RESUMO
Autophagy is essential for maintaining glucose homeostasis. However, the mechanism by which cells sense and respond to glucose starvation to induce autophagy remains incomplete. Here, we show that calcium serves as a fundamental triggering signal that connects environmental sensing to the formation of the autophagy initiation complex during glucose starvation. Mechanistically, glucose starvation instigates the release of vacuolar calcium into the cytoplasm, thus triggering the activation of Rck2 kinase. In turn, Rck2-mediated Atg11 phosphorylation enhances Atg11 interactions with Bmh1/2 bound to the Snf1-Sip1-Snf4 complex, leading to recruitment of vacuolar membrane-localized Snf1 to the PAS and subsequent Atg1 activation, thereby initiating autophagy. We also identified Glc7, a protein phosphatase-1, as a critical regulator of the association between Bmh1/2 and the Snf1 complex. We thus propose that calcium-triggered Atg11-Bmh1/2-Snf1 complex assembly initiates autophagy by controlling Snf1-mediated Atg1 activation in response to glucose starvation.
Assuntos
Autofagia , Glucose , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Cálcio/metabolismo , Glucose/metabolismo , Complexos Multiproteicos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Vacúolos/genéticaRESUMO
Together with its ß-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, "HOD syndrome"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.
Assuntos
Canais de Cloreto , Doenças por Armazenamento dos Lisossomos , Lisossomos , Humanos , Masculino , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Mutação com Ganho de Função , Células HEK293 , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/metabolismo , Lisossomos/genética , Proteínas de Membrana , Mutação de Sentido Incorreto , Fosfatos de Fosfatidilinositol/metabolismo , Ubiquitina-Proteína Ligases , Vacúolos/metabolismo , Vacúolos/genética , Vacúolos/patologiaRESUMO
Dominant mutations in CACNA1S gene mainly causes hypokalemic periodic paralysis (PP)(hypoPP). A 68-year-old male proband developed a progressive proximal weakness from the age of 35. Muscle biopsy showed atrophic fibers with vacuoles containing tubular aggregates. Exome sequencing revealed a heterozygous p.R528H (c.1583G>A) mutation in the CACNA1S gene. CACNA1S-related HypoPP evolving to persistent myopathy in late adulthood is a well-known clinical condition. However, isolated progressive myopathy (without PP) was only exceptionally reported and never with an early onset. Reporting a case of early onset CACNA1S-related myopathy in a patient with no HypoPP we intend to alert clinicians to consider it in the differential diagnosis of younger adult-onset myopathies especially when featuring vacuolar changes.
Assuntos
Canais de Cálcio Tipo L , Mutação , Humanos , Masculino , Idoso , Canais de Cálcio Tipo L/genética , Doenças Musculares/genética , Vacúolos/patologia , Vacúolos/genética , Músculo Esquelético/patologia , Idade de Início , Paralisia Periódica Hipopotassêmica/genéticaRESUMO
The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.
Assuntos
Proteínas R-SNARE , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Vacúolos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Vacúolos/genética , Proteínas R-SNARE/metabolismo , Proteínas R-SNARE/genética , Fusão de Membrana , Exocitose , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , MutaçãoRESUMO
Vacuolar malic acid accumulation largely determines fruit acidity, a key trait for the taste and flavor of apple and other fleshy fruits. Aluminum-activated malate transporter 9 (ALMT9/Ma1) underlies a major genetic locus, Ma, for fruit acidity in apple, but how the protein transports malate across the tonoplast is unclear. Here, it is shown that overexpression of the coding sequence of Ma1 (Ma1α) drastically decreases fruit acidity in "Royal Gala" apple, leading to uncovering alternative splicing underpins Ma1's function. Alternative splicing generates two isoforms: Ma1ß is 68 amino acids shorter with much lower expression than the full-length protein Ma1α. Ma1ß does not transport malate itself but interacts with the functional Ma1α to form heterodimers, creating synergy with Ma1α for malate transport in a threshold manner (When Ma1ß/Ma1α ≥ 1/8). Overexpression of Ma1α triggers feedback inhibition on the native Ma1 expression via transcription factor MYB73, decreasing the Ma1ß level well below the threshold that leads to significant reductions in Ma1 function and malic acid accumulation in fruit. Overexpression of Ma1α and Ma1ß or genomic Ma1 increases both isoforms proportionally and enhances fruit malic acid accumulation. These findings reveal an essential role of alternative splicing in ALMT9-mediated malate transport underlying apple fruit acidity.
