scholarly journals Role of Atg8 in the regulation of vacuolar membrane invagination

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Ayane Ishii ◽  
Kazuo Kurokawa ◽  
Miyuu Hotta ◽  
Suzuka Yoshizaki ◽  
Maki Kurita ◽  
...  
Keyword(s):  
Heat Stress ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Bar Domain ◽  
Escrt Machinery ◽  
Membrane Invagination ◽  
Cellular Factors ◽  
Vacuolar Membranes ◽  
Membrane Cell

Abstract Cellular heat stress can cause damage, and significant changes, to a variety of cellular structures. When exposed to chronically high temperatures, yeast cells invaginate vacuolar membranes. In this study, we found that the expression of Atg8, an essential autophagy factor, is induced after chronic heat stress. In addition, without Atg8, vacuolar invaginations are induced conspicuously, beginning earlier and invaginating vacuoles more frequently after heat stress. Our results indicate that Atg8’s invagination-suppressing functions do not require Atg8 lipidation, in contrast with autophagy, which requires Atg8 lipidation. Genetic analyses of vps24 and vps23 further suggest that full ESCRT machinery is necessary to form vacuolar invaginations irrespective of Atg8. In contrast, through a combined mutation with the vacuole BAR domain protein Ivy1, vacuoles show constitutively enhanced invaginated structures. Finally, we found that the atg8Δivy1Δ mutant is sensitive against agents targeting functions of the vacuole and/or plasma membrane (cell wall). Collectively, our findings revealed that Atg8 maintains vacuolar membrane homeostasis in an autophagy-independent function by coordinating with other cellular factors.

scholarly journals The Vacuolar Transporter Chaperone (VTC) Complex Is Required for Microautophagy

2007 ◽  
Vol 18 (1) ◽  
pp. 166-175 ◽  
Author(s):  
Andreas Uttenweiler ◽  
Heinz Schwarz ◽  
Heinz Neumann ◽  
Andreas Mayer
Keyword(s):  
Nutrient Limitation ◽  
Vacuolar Membrane ◽  
Lysosomal Membrane ◽  
Cell Periphery ◽  
Membrane Invagination ◽  
Reconstituted System ◽  
Chaperone Complex ◽  
Vacuolar Membranes ◽  
Important Constituent

Microautophagy involves direct invagination and fission of the vacuolar/lysosomal membrane under nutrient limitation. This occurs by an autophagic tube, a specialized vacuolar membrane invagination that pinches off vesicles into the vacuolar lumen. In this study we have identified the VTC (vacuolar transporter chaperone) complex as required for microautophagy. The VTC complex is present on the ER and vacuoles and at the cell periphery. On induction of autophagy by nutrient limitation the VTC complex is recruited to and concentrated on vacuoles. The VTC complex is inhomogeneously distributed within the vacuolar membranes, showing an enrichment on autophagic tubes. Deletion of the VTC complex blocks microautophagic uptake into vacuoles. The mutants still form autophagic tubes but the production of microautophagic vesicles from their tips is impaired. In line with this, affinity-purified antibodies to the Vtc proteins inhibit microautophagic uptake in a reconstituted system in vitro. Our data suggest that the VTC complex is an important constituent of autophagic tubes and that it is required for scission of microautophagic vesicles from these tubes.

Vacuolar Membranes: Isolation from Yeast Cells

1973 ◽  
Vol 28 (7-8) ◽  
pp. 417-424 ◽  
Author(s):  
W. van der Wilden ◽  
Ph. Matile ◽  
M. Schellenberg ◽  
J. Meyer ◽  
A. Wiemken
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sedimentation Velocity ◽  
Isolation Procedure ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Differential Centrifugation ◽  
Whole Cells ◽  
Vacuolar Membranes

α-Mannosidase was found associated with the vacuolar membranes of yeast. The vacuoles were isolated by flotation from osmotically disrupted spheroplasts of Saccharomyces cerevisiae. The enzyme was used as marker for isolating vacuolar membrane fragments directly from whole cells which were mechanically disintegrated. Over 90% of the total α-mannosidase was recovered in the particulate fraction. The enzyme was present in all of the fractions obtained upon differential centrifugation. Density gradient centrifugation in Urografin (5 - 20% w/v) of preparations obtained by differential centrifugation between 20 000 and 50 000 × g did not result in density equilibrium of the membrane. An isolation procedure involving a sedimentation velocity cut in Urografin gradients has, therefore, been worked out.

