RESUMEN

In this issue of Developmental Cell, Jiang et al. report that the Arabidopsis HOPS tethering complex subunit VPS41 acts to catalyze the formation of a degradation pathway composed of a hybrid of autophagosomes and late endosomes.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Autofagosomas , Autofagia , Endosomas , Vacuolas , Endosomas/metabolismo , Vacuolas/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Autofagosomas/metabolismo , Autofagia/fisiología , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética

RESUMEN

The Legionella pneumophila Sde family of translocated proteins promotes host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity is tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, prevents binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb results in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decays quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.

Asunto(s)

Autofagia , Proteínas Bacterianas , Retículo Endoplásmico , Legionella pneumophila , Ubiquitina , Ubiquitinación , Vacuolas , Legionella pneumophila/metabolismo , Vacuolas/metabolismo , Vacuolas/microbiología , Ubiquitina/metabolismo , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Retículo Endoplásmico/metabolismo , Animales , Enfermedad de los Legionarios/metabolismo , Enfermedad de los Legionarios/microbiología , Poliubiquitina/metabolismo , Ratones , Proteínas de la Membrana

RESUMEN

Ubiquitination is a posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila. While the Legionella-containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. Here, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on phosphoribosyl-Ub conjugated to host targets by Sde. Remarkably, Ub moieties within poly-Ub chains are either modified with a phosphoribosyl group by PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated phosphoribosyl-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors and therefore exclude host autophagy adaptors from the LCV. In this work, we shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.

Asunto(s)

Autofagia , Proteínas Bacterianas , Legionella pneumophila , Poliubiquitina , Ubiquitinación , Vacuolas , Legionella pneumophila/metabolismo , Legionella pneumophila/genética , Humanos , Vacuolas/metabolismo , Vacuolas/microbiología , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Poliubiquitina/metabolismo , Interacciones Huésped-Patógeno , Células HEK293 , Ubiquitina/metabolismo

RESUMEN

Plant vacuoles, play a crucial role in maintaining cellular stability, adapting to environmental changes, and responding to external pressures. The accurate identification of vacuolar proteins (PVPs) is crucial for understanding the biosynthetic mechanisms of intracellular vacuoles and the adaptive mechanisms of plants. In order to more accurately identify vacuole proteins, this study developed a new predictive model PEL-PVP based on ESM-2. Through this study, the feasibility and effectiveness of using advanced pre-training models and fine-tuning techniques for bioinformatics tasks were demonstrated, providing new methods and ideas for plant vacuolar protein research. In addition, previous datasets for vacuolar proteins were balanced, but imbalance is more closely related to the actual situation. Therefore, this study constructed an imbalanced dataset UB-PVP from the UniProt database，helping the model better adapt to the complexity and uncertainty in real environments, thereby improving the model's generalization ability and practicality. The experimental results show that compared with existing recognition techniques, achieving significant improvements in multiple indicators, with 6.08 %, 13.51 %, 11.9 %, and 5 % improvements in ACC, SP, MCC, and AUC, respectively. The accuracy reaches 94.59 %, significantly higher than the previous best model GraphIdn. This provides an efficient and precise tool for the study of plant vacuole proteins.

Asunto(s)

Proteínas de Plantas , Vacuolas , Vacuolas/metabolismo , Biología Computacional/métodos , Bases de Datos de Proteínas

RESUMEN

Coxiella burnetii is a globally distributed obligate intracellular pathogen. Although often asymptomatic, infections can cause acute Q fever with influenza-like symptoms and/or severe chronic Q fever. Coxiella burnetii develops a unique replicative niche within host cells called the Coxiella-containing vacuole (CCV), facilitated by the Dot/Icm type IV secretion system translocating a cohort of bacterial effector proteins into the host. The role of some effectors has been elucidated; however, the actions of the majority remain enigmatic and the list of true effectors is disputable. This study examined CBU2016, a unique C. burnetii protein previously designated as an effector with a role in infection. We were unable to validate CBU2016 as a translocated effector protein. Employing targeted knock-out and complemented strains, we found that the loss of CBU2016 did not cause a replication defect within Hela, THP-1, J774, or iBMDM cells or in axenic media, nor did it affect the pathogenicity of C. burnetii in the Galleria mellonella infection model. The absence of CBU2016 did, however, result in a consistent decrease in the size of CCVs in HeLa cells. These results suggest that although CBU2016 may not be a Dot/Icm effector, it is still able to influence the host environment during infection.