Assuntos
Processamento Alternativo , Malatos , Malus , Malatos/metabolismo , Processamento Alternativo/genética , Malus/genética , Malus/metabolismo , Frutas/metabolismo , Frutas/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Vacúolos/metabolismo , Vacúolos/genética , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
Contractile vacuoles (CVs), enigmatic osmoregulatory organelles, share common characteristics, such as a requirement for RAB11 and high levels of V-ATPase. These commonalities suggest a conserved evolutionary origin for the CVs with implications for understanding of the last common ancestor of eukaryotes and eukaryotic diversification more broadly. A taxonomically broader sampling of CV-associated machinery is required to address this question further. We used a transcriptomics-based approach to identify CV-associated gene products in Dictyostelium discoideum. This approach was first validated by assessing a set of known CV-associated gene products, which were significantly upregulated following hypo-osmotic exposure. Moreover, endosomal and vacuolar gene products were enriched in the upregulated gene set. An upregulated SNARE protein (NPSNB) was predominantly plasma membrane localised and enriched in the vicinity of CVs, supporting the association with this organelle found in the transcriptomic analysis. We therefore confirm that transcriptomic approaches can identify known and novel players in CV function, in our case emphasizing the role of endosomal vesicle fusion machinery in the D. discoideum CV and facilitating future work to address questions regarding the deep evolution of eukaryotic organelles.
Assuntos
Dictyostelium , Vacúolos , Vacúolos/genética , Vacúolos/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Endossomos/genética , Endossomos/metabolismo , Transporte Biológico , Membrana Celular/metabolismoRESUMO
It is widely stated in the literature that closed mature autophagosomes (APs) fuse with lysosomes/vacuoles during macroautophagy/autophagy. Previously, we showed that unclosed APs accumulated as clusters outside vacuoles in Vps21/Rab5 and ESCRT mutants after a short period of nitrogen starvation. However, the fate of such unclosed APs remains unclear. In this study, we used a combination of cellular and biochemical approaches to show that unclosed double-membrane APs entered vacuoles and formed unclosed single-membrane autophagic bodies after prolonged nitrogen starvation or rapamycin treatment. Vacuolar hydrolases, vacuolar transport chaperon (VTC) proteins, Ypt7, and Vam3 were all involved in the entry of unclosed double-membrane APs into vacuoles in Vps21-mutant cells. Overexpression of the vacuolar hydrolases, Pep4 or Prb1, or depletion of most VTC proteins promoted the entry of unclosed APs into vacuoles in Vps21-mutant cells, whereas depletion of Pep4 and/or Prb1 delayed the entry into vacuoles. In contrast to the complete infertility of diploid cells of typical autophagy mutants, diploid cells of Vps21 mutant progressed through meiosis to sporulation, benefiting from the entry of unclosed APs into vacuoles after prolonged nitrogen starvation. Overall, these data represent a new observation that unclosed double-membrane APs can enter vacuoles after prolonged autophagy induction, most likely as a survival strategy.
Assuntos
Proteínas de Saccharomyces cerevisiae , Vacúolos , Autofagossomos/metabolismo , Autofagia/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Hidrolases/metabolismo , Chaperonas Moleculares/metabolismo , Nitrogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirolimo/metabolismo , Sirolimo/farmacologia , Vacúolos/genética , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Diverse physiology relies on receptor and transporter protein down-regulation and degradation mediated by ESCRTs. Loss-of-function mutations in human ESCRT genes linked to cancers and neurological disorders are thought to block this process. However, when homologous mutations are introduced into model organisms, cells thrive and degradation persists, suggesting other mechanisms compensate. To better understand this secondary process, we studied degradation of transporter (Mup1) or receptor (Ste3) proteins when ESCRT genes (VPS27, VPS36) are deleted in Saccharomyces cerevisiae using live-cell imaging and organelle biochemistry. We find that endocytosis remains intact, but internalized proteins aberrantly accumulate on vacuolar lysosome membranes within cells. Here they are sorted for degradation by the intralumenal fragment (ILF) pathway, constitutively or when triggered by substrates, misfolding or TOR activation in vivo and in vitro. Thus, the ILF pathway functions as fail-safe layer of defense when ESCRTs disregard their clients, representing a two-tiered system that ensures degradation of surface polytopic proteins.
