Yeast Vacuole Research Articles

Acetate and hypertonic stress stimulate vacuole membrane fission using distinct mechanisms.

Vacuoles in plants and fungi play critical roles in cell metabolism and osmoregulation. To support these functions, vacuoles change their morphology, e.g. they fragment when these organisms are challenged with draught, high salinity or metabolic stress (e.g. acetate accumulation). In turn, morphology reflects an equilibrium between membrane fusion and fission that determines size, shape and copy number. By studying Saccharomyces cerevisiae and its vacuole as models, conserved molecular mechanisms responsible for fusion have been revealed. However, a detailed understanding of vacuole fission and how these opposing processes respond to metabolism or osmoregulation remain elusive. Herein we describe a new fluorometric assay to measure yeast vacuole fission in vitro. For proof–of–concept, we use this assay to confirm that acetate, a metabolic stressor, triggers vacuole fission and show it blocks homotypic vacuole fusion in vitro. Similarly, hypertonic stress induced by sorbitol or glucose caused robust vacuole fission in vitro whilst inhibiting fusion. Using wortmannin to inhibit phosphatidylinositol (PI) -kinases or rGyp1-46 to inactivate Rab–GTPases, we show that acetate stress likely targets PI signaling, whereas osmotic stress affects Rab signaling on vacuole membranes to stimulate fission. This study sets the stage for further investigation into the mechanisms that change vacuole morphology to support cell metabolism and osmoregulation.

Expanding the view of the molecular mechanisms of autophagy pathway.

Autophagy is an evolutionarily conserved multistep degradation mechanism in eukaryotes, that maintains cellular homoeostasis by replenishing cells with nutrients through catabolic lysis of the cytoplasmic components. This critically coordinated pathway involves sequential processing events that begin with initiation, nucleation, and elongation of phagophores, followed by the formation of double-membrane vesicles known as autophagosomes. Finally, autophagosomes migrate towards and fuse with lysosomes in mammals and vacuoles in yeast and plants, for the eventual degradation of the intravesicular cargo. Here, we review the recent advances in our understanding of the molecular events that define the process of autophagy.

Cvm1 is a component of multiple vacuolar contact sites required for sphingolipid homeostasis.

Membrane contact sites are specialized platforms formed between most organelles that enable them to exchange metabolites and influence the dynamics of each other. The yeast vacuole is a degradative organelle equivalent to the lysosome in higher eukaryotes with important roles in ion homeostasis and metabolism. Using a high-content microscopy screen, we identified Ymr160w (Cvm1, for contact of the vacuole membrane 1) as a novel component of three different contact sites of the vacuole: with the nuclear endoplasmic reticulum, the mitochondria, and the peroxisomes. At the vacuole-mitochondria contact site, Cvm1 acts as a tether independently of previously known tethers. We show that changes in Cvm1 levels affect sphingolipid homeostasis, altering the levels of multiple sphingolipid classes and the response of sphingolipid-sensing signaling pathways. Furthermore, the contact sites formed by Cvm1 are induced upon a decrease in sphingolipid levels. Altogether, our work identifies a novel protein that forms multiple contact sites and supports a role of lysosomal contacts in sphingolipid homeostasis.

Characterization of Protein-Membrane Interactions in Yeast Autophagy.

Cells rely on autophagy to degrade cytosolic material and maintain homeostasis. During autophagy, content to be degraded is encapsulated in double membrane vesicles, termed autophagosomes, which fuse with the yeast vacuole for degradation. This conserved cellular process requires the dynamic rearrangement of membranes. As such, the process of autophagy requires many soluble proteins that bind to membranes to restructure, tether, or facilitate lipid transfer between membranes. Here, we review the methods that have been used to investigate membrane binding by the core autophagy machinery and additional accessory proteins involved in autophagy in yeast. We also review the key experiments demonstrating how each autophagy protein was shown to interact with membranes.

Vacuole-Specific Lipid Release for Tracking Intracellular Lipid Metabolism and Transport in Saccharomyces cerevisiae.

Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here, we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in Saccharomyces cerevisiae. Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the endoplasmic reticulum-vacuole-tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.

Metabolism and autophagy in plants-a perfect match.