scholarly journals Endocytosis against high turgor pressure is made easier by partial protein coating and a freely rotating base

2019 ◽  
Author(s):  
Rui Ma ◽  
Julien Berro
Keyword(s):  
Mammalian Cells ◽  
Turgor Pressure ◽  
Yeast Cells ◽  
Large Protein ◽  
Biologically Relevant ◽  
Membrane Invagination ◽  
Membrane Tube ◽  
Membrane Patch ◽  
Theoretical Results

ABSTRACTDuring clathrin-mediated endocytosis, a patch of flat plasma membrane is deformed into a vesicle. In walled cells, such as plants and fungi, the turgor pressure is high and pushes the membrane against the cell wall, thus hindering membrane internalization. In this paper, we study how a patch of membrane is deformed against turgor pressure by force and by curvature-generating proteins. We show that a large amount of force is needed to merely start deforming the membrane and an even larger force is needed to pull a membrane tube. The magnitude of these forces strongly depends on how the base of the membrane is constrained and how the membrane is coated with curvature-generating proteins. In particular, these forces can be reduced by partially but not fully coating the membrane patch with curvature-generating proteins. Our theoretical results show excellent agreement with experimental data.SIGNIFICANCEYeast cells have been widely used as a model system to study clathrin-mediated endocytosis. The mechanics of membrane during endocytosis has been extensively studied mostly in low turgor pressure condition, which is relevant for mammalian cells but not for yeast cells. It has been suggested that as a result of high turgor pressure in yeast cells, a large amount of force is needed to drive the progress of the membrane invagination. In this paper, we investigated biologically relevant mechanisms to reduce the force requirement. We highlight the role of boundary conditions at the membrane base, which is a factor that has been largely ignored in previous studies. We also investigate the role of curvature-generating proteins and show that a large protein coat does not necessarily reduce the force barrier for endocytosis.

scholarly journals ESCRT machinery plays a role in microautophagy in yeast

2020 ◽  
Author(s):  
Shamsul Morshed ◽  
Most Naoshia Tasnin ◽  
Takashi Ushimaru
Keyword(s):  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Vacuolar Membrane ◽  
Nutrient Starvation ◽  
Direct Role ◽  
Endosomal Sorting ◽  
Vacuolar Sorting ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract Background: Microautophagy, which degrades cargos by direct lysosomal/vacuolar engulfment of cytoplasmic cargos, is promoted after nutrient starvation and the inactivation of target of rapamycin complex 1 (TORC1) protein kinase. In budding yeast, microautophagy has been commonly assessed using processing assays with green fluorescent protein (GFP)-tagged vacuolar membrane proteins, such as Vph1 and Pho8. The endosomal sorting complex required for transport (ESCRT) system is proposed to be required for microautophagy, because degradation of vacuolar membrane protein Vph1 was compromised in ESCRT-defective mutants. However, ESCRT is also critical for the vacuolar sorting of most vacuolar proteins, and hence reexamination of the involvement of ESCRT in microautophagic processes is required.Results: Here, we show that the Vph1-GFP processing assay is unsuitable for estimating the involvement of ESCRT in microautophagy, because Vph1-GFP accumulated highly in the prevacuolar class E compartment in ESCRT mutants. In contrast, GFP-Pho8 and Sna4-GFP destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, were properly localized on vacuolar membranes in ESCRT-deficient cells. Nevertheless, microautophagic degradation of GFP-Pho8 and Sna4-GFP after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation.Conclusions: These findings provide evidence for the direct role of ESCRT in microautophagy induction.

scholarly journals HIV Assembly and Budding: Ca2+ Signaling and Non-ESCRT Proteins Set the Stage

