , Cadenas Ligeras de Clatrina/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/fisiología , Sitios de Unión , Cadenas Pesadas de Clatrina/genética , Cadenas Ligeras de Clatrina/genética , Vesículas Cubiertas por Clatrina/fisiología , Proteínas del Citoesqueleto , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The yeast myosins I Myo3p and Myo5p have well established functions in the polarization of the actin cytoskeleton and in the endocytic uptake of the G protein-coupled receptor Ste2p. A number of results suggest that phosphorylation of the conserved TEDS serine of the myosin I motor head by the Cdc42p activated p21-activated kinases Ste20p and Cla4p is required for the organization of the actin cytoskeleton. However, the role of this signaling cascade in the endocytic uptake has not been investigated. Interestingly, we find that Myo5p TEDS site phosphorylation is not required for slow, constitutive endocytosis of Ste2p, but it is essential for rapid, ligand-induced internalization of the receptor. Our results strongly suggest that a kinase activates the myosins I to sustain fast endocytic uptake. Surprisingly, however, despite the fact that only p21-activated kinases are known to phosphorylate the conserved TEDS site, we find that these kinases are not essential for ligand-induced internalization of Ste2p. Our observations indicate that a different signaling cascade, involving the yeast homologues of the mammalian PDK1 (3-phosphoinositide-dependent-protein kinase-1), Phk1p and Pkh2p, and serum and glucocorticoid-induced kinase, Ypk1p and Ypk2p, activate Myo3p and Myo5p for their endocytic function.
Asunto(s)
Miosinas/química , Receptores del Factor de Conjugación/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Actinas/química , Sitios de Unión , Catepsina A/metabolismo , Citoesqueleto/metabolismo , ADN/metabolismo , Endocitosis , Genotipo , Glucocorticoides/metabolismo , Immunoblotting , Inmunoprecipitación , Ligandos , Espectrometría de Masas , Microscopía Fluorescente , Modelos Biológicos , Fenotipo , Fosforilación , Plásmidos/metabolismo , Unión Proteica , Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/química , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , Factores de Tiempo , Proteína de Unión al GTP cdc42/metabolismoRESUMEN
Mutations in the budding yeast myosins-I (MYO3 and MYO5) cause defects in the actin cytoskeleton and in the endocytic uptake. Robust evidence also indicates that these proteins induce Arp2/3-dependent actin polymerization. Consistently, we have recently demonstrated, using fluorescence microscopy, that Myo5p is able to induce cytosol-dependent actin polymerization on the surface of Sepharose beads. Strikingly, we now observed that, at short incubation times, Myo5p induced the formation of actin foci that resembled the yeast cortical actin patches, a plasma membrane-associated structure that might be involved in the endocytic uptake. Analysis of the machinery required for the formation of the Myo5p-induced actin patches in vitro demonstrated that the Arp2/3 complex was necessary but not sufficient in the assay. In addition, we found that cofilin was directly involved in the process. Strikingly though, the cofilin requirement seemed to be independent of its ability to disassemble actin filaments and profilin, a protein that closely cooperates with cofilin to maintain a rapid actin filament turnover, was not needed in the assay. In agreement with these observations, we found that like the Arp2/3 complex and the myosins-I, cofilin was essential for the endocytic uptake in vivo, whereas profilin was dispensable.