Autophagy is a eukaryotic cellular transport mechanism that delivers intracellular macromolecules, proteins, and even organelles to a lytic organelle (vacuole in yeast and plants/lysosome in animals) for degradation and nutrient recycling. The process is mediated by highly conserved autophagy-related (ATG) proteins. In plants, autophagy maintains cellular homeostasis under favorable conditions, guaranteeing normal plant growth and fitness. Severe stress such as nutrient starvation and plant senescence further induce it, thus ensuring plant survival under unfavorable conditions by providing nutrients through the removal of damaged or aged proteins, or organelles. In this article, we examine the interplay between metabolism and autophagy, focusing on the different aspects of this reciprocal relationship. We show that autophagy has a strong influence on a range of metabolic processes, whereas at the same time, even single metabolites can activate autophagy. We highlight the involvement of ATG genes in metabolism, examine the role of the macronutrients carbon and nitrogen, and various micronutrients, and take a closer look at how the interaction between autophagy and metabolism impacts on plant phenotypes and yield.

CROP: a retromer‐PROPPIN complex mediating membrane fission in the endo‐lysosomal system

Endo‐lysosomal compartments exchange proteins by fusing, fissioning, and through endosomal transport carriers. Thereby, they sort many plasma membrane receptors and transporters and control cellular signaling and metabolism. How the membrane fission events are catalyzed is poorly understood. Here, we identify the novel CROP complex as a factor acting at this step. CROP joins members of two protein families: the peripheral subunits of retromer, a coat forming endosomal transport carriers, and membrane inserting PROPPINs. Integration into CROP potentiates the membrane fission activity of the PROPPIN Atg18 on synthetic liposomes and confers strong preference for binding PI(3,5)P2, a phosphoinositide required for membrane fission activity. Disrupting CROP blocks fragmentation of lysosome‐like yeast vacuoles in vivo. CROP‐deficient mammalian endosomes accumulate micrometer‐long tubules and fail to export cargo, suggesting that carriers attempt to form but cannot separate from these organelles. PROPPINs compete for retromer binding with the SNX‐BAR proteins, which recruit retromer to the membrane during the formation of endosomal carriers. Transition from retromer–SNX‐BAR complexes to retromer–PROPPIN complexes might hence switch retromer activities from cargo capture to membrane fission.

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

SUMMARYEukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation.

The general mechanisms by which ESCRTs (Endosomal Sorting Complexes Required for Transport) are specifically recruited to various membranes, and how ESCRT subunits are spatially organized remain central questions in cell biology. At the endosome and lysosomes, ubiquitination of membrane proteins triggers ESCRT-mediated substrate recognition and degradation. Using the yeast lysosome/vacuole, we define the principles by which substrate engagement by ESCRTs occurs at this organelle. We find that multivalent interactions between ESCRT-0 and polyubiquitin are critical for substrate recognition at yeast vacuoles, with a lower-valency requirement for cargo engagement at endosomes. Direct recruitment of ESCRT-0 induces dynamic foci on the vacuole membrane and forms fluid condensates in vitro with polyubiquitin. We propose that self-assembly of early ESCRTs induces condensation, an initial step in ESCRT assembly/nucleation at membranes. This property can be tuned specifically at various organelles by modulating the number of binding interactions.

Porin 1 Modulates Autophagy in Yeast.

Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.

The Role of Exosome and the ESCRT Pathway on Enveloped Virus Infection.

The endosomal sorting complex required for transport (ESCRT) system consists of peripheral membrane protein complexes ESCRT-0, -I, -II, -III VPS4-VTA1, and ALIX homodimer. This system plays an important role in the degradation of non-essential or dangerous plasma membrane proteins, the biogenesis of lysosomes and yeast vacuoles, the budding of most enveloped viruses, and promoting membrane shedding of cytokinesis. Recent results show that exosomes and the ESCRT pathway play important roles in virus infection. This review mainly focuses on the roles of exosomes and the ESCRT pathway in virus assembly, budding, and infection of enveloped viruses. The elaboration of the mechanism of exosomes and the ESCRT pathway in some enveloped viruses provides important implications for the further study of the infection mechanism of other enveloped viruses.

Selective increment of phosphatidylserine on the autophagic body membrane in the yeast vacuole.

In yeast cells, the autophagosome is a double-membrane structure; the inner membrane becomes the autophagic body membrane in the vacuole. Vacuolar enzymes degrade the autophagic body. There is no critical information regarding its selective degradation. Using the electron microscopy method, distributions of four phospholipids were examined in the autophagosomal and autophagic body membranes upon autophagy induction. The labeling of phosphatidylserine (PtdSer) in the autophagic body membrane dramatically increased after it converted from the autophagosome, but remained low in the vacuolar membrane. PtdSer in the autophagic body membrane also increased in atg15∆ yeast. These results suggest that the selective increment of PtdSer in the autophagic body, but not the vacuolar, membrane, can explain the selective degradation of the autophagic membrane.