Asunto(s)

Proteínas Bacterianas , Coxiella burnetii , Fiebre Q , Vacuolas , Coxiella burnetii/genética , Coxiella burnetii/metabolismo , Coxiella burnetii/patogenicidad , Humanos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Vacuolas/microbiología , Vacuolas/metabolismo , Animales , Fiebre Q/microbiología , Células HeLa , Línea Celular , Factores de Virulencia/metabolismo , Factores de Virulencia/genética , Técnicas de Inactivación de Genes , Mariposas Nocturnas/microbiología , Interacciones Huésped-Patógeno , Células THP-1

RESUMEN

The selective degradation of nuclear components via autophagy, termed nucleophagy, is an essential process observed from yeasts to mammals and crucial for maintaining nucleus homeostasis and regulating nucleus functions. In the budding yeast Saccharomyces cerevisiae, nucleophagy occurs in two different manners: one involves autophagosome formation for the sequestration and vacuolar transport of nucleus-derived vesicles (NDVs), and the other proceeds with the invagination of the vacuolar membrane for the uptake of NDVs into the vacuole, termed macronucleophagy and micronucleophagy, respectively. This chapter describes methods to analyze and quantify activities of these nucleophagy pathways in yeast.

Asunto(s)

Autofagia , Núcleo Celular , Saccharomyces cerevisiae , Vacuolas , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Núcleo Celular/metabolismo , Autofagia/fisiología , Autofagosomas/metabolismo

RESUMEN

Protein secretion and vacuole formation are vital processes in plant cells, playing crucial roles in various aspects of plant development, growth, and stress responses. Multiple regulators have been uncovered to be involved in these processes. In animal cells, the transcription factor TFEB has been extensively studied and its role in lysosomal biogenesis is well understood. However, the transcription factors governing protein secretion and vacuole formation in plants remain largely unexplored. In recent years, an increasing number of bioinformatics databases and tools have been developed, facilitating computational prediction and analysis of the function of genes or proteins in specific cellular processes. Leveraging these resources, this chapter aims to provide practical guidance on how to effectively utilize these existing databases and tools for the analysis of key transcription factors involved in regulating protein secretion and vacuole formation in plants, with a particular focus on Arabidopsis and other higher plants. The findings from this analysis can serve as a valuable resource for future experimental investigations and the development of targeted strategies to manipulate protein secretion and vacuole formation in plants.

Asunto(s)

Biología Computacional , Factores de Transcripción , Vacuolas , Vacuolas/metabolismo , Biología Computacional/métodos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Transporte de Proteínas , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética

RESUMEN

Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.

Asunto(s)

Arabidopsis , Tomografía con Microscopio Electrónico , Vacuolas , Tomografía con Microscopio Electrónico/métodos , Vacuolas/ultraestructura , Vacuolas/metabolismo , Arabidopsis/ultraestructura , Arabidopsis/metabolismo , Raíces de Plantas/ultraestructura , Raíces de Plantas/metabolismo , Imagenología Tridimensional/métodos , Substitución por Congelación/métodos , Biogénesis de Organelos

RESUMEN

The pistil is the most important organ for fertilization in flowering plants, and the stigmatic papilla cells are responsible for pollen acceptance and pollen tube germination. Arabidopsis plants possess dry stigmas exhibiting high selectivity for compatible pollen. When compatible pollens are recognized and accepted by stigmatic papilla cells, water and nutrients are then transported from the stigma to pollen grains through the secretory pathway. Here, we present light microscopy-based methods for investigating autophagy and senescence of stigmatic papilla cells. These methods include the assessment of viability of stigmatic papilla cells using dual staining with fluorescein diacetate/propidium iodide, as well as the examination of vacuolar-accumulated proteins during stigma senescence. These methods can be used to understand the functions of the stigma tissue from a subcellular perspective.

Asunto(s)

Arabidopsis , Autofagia , Arabidopsis/fisiología , Arabidopsis/citología , Autofagia/fisiología , Senescencia Celular , Flores/crecimiento & desarrollo , Flores/citología , Vacuolas/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo

RESUMEN

Autophagy is a universal degradation system in eukaryotic cells. In plants, although autophagosome biogenesis has been extensively studied, the mechanism of how autophagosomes are transported to the vacuole for degradation remains largely unexplored. In this study, we demonstrated that upon autophagy induction, Arabidopsis homotypic fusion and protein sorting (HOPS) subunit VPS41 converts first from condensates to puncta, then to ring-like structures, termed VPS41-associated phagic vacuoles (VAPVs), which enclose autophagy-related gene (ATG)8s for vacuolar degradation. This process is initiated by ADP ribosylation factor (ARF)-like GTPases ARLA1s and occurs concurrently with autophagy progression through coupling with the synaptic-soluble N-ethylmaleimide-sensitive factor attachment protein rmleceptor (SNARE) proteins. Unlike in other eukaryotes, autophagy degradation in Arabidopsis is largely independent of the RAB7 pathway. By contrast, dysfunction in the condensates-to-VAPVs conversion process impairs autophagosome structure and disrupts their vacuolar transport, leading to a significant reduction in autophagic flux and plant survival rate. Our findings suggest that the conversion pathway might be an integral part of the autophagy program unique to plants.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Autofagosomas , Autofagia , Vacuolas , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vacuolas/metabolismo , Autofagosomas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Transporte Vesicular/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Proteínas de Unión a GTP rab7 , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética

RESUMEN

Aluminum (Al) stress is a significant issue in acidic soils, severely affecting crop growth and yield. Rice is notably resilient to Al toxicity, yet the internal tolerance mechanisms remain inadequately addressed. Here, we examined the role of OsTIP2;1, a tonoplast-bound intrinsic protein (TIP), in rice's internal Al detoxification. Our findings reveal that OsTIP2;1 expression was quickly and explicitly activated by Al ions in roots but not in shoots. The OsTIP2;1-GFP protein localizes to the tonoplast in plant and yeast cells. Non-functional ostip2;1 rice mutants were more vulnerable to Al toxicity. In the roots, the ostip2;1 mutants exhibited considerably lower levels of Al in the cell sap, primarily the vacuolar contents, than in the wild-type plant. Moreover, the ostip2;1 mutants showed reduced Al accumulation in the roots but increased translocation to the shoots. Heterologous expression of tonoplast-localized OsTIP2;1 in yeast led to enhanced Al tolerance, suggesting that OsTIP2;1 facilitates Al sequestration to the vacuole. These findings indicate that OsTIP2;1 mediates internal detoxification by transporting Al into the vacuole in the root and restricting its transport to above-ground tissues, thus contributing to Al resistance in rice.

Asunto(s)

Aluminio , Oryza , Proteínas de Plantas , Raíces de Plantas , Oryza/metabolismo , Oryza/genética , Aluminio/toxicidad , Aluminio/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/genética , Vacuolas/metabolismo , Regulación de la Expresión Génica de las Plantas , Mutación , Inactivación Metabólica

RESUMEN

Phagocytosis is an essential physiological mechanism; its impairment is associated with many diseases. A highly smart particle is required for understanding detailed sequential cellular events in phagocytosis. Recently, we identified an Indian traditional medicine named Godanti Bhasma (GB), a bioactive calcium sulfate particle prepared by thermo-transformation ofgypsum. Thermal processing of the gypsum transforms its native physicochemical properties by removing water molecules into the anhydrous GB, which was confirmed by Raman and FT-IR spectroscopy. GB particle showed a 0.5-5 µm size range and a neutral surface charge. Exposure of mammalian cells to GB particles showed a rapid cellular uptake through phagocytosis and induced massive cytoplasmic vacuolation in cells. Interestingly, no cellular uptake and cytoplasmic vacuolation were observed with the parent gypsum particle. The presence of the GB particles in intra-vacuolar space was confirmed using FESEM coupled with EDX. Flow cytometry analysis and live tracking of GB-treated cells showed particle internalization, vacuole formation, particle dissolution, and later vacuolar turnover. Quantification of GB-induced vacuolation was done using neutral red uptake assay in cells. Treatment of lysosomal inhibitors (BFA1 or CQ) with GB could not induce vacuolation, suggesting the requirement of an acidic environment for the vacuolation. In the mimicking experiment, GB particle dissolution in acidic cell-free solution suggested that degradation of GB occurs by acidic pH inside the cell vacuole. Vacuole formation generally accompanies with cell death, whereas GB-induced massive vacuolation does not cause cell death. Moreover, the cell divides and proliferates with the vacuolar process, intra-vacuolar cargo degradation, and eventually vacuolar turnover. Taken together, the sequential cellular events in this study suggest that GB can be used as a smart particle for phagocytosis assay development in animal cells.