Assuntos
Proteínas de Saccharomyces cerevisiae , Humanos , Proteólise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte/metabolismoRESUMO
In Arabidopsis, vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein-protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide (468RVAAA472) of cruciferin 1, an isoform of 12S globulins. The 468RVA470 motif forms a parallel ß-sheet with the switch III residues (127TMD129) of VSR1-PA, and the 471AA472 motif docks to a cradle formed by the cargo-binding loop (95RGDCYF100), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor-cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge-charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Substituição de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cristalografia por Raios X , Mutação de Sentido Incorreto , Conformação Proteica em Folha beta , Domínios Proteicos , Transporte Proteico , Protoplastos/química , Protoplastos/metabolismo , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Proteínas de Armazenamento de Sementes/metabolismo , Relação Estrutura-Atividade , Vacúolos/química , Vacúolos/genética , Vacúolos/metabolismoAssuntos
Mutação de Sentido Incorreto , Células Progenitoras Mieloides , Síndrome de Turner , Enzimas Ativadoras de Ubiquitina , Vacúolos , Idoso , Substituição de Aminoácidos , Feminino , Humanos , Células Progenitoras Mieloides/metabolismo , Células Progenitoras Mieloides/patologia , Síndrome de Turner/genética , Síndrome de Turner/metabolismo , Síndrome de Turner/patologia , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismo , Vacúolos/genética , Vacúolos/metabolismo , Vacúolos/patologiaRESUMO
Sorting nexin 10 (SNX10) induces formation of vacuoles participating in the endosome morphogenesis in mammalian cells, but the key amino acids involved in this function have not been fully identified. In this study, point mutations were introduced to the conserved region of the SNX10 PX domain to elucidate the function of these key amino acid residues. The number of vacuoles in the R53A mutant was partially decreased, while the R52A and R51A mutants completely lacked the vacuoles. All mutant proteins lost the phosphatidylinositol 3-phosphate (PtdIns3P)-binding ability and endosomal localization. Retargeting the mutants to the endosomes rescued partially or fully the vacuole-inducing ability in the R51A and R53A mutants, respectively, but not in the R52A mutant. No vacuoles were induced when the R51A mutant was targeted to other organelles. Structural analysis showed that Arg53 is responsible for the PtdIns(3)P binding, whereas Arg51 and Arg52 contribute to the structural integrity of SNX10. We conclude that the disruption of the key residues affects the structure and function of SNX10 and that induction of vacuole formation by SNX10 depends on its endosomal location.
Assuntos
Endossomos/metabolismo , Mutação de Sentido Incorreto , Nexinas de Classificação/metabolismo , Vacúolos/metabolismo , Substituição de Aminoácidos , Endossomos/genética , Células HeLa , Humanos , Domínios Proteicos , Nexinas de Classificação/genética , Vacúolos/genéticaRESUMO
Toxoplasma gondii is a ubiquitous, intracellular parasite that envelops its parasitophorous vacuole with a protein-laden membrane (PVM). The PVM is critical for interactions with the infected host cell, such as nutrient transport and immune defense. Only a few parasite and host proteins have so far been identified on the host-cytosolic side of the Toxoplasma PVM. We report here the use of human foreskin fibroblasts expressing the proximity-labeling enzyme miniTurbo, fused to a domain that targets it to this face of the PVM, in combination with quantitative proteomics to specifically identify proteins present at this interface. Out of numerous human and parasite proteins with candidate PVM localization, we validate three parasite proteins (TGGT1_269950 [GRA61], TGGT1_215360 [GRA62], and TGGT1_217530 [GRA63]) and four new host proteins (PDCD6IP/ALIX, PDCD6, CC2D1A, and MOSPD2) as localized to the PVM in infected human cells through immunofluorescence microscopy. These results significantly expand our knowledge of proteins present at the Toxoplasma PVM and, given that three of the validated host proteins are components of the ESCRT (endosomal sorting complexes required for transport) machinery, they further suggest that novel biology is operating at this crucial host-pathogen interface. IMPORTANCEToxoplasma is an intracellular pathogen which resides and replicates inside a membrane-bound vacuole in infected cells. This vacuole is modified by both parasite and host proteins which participate in a variety of host-parasite interactions at this interface, including nutrient exchange, effector transport, and immune modulation. Only a small number of parasite and host proteins present at the vacuolar membrane and exposed to the host cytosol have thus far been identified. Here, we report the identification of several novel parasite and host proteins present at the vacuolar membrane using enzyme-catalyzed proximity-labeling, significantly increasing our knowledge of the molecular players present and novel biology occurring at this crucial interface.