How the vacuole ESCRTs its own proteins to their final destination.

Lysosomes (vacuoles in yeast) are master regulators of metabolism and protein turnover, but how they degrade their own resident proteins is unclear. Recently, multiple models have been proposed explaining yeast vacuole protein sorting, but the role of the ESCRT pathway was unclear. In this JCB issue, work from Yang et al. (https://doi.org/10.1083/jcb.202012104) highlights how the ESCRT pathway localizes to the vacuole surface to execute protein sorting of its resident proteins.

The Effect of Phosphorus Metabolism on the Motion of Saccharomyces cerevisiae Volutin Granules

It is known that moving volutin granules (“dancing bodies”), mechanism of which occurrence remains poorly understood, can be observed in yeast vacuoles. This study was performed to reveal the presence of a connection between moving volutin granules of Saccharomyces cerevisiae and polyphosphate metabolism in conditions of phosphoric starvation and hypercompensation. Methods. Cytological, biochemical, statistical methods were used in the study. Results. It was observed that the inactivation of the PPN1 gene, which encodes exopolyphosphatase Ppn1, resulted in a change in the number of cells with moving volutin granules (“dancing bodies” index) in the studied conditions. The index of “dancing bodies” was almost always lower in mutant CRN strain than in parent CRY strain. Using linear correlation analysis and factor analysis with the method of principal component, it was established that the “dancing bodies” index in both strains had significant correlation coefficients with exopolyphosphatase activity (EPPA) and the content of polyphosphate fractions (polyP). The difference was that this index in parent strain correlated better with the first three fractions of inorganic polyphosphates, while in mutant strain – with polyP4 and EPPA. Conclusions. Obtained data indicated the direct connection of motion of volutin granules with phosphoric metabolism in the studied conditions. It is assumed that the phenomenon of “dancing bodies” may be a consequence of the activity of vacuolar polyphosphatases.

Let it go: mechanisms that detach myosin V from the yeast vacuole.

A major question in cell biology is, how are organelles and macromolecular machines moved within a cell? The delivery of cargoes to the right place at the right time within a cell is critical to cellular health. Failure to do so is often catastrophic for animal physiology and results in diseases of the gut, brain, and skin. In budding yeast, a myosin V motor, Myo2, moves cellular materials from the mother cell into the growing daughter bud. Myo2-based transport ensures that cellular contents are shared during cell division. During transport, Myo2 is often linked to its cargo via cargo-specific adaptor proteins. This simple organism thus serves as a powerful tool to study how myosin V moves cargo, such as organelles. Some critical questions include how myosin V moves along the actin cytoskeleton, or how myosin V attaches to cargo in the mother. Other critical questions include how the cargo is released from myosin V when it reaches its final destination in the bud. Here, we review the mechanisms that regulate the vacuole-specific adaptor protein, Vac17, to ensure that Myo2 delivers the vacuole to the bud and releases it at the right place and the right time. Recent studies have revealed that Vac17 is regulated by ubiquitylation and phosphorylation events that coordinate its degradation and the detachment of the vacuole from Myo2. Thus, multiple post-translational modifications tightly coordinate cargo delivery with cellular events. It is tempting to speculate that similar mechanisms regulate other cargoes and molecular motors.

ESCRT, not intralumenal fragments, sorts ubiquitinated vacuole membrane proteins for degradation.

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.

Partitioning of the mevalonate synthesizing enzymes at ER‐lysosome contacts influences their catalytic efficiency

The last decade of molecular biology is hallmarked by an extraordinary increase in our understanding of previously unappreciated spatial scales in biology. Enzymes and metabolites are partitioned into distinct membrane-bound organelles to spatially regulate metabolism and signaling. More recent discoveries have uncovered that enzymes can also inducibly or permanently compartmentalize within sub-organelle domains such as inter-organelle contact sites (1). However, a key knowledge gap is understanding role that enzyme partitioning plays on regulating enzyme function or pathway flux. Here, we performed a GFP-based screen in Saccharomyces cerevisiae to identify metabolic enzymes that may enrich at sub-organelle domains. We found that during Acute Glucose Restriction (AGR) the rate-limiting enzymes of sterol biogenesis, Hmg1 and Hmg2, partition at the Nucleus-Vacuole Junction (NVJ), an inter-organelle contact site between the nuclear envelope and yeast vacuole. NVJ partitioning of Hmg1/2 correlates with increased de-novo synthesis of sterol-esters. Additionally, Hmg1/2 partitioning can be blocked by deletion of the NVJ tether protein Nvj1. Remarkably, Hmg1/2 is less catalytically efficient in Nvj1 knock-out cells, suggesting a potential link between its spatial compartmentalization and increased enzymatic activity. We further demonstrate that Nvj1-mediated partitioning of Hmg1/2 during AGR contributes to cellular fitness, and coincides with ability to resume growth following glucose starvation. Overall, we have uncovered a previously underappreciated level of spatial regulation for Hmg1/2, and also identified a novel role for inter-organelle contact sites in the regulation of mevalonate metabolism.