Asunto(s)

Fagocitosis , Vacuolas , Fagocitosis/efectos de los fármacos , Vacuolas/efectos de los fármacos , Vacuolas/metabolismo , Animales , Humanos , Ratones , Citoplasma/metabolismo , Citoplasma/efectos de los fármacos , Lisosomas/metabolismo , Lisosomas/efectos de los fármacos

RESUMEN

We previously reported autophagy-mediated degradation of nuclei, nucleophagy, in the filamentous fungus Aspergillus oryzae. In this study, we examined whether nuclei are degraded as a whole. We generated A. oryzae mutants deleted for orthologs of Saccharomyces cerevisiae YPT7 and ATG15 which are required, respectively, for autophagosome-vacuole fusion and vacuolar degradation of autophagic bodies. Degradation of histone H2B-EGFP under starvation conditions was greatly decreased in the ΔAoypt7 and ΔAoatg15 mutants. Fluorescence and electron microscopic observations showed that autophagosomes and autophagic bodies surrounding the entire nuclei were accumulated in the cytoplasm of ΔAoypt7 and the vacuole of ΔAoatg15, respectively. These results indicate that nuclei are engulfed in the autophagosomes as a whole and transported/released into the vacuolar lumen where they are degraded.

Asunto(s)

Aspergillus oryzae , Autofagosomas , Proteínas Fúngicas , Vacuolas , Vacuolas/metabolismo , Aspergillus oryzae/genética , Aspergillus oryzae/metabolismo , Autofagosomas/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Núcleo Celular/metabolismo , Núcleo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Autofagia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Proteínas de Unión al GTP rab

RESUMEN

Plants have developed precise defense mechanisms against cadmium (Cd) stress, with vacuolar compartmentalization of Cd2+ being a crucial process in Cd detoxification. The transport of Cd into vacuoles by these cation / H+ antiporters is powered by the pH gradient created by proton pumps. In this study, the full-length cDNA of a vacuolar H+-pyrophosphatase (V-PPase) gene from Boehmeria nivea (ramie), BnVP1, was isolated using the rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of BnVP1 is 2292 bp, encoding a 763 amino acid V-PPase protein with 15 predicted transmembrane domains. Sequence alignment and phylogenetic analysis revealed that BnVP1 belongs to the Type I V-PPase family. Quantitative RT-PCR assays demonstrated that BnVP1 expression was significantly higher in ramie roots than in shoots. Cd treatments markedly induced BnVP1 expression in both roots and leaves of ramie seedlings, with a more pronounced effect in roots. Additionally, BnVP1 expression was significantly upregulated by the plant hormone methyl jasmonate (MeJA). Heterologous expression of BnVP1 in transgenic Arabidopsis significantly enhanced V-PPase activity in the roots. The growth performance, root elongation, and total chlorophyll content of transgenic plants with high tonoplast H+-PPase (V-PPase) activity were superior to those of wild-type plants. Overexpression of BnVP1 reduced membrane lipid peroxidation and ion leakage, and significantly increased Cd accumulation in the roots of transgenic Arabidopsis seedlings. This study provides new genetic resources for the phytoremediation of Cd-contaminated farmland.

Asunto(s)

Arabidopsis , Boehmeria , Cadmio , Regulación de la Expresión Génica de las Plantas , Pirofosfatasa Inorgánica , Filogenia , Plantas Modificadas Genéticamente , Vacuolas , Arabidopsis/genética , Cadmio/metabolismo , Cadmio/toxicidad , Plantas Modificadas Genéticamente/genética , Pirofosfatasa Inorgánica/genética , Pirofosfatasa Inorgánica/metabolismo , Vacuolas/metabolismo , Boehmeria/genética , Boehmeria/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/efectos de los fármacos , Secuencia de Aminoácidos , Ciclopentanos/farmacología , Ciclopentanos/metabolismo , Oxilipinas/farmacología , Oxilipinas/metabolismo , Acetatos

RESUMEN

Monoterpenoid indole alkaloid (MIA) biosynthesis in Catharanthus roseus is a paragon of the spatiotemporal complexity achievable by plant specialized metabolism. Spanning a range of tissues, four cell types, and five cellular organelles, MIA metabolism is intricately regulated and organized. This high degree of metabolic differentiation requires inter-cellular and organellar transport, which remains understudied. Here, we have characterized a vacuolar importer of secologanin belonging to the multidrug and toxic compound extrusion (MATE) family, named CrMATE1. Phylogenetic analyses of MATEs suggested a role in alkaloid transport for CrMATE1, and in planta silencing in two varieties of C. roseus resulted in a shift in the secoiridoid and MIA profiles. Subcellular localization of CrMATE1 confirmed tonoplast localization. Biochemical characterization was conducted using the Xenopus laevis oocyte expression system to determine substrate range, directionality, and rate. We can confirm that CrMATE1 is a vacuolar importer of secologanin, translocating 1 mM of substrate within 25 min. The transporter displayed strict directionality and specificity for secologanin and did not accept other secoiridoid substrates. The unique substrate-specific activity of CrMATE1 showcases the utility of transporters as gatekeepers of pathway flux, mediating the balance between a defense arsenal and cellular homeostasis.