2012 ◽  
Vol 2012 ◽  
pp. 1-12 ◽  
Author(s):  
Lorna S. Ehrlich ◽  
Carol A. Carter
Keyword(s):  
Protein Trafficking ◽  
Endocytic Pathway ◽  
Host Proteins ◽  
Virus Budding ◽  
Endosomal Sorting ◽  
The Past ◽  
Escrt Machinery ◽  
Cellular Factors ◽  
Hiv 1

More than a decade has elapsed since the link between the endosomal sorting complex required for transport (ESCRT) machinery and HIV-1 protein trafficking and budding was first identified. L domains in HIV-1 Gag mediate recruitment of ESCRT which function in bud abscission releasing the viral particle from the host cell. Beyond virus budding, the ESCRT machinery is also involved in the endocytic pathway, cytokinesis, and autophagy. In the past few years, the number of non-ESCRT host proteins shown to be required in the assembly process has also grown. In this paper, we highlight the role of recently identified cellular factors that link ESCRT machinery to calcium signaling machinery and we suggest that this liaison contributes to setting the stage for productive ESCRT recruitment and mediation of abscission. Parallel paradigms for non-ESCRT roles in virus budding and cytokinesis will be discussed.

scholarly journals ESCRT machinery plays a role in microautophagy in yeast

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Shamsul Morshed ◽  
Most Naoshia Tasnin ◽  
Takashi Ushimaru
Keyword(s):  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Vacuolar Membrane ◽  
Nutrient Starvation ◽  
Direct Role ◽  
Endosomal Sorting ◽  
Vacuolar Sorting ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract Background Microautophagy, which degrades cargos by direct lysosomal/vacuolar engulfment of cytoplasmic cargos, is promoted after nutrient starvation and the inactivation of target of rapamycin complex 1 (TORC1) protein kinase. In budding yeast, microautophagy has been commonly assessed using processing assays with green fluorescent protein (GFP)-tagged vacuolar membrane proteins, such as Vph1 and Pho8. The endosomal sorting complex required for transport (ESCRT) system is proposed to be required for microautophagy, because degradation of vacuolar membrane protein Vph1 was compromised in ESCRT-defective mutants. However, ESCRT is also critical for the vacuolar sorting of most vacuolar proteins, and hence reexamination of the involvement of ESCRT in microautophagic processes is required. Results Here, we show that the Vph1-GFP processing assay is unsuitable for estimating the involvement of ESCRT in microautophagy, because Vph1-GFP accumulated highly in the prevacuolar class E compartment in ESCRT mutants. In contrast, GFP-Pho8 and Sna4-GFP destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, were properly localized on vacuolar membranes in ESCRT-deficient cells. Nevertheless, microautophagic degradation of GFP-Pho8 and Sna4-GFP after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation. Conclusions These findings provide evidence for the direct role of ESCRT in microautophagy induction.

scholarly journals Lipids and Trehalose Actively Cooperate in Heat Stress Management of Schizosaccharomyces pombe

2021 ◽  
Vol 22 (24) ◽  
pp. 13272
Author(s):  
Mária Péter ◽  
Péter Gudmann ◽  
Zoltán Kóta ◽  
Zsolt Török ◽  
László Vígh ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Heat Stress ◽  
Schizosaccharomyces Pombe ◽  
De Novo ◽  
Acyl Chain ◽  
Membrane Lipid ◽  
Yeast Cells ◽  
Storage Lipids ◽  
Acyl Chain Length

Homeostatic maintenance of the physicochemical properties of cellular membranes is essential for life. In yeast, trehalose accumulation and lipid remodeling enable rapid adaptation to perturbations, but their crosstalk was not investigated. Here we report about the first in-depth, mass spectrometry-based lipidomic analysis on heat-stressed Schizosaccharomyces pombe mutants which are unable to synthesize (tps1Δ) or degrade (ntp1Δ) trehalose. Our experiments provide data about the role of trehalose as a membrane protectant in heat stress. We show that under conditions of trehalose deficiency, heat stress induced a comprehensive, distinctively high-degree lipidome reshaping in which structural, signaling and storage lipids acted in concert. In the absence of trehalose, membrane lipid remodeling was more pronounced and increased with increasing stress dose. It could be characterized by decreasing unsaturation and increasing acyl chain length, and required de novo synthesis of stearic acid (18:0) and very long-chain fatty acids to serve membrane rigidification. In addition, we detected enhanced and sustained signaling lipid generation to ensure transient cell cycle arrest as well as more intense triglyceride synthesis to accommodate membrane lipid-derived oleic acid (18:1) and newly synthesized but unused fatty acids. We also demonstrate that these changes were able to partially substitute for the missing role of trehalose and conferred measurable stress tolerance to fission yeast cells.