Autophagy in normal pituitary and pituitary tumor cells and its potential role in the actions of somatostatin receptor ligands in acromegaly.

Autophagy is an evolutionary conserved process for the self-degradation and recycling of cellular components in the cytoplasm. It is involved in both physiological and pathological conditions. In detail, the term "autophagy" refers to intracellular degradative pathways that lead to packaging and deliver of cellular components to lysosomes or to plant and yeast vacuoles. Autophagy is triggered by a variety of stimuli like nutrient deprivation, hypoxia, mitochondrial dysfunction, endoplasmic reticulum stress, and is regulated by immune- and hormonal factors. The role of autophagy in tumor cells is complex. Indeed, autophagy may act as a tumor suppressor as well as a tumor survival factor, in a context-dependent manner. The research into autophagy in normal pituitary and pituitary tumors has not gained great consideration, yet. Nevertheless, some recent articles joint to previous case studies, suggest that this process plays a role in the modulation and fluctuation of normal pituitary cell functions and in the response of pituitary tumor cells to drug therapy, including the response to somatostatin receptor ligand (SRLs), the first-line medical therapy of acromegaly. Although it is not possible to draw any conclusion, the aim of this review was to highlight some considerations and perspectives in this research field. Reports on the effects of octreotide on autophagy induction and autophagic flux in extra-pituitary target tissues, have also been discussed.

Mitochondrial clearance: mechanisms and roles in cellular fitness.

Mitophagy is one of the selective autophagy pathways that catabolizes dysfunctional or superfluous mitochondria. Under mitophagy-inducing conditions, mitochondria are labeled with specific molecular landmarks that recruit the autophagy machinery to the surface of mitochondria, enclosed into autophagosomes, and delivered to lysosomes (vacuoles in yeast) for degradation. As damaged mitochondria are the major sources of reactive oxygen species, mitophagy is critical for mitochondrial quality control and cellular health. Moreover, appropriate control of mitochondrial quantity via mitophagy is vital for the energy supply-demand balance in cells and whole organisms, cell differentiation, and developmental programs. Thus, it seems conceivable that defects in mitophagy could elicit pleiotropic pathologies such as excess inflammation, tissue injury, neurodegeneration, and aging. In this review, we will focus on the molecular basis and physiological relevance of mitophagy, and potential of mitophagy as a therapeutic target to overcome such disorders.

The Role of Growth Temperature and Lipid Composition in Phase Separation of Yeast Vacuole Membranes

Membranes of vacuoles, the lysosomal organelle in yeast, phase separate into two coexisting liquid phases. This phenomenon provides a physical mechanism for lateral organization of biological membranes. Recent studies show that these domains play a key role in a central eukaryotic signaling pathway. Howthe cell adapts and tunes its vacuole membrane to invoke phase separation is still largely unknown. Here we examine how membrane composition changes due to two different environmental stimuli: growth temperature and nutrient depletion. If it is important for cells to regulate phase separation of their membranes, then we expect yeast cells to acclimate to external conditions. We test this hypothesis by growing yeast at 25°C and 30°C and find that the yeast adapts to this change by dramatically shifting the demixing temperature of vacuole membranes (to 29.7°C and 42.7°C respectively). This stark change in vacuole miscibility temperatures scales with growth temperature both in live cells and in isolated vacuoles, showing that yeast alter their membrane compositions to maintain phase separation. We also directly investigate the role of ergosterol, the main sterol in yeast, in domain formation in vacuoles. The yeast growth cycle starts with a logarithmic phase of rapid growth followed by a stationary phase in which glucose becomes scarce and vacuole membranes exhibit coexisting liquid domains. We isolate vacuoles from yeast in the logarithmic phase of growth, in which the membranes do not natively exhibit domains. Using fluorescence microscopy, we observe changes in vacuole phase separation as a function of ergosterol content. We find the surprising result that domains appear in vacuole membranes with reduced ergosterol.