Assuntos
Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Proteínas de Protozoários/metabolismo , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Vacúolos/parasitologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Interações Hospedeiro-Parasita , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transporte Proteico , Proteínas de Protozoários/genética , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Toxoplasma/genética , Toxoplasmose/genética , Vacúolos/genética , Vacúolos/metabolismoRESUMO
Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.
Assuntos
Proteínas de Bactérias/metabolismo , Endossomos/enzimologia , Flavoproteínas/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/enzimologia , Macrófagos/enzimologia , Monoéster Fosfórico Hidrolases/metabolismo , Vacúolos/enzimologia , Animais , Proteínas de Bactérias/genética , Células COS , Chlorocebus aethiops , Endocitose , Endossomos/genética , Endossomos/microbiologia , Evolução Molecular , Feminino , Flavoproteínas/genética , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Legionella pneumophila/genética , Legionella pneumophila/crescimento & desenvolvimento , Doença dos Legionários/microbiologia , Macrófagos/microbiologia , Camundongos , Mutação , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolases/genética , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Células U937 , Vacúolos/genética , Vacúolos/microbiologia , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
The hexameric HOPS (homotypic fusion and protein sorting) complex is a conserved tethering complex at the lysosome-like vacuole, where it mediates tethering and promotes all fusion events involving this organelle. The Vps39 subunit of this complex also engages in a membrane contact site between the vacuole and the mitochondria, called vCLAMP. Additionally, four subunits of HOPS are also part of the endosomal CORVET tethering complex. Here, we analyzed the partition of HOPS and CORVET subunits between the different complexes by tracing their localization and function. We find that Vps39 has a specific role in vCLAMP formation beyond tethering, and that vCLAMPs and HOPS compete for the same pool of Vps39. In agreement, we find that the CORVET subunit Vps3 can take the position of Vps39 in HOPS. This endogenous pool of a Vps3-hybrid complex is affected by Vps3 or Vps39 levels, suggesting that HOPS and CORVET assembly is dynamic. Our data shed light on how individual subunits of tethering complexes such as Vps39 can participate in other functions, while maintaining the remaining subcomplex available for its function in tethering and fusion.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Lisossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Lisossomos/genética , Mitocôndrias/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/genética , Proteínas de Transporte Vesicular/genéticaRESUMO
Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. Plants' responses/adaptations to stress often involve changes in the distribution and sorting of specific proteins and molecules. Still, little attention has been given to the molecular mechanisms controlling these rearrangements. We tested the hypothesis that plants respond to stress by remodelling their endomembranes and adapting their trafficking pathways. We focused on the molecular machinery behind organelle biogenesis and protein trafficking under abiotic stress conditions, evaluating their effects at the subcellular level, by looking at ultrastructural changes and measuring the expression levels of genes involved in well-known intracellular routes. The results point to a differential response of the endomembrane system, showing that the genes involved in the pathway to the Protein Storage Vacuole and the exocyst-mediated routes are upregulated. In contrast, the ones involved in the route to the Lytic Vacuole are downregulated. These changes are accompanied by morphological alterations of endomembrane compartments. The data obtained demonstrate that plants' response to abiotic stress involves the differential expression of genes related to protein trafficking machinery, which can be connected to the activation/deactivation of specific intracellular sorting pathways and lead to alterations in the cell ultrastructure.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Estresse Fisiológico/genética , Regulação para Cima/genética , Vacúolos/genética , Proteínas de Transporte Vesicular/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Parede Celular/metabolismo , Citoplasma/metabolismo , Expressão Gênica , Membranas Intracelulares/metabolismo , Biogênese de Organelas , Organelas/genética , Organelas/metabolismo , Transporte Proteico/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismoAssuntos
Genes Ligados ao Cromossomo X , Doenças Genéticas Ligadas ao Cromossomo X/genética , Enzimas Ativadoras de Ubiquitina/genética , Idoso , Doenças Genéticas Ligadas ao Cromossomo X/patologia , Humanos , Masculino , Mutação de Sentido Incorreto , Mutação Puntual , Síndrome , Vacúolos/genética , Vacúolos/patologiaRESUMO
Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca2+ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca2+ concentration. However, the mechanism(s) of Ca2+-dependent regulation of TRPY1 and possible contribution(s) of Ca2+-binding proteins are yet not well understood. Our results demonstrate a Ca2+-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1-6 yeast strain, carrying a non-Ca2+-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1-6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca2+ influx and Ca2+ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca2+-CaM required the cytoplasmic amino acid stretch E33-Y92. In summary, our results show that TRPY1 is under inhibitory control of Ca2+-CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca2+-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca2+-CaM.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Canais de Cátion TRPC/metabolismo , Vacúolos/metabolismo , Cálcio/química , Calmodulina/antagonistas & inibidores , Calmodulina/química , Calmodulina/genética , Células HEK293 , Humanos , Domínios Proteicos , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Sesterterpenos/farmacologia , Canais de Cátion TRPC/química , Canais de Cátion TRPC/genética , Vacúolos/química , Vacúolos/genéticaRESUMO
ABCC multidrug resistance-associated proteins (ABCCs/MRPs), a subfamily of ABC transporters, are involved in multiple physiological processes. Although these proteins have been characterized in some plants, limited efforts have been made to address their possible roles in Rehmannia glutinosa, a medicinal plant. Here, we scanned R. glutinosa transcriptome sequences and identified 18 RgABCC genes by in silico analysis. Sequence alignment revealed that the RgABCCs were closely phylogenetically related and highly conserved with other plant ABCCs/MRPs. Subcellular localization revealed that most of the RgABCCs were deposited in vacuoles and a few in plasma membranes. Tissue-specific expression of the RgABCCs indicated significant specific accumulation patterns, implicating their roles in the respective tissues. Differential temporal expression patterns of the RgABCCs exhibited their potential roles during root development. Various abiotic stress and hormone treatment experiments indicated that some RgABCCs could be transcriptionally regulated in roots. Furthermore, the transcription of several RgABCCs in roots was strongly activated by cadmium (Cd), suggesting possible roles under heavy metal stresses. Functional analysis of RgABCC1 heterologous expression revealed that it may increase the tolerance to Cd in yeast, implying its Cd transport activity. Our study provides a detailed inventory and molecular characterization of the RgABCCs and valuable information for exploring their functions in R. glutinosa.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/biossíntese , Raízes de Plantas/metabolismo , Rehmannia/metabolismo , Transcriptoma , Transportadores de Cassetes de Ligação de ATP/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/genética , Rehmannia/genética , Estresse Fisiológico/fisiologia , Vacúolos/genética , Vacúolos/metabolismoRESUMO
The lysosome (or vacuole in fungi and plants) is an essential organelle for nutrient sensing and cellular homeostasis. In response to environmental stresses such as starvation, the yeast vacuole can adjust its membrane composition by selectively internalizing membrane proteins into the lumen for degradation. Regarding the selective internalization mechanism, two competing models have been proposed. One model suggests that the ESCRT machinery is responsible for the sorting. In contrast, the ESCRT-independent intralumenal fragment (ILF) pathway proposes that the fragment generated by homotypic vacuole fusion is responsible for the sorting. Here, we applied a microfluidics-based imaging method to capture the complete degradation process in vivo. Combining live-cell imaging with a synchronized ubiquitination system, we demonstrated that ILF cargoes are not degraded through intralumenal fragments. Instead, ESCRTs function on the vacuole membrane to sort them into the lumen for degradation. We further discussed challenges in reconstituting vacuole membrane protein degradation.