Asunto(s)

Catharanthus , Proteínas de Plantas , Alcaloides de Triptamina Secologanina , Vacuolas , Catharanthus/metabolismo , Catharanthus/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Vacuolas/metabolismo , Alcaloides de Triptamina Secologanina/metabolismo , Animales , Filogenia , Xenopus laevis/metabolismo , Transporte Biológico , Oocitos/metabolismo , Glucósidos Iridoides

RESUMEN

The plasma membrane (PM) is constantly exposed to various stresses from the extracellular environment, such as heat and oxidative stress. These stresses often cause the denaturation of membrane proteins and destabilize PM integrity, which is essential for normal cell viability and function. For maintenance of PM integrity, most eukaryotic cells have the PM quality control (PMQC) system, which removes damaged membrane proteins by endocytosis. Removal of damaged proteins from the PM by ubiquitin-mediated endocytosis is a key mechanism for the maintenance of PM integrity, but the importance of the early endosome in the PMQC system is still not well understood. Here we show that key proteins in early/sorting endosome function, Vps21p (yeast Rab5), Vps15p (phosphatidylinositol-3 kinase subunit), and Vps3p/8p (CORVET complex subunits), are involved in maintaining PM integrity. We found that Vps21p-enriched endosomes change the localization in the vicinity of the PM in response to heat stress and then rapidly fuse and form the enlarged compartments to efficiently transport Can1p to the vacuole. Additionally, we show that the deubiquitinating enzyme Doa4p is also involved in the PM integrity and its deletion causes the mislocalization of Vps21p to the vacuolar lumen. Interestingly, in cells lacking Doa4p or Vps21p, the amounts of free ubiquitin are decreased, and overexpression of ubiquitin restored defective cargo internalization in vps9Δ cells, suggesting that defective PM integrity in vps9Δ cells is caused by lack of free ubiquitin.

Asunto(s)

Membrana Celular , Endocitosis , Endosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Unión al GTP rab5 , Endocitosis/fisiología , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab5/metabolismo , Proteínas de Unión al GTP rab5/genética , Membrana Celular/metabolismo , Endosomas/metabolismo , Respuesta al Choque Térmico/fisiología , Vacuolas/metabolismo , Vacuolas/genética , Calor , Ubiquitina/metabolismo , Proteínas de Unión al GTP rab

RESUMEN

Here, we report a novel role for the yeast lysine acetyltransferase NuA4 in regulating phospholipid availability for organelle morphology. Disruption of the NuA4 complex results in 70% of cells displaying nuclear deformations and nearly 50% of cells exhibiting vacuolar fragmentation. Cells deficient in NuA4 also show severe defects in the formation of nuclear-vacuole junctions (NJV), as well as a decrease in piecemeal microautophagy of the nucleus (PMN). To determine the cause of these defects we focused on Pah1, an enzyme that converts phosphatidic acid into diacylglycerol, favoring accumulation of lipid droplets over phospholipids that are used for membrane expansion. NuA4 subunit Eaf1 was required for Pah1 localization to the inner nuclear membrane and artificially tethering of Pah1 to the nuclear membrane rescued nuclear deformation and vacuole fragmentation defects, but not defects related to the formation of NVJs. Mutation of a NuA4-dependent acetylation site on Pah1 also resulted in aberrant Pah1 localization and defects in nuclear morphology and NVJ. Our work suggests a critical role for NuA4 in organelle morphology that is partially mediated through the regulation of Pah1 subcellular localization.