scholarly journals ESCRT machinery plays a role in microautophagy in yeast

2020 ◽  
Author(s):  
Shamsul Morshed ◽  
Most Naoshia Tasnin ◽  
Takashi Ushimaru
Keyword(s):  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Vacuolar Membrane ◽  
Nutrient Starvation ◽  
Direct Role ◽  
Endosomal Sorting ◽  
Vacuolar Sorting ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract Background: Microautophagy, which degrades cargos by direct lysosomal/vacuolar engulfment of cytoplasmic cargos, is promoted after nutrient starvation and the inactivation of target of rapamycin complex 1 (TORC1) protein kinase. In budding yeast, microautophagy has been commonly assessed using processing assays with green fluorescent protein (GFP)-tagged vacuolar membrane proteins, such as Vph1 and Pho8. The endosomal sorting complex required for transport (ESCRT) system is proposed to be required for microautophagy, because degradation of vacuolar membrane protein Vph1 was compromised in ESCRT-defective mutants. However, ESCRT is also critical for the vacuolar sorting of most vacuolar proteins, and hence reexamination of the involvement of ESCRT in microautophagic processes is required.Results: Here, we show that the Vph1-GFP processing assay is unsuitable for estimating the involvement of ESCRT in microautophagy, because Vph1-GFP accumulated highly in the prevacuolar class E compartment in ESCRT mutants. In contrast, GFP-Pho8 and Sna4-GFP destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, were properly localized on vacuolar membranes in ESCRT-deficient cells. Nevertheless, microautophagic degradation of GFP-Pho8 and Sna4-GFP after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation.Conclusions: These findings provide evidence for the direct role of ESCRT in microautophagy induction.

scholarly journals ESCRT machinery plays a role in microautophagy in yeast

2020 ◽  
Author(s):  
Shamsul Morshed ◽  
Most Naoshia Tasnin ◽  
Takashi Ushimaru
Keyword(s):  
Fluorescent Protein ◽  
Adaptor Protein ◽  
Vacuolar Membrane ◽  
Nutrient Starvation ◽  
Direct Role ◽  
Endosomal Sorting ◽  
Vacuolar Sorting ◽  
Green Fluorescent ◽  
Vacuolar Membranes

Abstract Background: Microautophagy, which degrades cargos by direct lysosomal/vacuolar engulfment of cytoplasmic cargos, is promoted after nutrient starvation and the inactivation of target of rapamycin complex 1 (TORC1) protein kinase. In budding yeast, microautophagy has been commonly assessed using processing assays with green fluorescent protein (GFP)-tagged vacuolar membrane proteins, such as Vph1 and Pho8. The endosomal sorting complex required for transport (ESCRT) system is proposed to be required for microautophagy, because degradation of vacuolar membrane protein Vph1 was compromised in ESCRT-defective mutants. However, ESCRT is also critical for the vacuolar sorting of most vacuolar proteins, and hence reexamination of the involvement of ESCRT in microautophagic processes is required.Results: Here, we show that the Vph1-GFP processing assay is unsuitable for estimating the involvement of ESCRT in microautophagy, because Vph1-GFP accumulated highly in the prevacuolar class E compartment in ESCRT mutants. In contrast, GFP-Pho8 and Sna4-GFP destined for vacuolar membranes via an alternative adaptor protein-3 (AP-3) pathway, were properly localized on vacuolar membranes in ESCRT-deficient cells. Nevertheless, microautophagic degradation of GFP-Pho8 and Sna4-GFP after TORC1 inactivation was hindered in ESCRT mutants, indicating that ESCRT is indeed required for microautophagy after nutrient starvation and TORC1 inactivation. Conclusion: These findings provide evidence for the direct role of ESCRT in microautophagy induction.

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