Asunto(s)

Núcleo Celular , Metabolismo de los Lípidos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacuolas , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Núcleo Celular/metabolismo , Histona Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Fosfatidato Fosfatasa/metabolismo , Fosfatidato Fosfatasa/genética , Acetilación , Membrana Nuclear/metabolismo , Fosfolípidos/metabolismo , Mutación

RESUMEN

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high-resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and various membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label-free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

Asunto(s)

Debaryomyces , Proteómica/métodos , Vacuolas/ultraestructura , Vacuolas/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Saccharomyces cerevisiae/ultraestructura

RESUMEN

Fusarium graminearum is an important plant pathogen that causes head blight in cereal crops such as wheat, barley, and rice worldwide. In this study, we identified and functionally characterized FgVAC1, an essential gene in F. graminearum that encodes a Rab5 effector involved in membrane tethering functions. The essentiality of FgVAC1 was confirmed through a conditional promoter replacement strategy using the zearalenone-inducible promoter (PZEAR). Cytological analyses revealed that FgVac1 colocalizes with FgRab51 on early endosomes and regulates the proper transport of the vacuolar hydrolase FgCpy1 to the vacuole. Suppression of FgVAC1 led to inhibited vegetative growth, reduced asexual and sexual reproduction, decreased deoxynivalenol (DON) biosynthesis, and diminished pathogenicity. Our findings highlight the significant role of FgVac1 in vacuolar protein sorting, fungal development, and plant infection in F. graminearum.

Asunto(s)

Proteínas Fúngicas , Fusarium , Aparato de Golgi , Enfermedades de las Plantas , Vacuolas , Fusarium/genética , Fusarium/patogenicidad , Fusarium/metabolismo , Fusarium/crecimiento & desarrollo , Enfermedades de las Plantas/microbiología , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Vacuolas/metabolismo , Vacuolas/microbiología , Aparato de Golgi/metabolismo , Tricotecenos/metabolismo , Triticum/microbiología , Genes Esenciales , Transporte de Proteínas , Oryza/microbiología

RESUMEN

Coxiella burnetii is a highly infectious, Gram-negative, obligate intracellular bacterium and the causative agent of human Q fever. The Coxiella Containing Vacuole (CCV) is a modified phagolysosome that forms through fusion with host endosomes and lysosomes. While an initial acidic pH < 4.7 is essential to activate Coxiella metabolism, the mature, growth-permissive CCV has a luminal pH of ~5.2 that remains stable throughout infection. Inducing CCV acidification to a lysosomal pH (~4.7) causes Coxiella degradation, suggesting that Coxiella regulates CCV pH. Supporting this hypothesis, Coxiella blocks host lysosomal biogenesis, leading to fewer host lysosomes available to fuse with the CCV. Host cell lysosome biogenesis is primarily controlled by the transcription factor EB (TFEB), which binds Coordinated Lysosomal Expression And Regulation (CLEAR) motifs upstream of genes involved in lysosomal biogenesis and function. TFEB is a member of the microphthalmia/transcription factor E (MiT/TFE) protein family, which also includes MITF, TFE3, and TFEC. This study examines the roles of MiT/TFE proteins during Coxiella infection. We found that in cells lacking TFEB, both Coxiella growth and CCV size increase. Conversely, TFEB overexpression or expression in the absence of other family members leads to significantly less bacterial growth and smaller CCVs. TFE3 and MITF do not appear to play a significant role during Coxiella infection. Surprisingly, we found that Coxiella actively blocks TFEB nuclear translocation in a Type IV Secretion System-dependent manner, thus decreasing lysosomal biogenesis. Together, these results suggest that Coxiella inhibits TFEB nuclear translocation to limit lysosomal biogenesis, thus avoiding further CCV acidification through CCV-lysosomal fusion. IMPORTANCE: The obligate intracellular bacterial pathogen Coxiella burnetii causes the zoonotic disease Q fever, which is characterized by a debilitating flu-like illness in acute cases and life-threatening endocarditis in patients with chronic disease. While Coxiella survives in a unique lysosome-like vacuole called the Coxiella Containing Vacuole (CCV), the bacterium inhibits lysosome biogenesis as a mechanism to avoid increased CCV acidification. Our results establish that transcription factor EB (TFEB), a member of the microphthalmia/transcription factor E (MiT/TFE) family of transcription factors that regulate lysosomal gene expression, restricts Coxiella infection. Surprisingly, Coxiella blocks TFEB translocation from the cytoplasm to the nucleus, thus downregulating the expression of lysosomal genes. These findings reveal a novel bacterial mechanism to regulate lysosomal biogenesis.

Asunto(s)

Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Coxiella burnetii , Lisosomas , Fiebre Q , Coxiella burnetii/genética , Coxiella burnetii/metabolismo , Coxiella burnetii/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Lisosomas/metabolismo , Humanos , Fiebre Q/microbiología , Animales , Vacuolas/metabolismo , Vacuolas/microbiología , Ratones , Núcleo Celular/metabolismo , Transporte de Proteínas