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1.
Methods Mol Biol ; 2845: 15-25, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39115654

RESUMO

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.


Assuntos
Autofagia , Núcleo Celular , Saccharomyces cerevisiae , Vacúolos , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Núcleo Celular/metabolismo , Autofagia/fisiologia , Autofagossomos/metabolismo
2.
Nat Struct Mol Biol ; 31(1): 170-178, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38057553

RESUMO

Atg8, a ubiquitin-like protein, is conjugated with phosphatidylethanolamine (PE) via Atg7 (E1), Atg3 (E2) and Atg12-Atg5-Atg16 (E3) enzymatic cascade and mediates autophagy. However, its molecular roles in autophagosome formation are still unclear. Here we show that Saccharomyces cerevisiae Atg8-PE and E1-E2-E3 enzymes together construct a stable, mobile membrane scaffold. The complete scaffold formation induces an in-bud in prolate-shaped giant liposomes, transforming their morphology into one reminiscent of isolation membranes before sealing. In addition to their enzymatic roles in Atg8 lipidation, all three proteins contribute nonenzymatically to membrane scaffolding and shaping. Nuclear magnetic resonance analyses revealed that Atg8, E1, E2 and E3 together form an interaction web through multivalent weak interactions, where the intrinsically disordered regions in Atg3 play a central role. These data suggest that all six Atg proteins in the Atg8 conjugation machinery control membrane shaping during autophagosome formation.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas Relacionadas à Autofagia/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Membranas/metabolismo , Autofagia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo
3.
J Biochem ; 175(2): 155-165, 2024 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-37983716

RESUMO

Autophagy is a highly conserved intracellular degradation mechanism. The most distinctive feature of autophagy is the formation of double-membrane structures called autophagosomes, which compartmentalize portions of the cytoplasm. The outer membrane of the autophagosome fuses with the vacuolar/lysosomal membrane, leading to the degradation of the contents of the autophagosome. Approximately 30 years have passed since the identification of autophagy-related (ATG) genes and Atg proteins essential for autophagosome formation, and the primary functions of these Atg proteins have been elucidated. These achievements have significantly advanced our understanding of the mechanism of autophagosome formation. This article summarizes our current knowledge on how the autophagosome precursor is generated, and how the membrane expands and seals to complete the autophagosome.


Assuntos
Autofagossomos , Autofagia , Autofagossomos/metabolismo , Vacúolos/metabolismo , Lisossomos/metabolismo , Lipídeos
4.
Nat Commun ; 14(1): 5815, 2023 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-37726301

RESUMO

In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation.


Assuntos
Autofagossomos , Autofagia , Citosol , Macroautofagia , Ribossomos
5.
J Cell Biol ; 222(8)2023 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-37436710

RESUMO

In macroautophagy, cellular components are sequestered within autophagosomes and transported to lysosomes/vacuoles for degradation. Although phosphatidylinositol 3-kinase complex I (PI3KCI) plays a pivotal role in the regulation of autophagosome biogenesis, little is known about how this complex localizes to the pre-autophagosomal structure (PAS). In Saccharomyces cerevisiae, PI3KCI is composed of PI3K Vps34 and conserved subunits Vps15, Vps30, Atg14, and Atg38. In this study, we discover that PI3KCI interacts with the vacuolar membrane anchor Vac8, the PAS scaffold Atg1 complex, and the pre-autophagosomal vesicle component Atg9 via the Atg14 C-terminal region, the Atg38 C-terminal region, and the Vps30 BARA domain, respectively. While the Atg14-Vac8 interaction is constitutive, the Atg38-Atg1 complex interaction and the Vps30-Atg9 interaction are enhanced upon macroautophagy induction depending on Atg1 kinase activity. These interactions cooperate to target PI3KCI to the PAS. These findings provide a molecular basis for PAS targeting of PI3KCI during autophagosome biogenesis.


Assuntos
Autofagossomos , Proteínas Relacionadas à Autofagia , Proteínas de Membrana , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Autofagossomos/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
6.
Autophagy Rep ; 2(1)2023 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-38214012

RESUMO

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.

7.
EMBO Rep ; 23(8): e55192, 2022 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-35758175

RESUMO

Eukaryotic cells adequately control the mass and functions of organelles in various situations. Autophagy, an intracellular degradation system, largely contributes to this organelle control by degrading the excess or defective portions of organelles. The endoplasmic reticulum (ER) is an organelle with distinct structural domains associated with specific functions. The ER dynamically changes its mass, components, and shape in response to metabolic, developmental, or proteotoxic cues to maintain or regulate its functions. Therefore, elaborate mechanisms are required for proper degradation of the ER. Here, we review our current knowledge on diverse mechanisms underlying selective autophagy of the ER, which enable efficient degradation of specific ER subdomains according to different demands of cells.


Assuntos
Estresse do Retículo Endoplasmático , Retículo Endoplasmático , Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Macroautofagia
8.
Autophagy ; 18(12): 3046-3047, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35468041

RESUMO

Recent studies have revealed that even the nucleus can be degraded by selective macroautophagy (hereafter macronucleophagy). In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a macronucleophagy receptor that mediates sequestration of nucleus-derived double-membrane vesicles (NDVs) into phagophores. The outer and inner membranes of these NDVs are derived from the outer and inner nuclear membranes (ONM and INM), respectively, and the lumen contains nucleoplasmic material. Little was known about the mechanisms underlying macronucleophagy, including how the two nuclear membranes are coordinately deformed to generate NDVs and what nuclear components are preferentially loaded into or rather eliminated from NDVs. We found that Atg39 links the ONM and INM through the ONM-embedded transmembrane domain and INM-associated amphipathic helices (APHs). These APHs are important for Atg39 anchoring to the NE and autophagosome formation-coupled Atg39 clustering in the NE. In addition, the overaccumulation of Atg39 in the NE caused NE protrusion toward the cytoplasm depending on the APHs. These results allowed us to propose the mechanism by which Atg39 conducts NDV formation in coordination with autophagosome formation during macronucleophagy.


Assuntos
Membrana Nuclear , Proteínas de Saccharomyces cerevisiae , Membrana Nuclear/metabolismo , Autofagia , Núcleo Celular/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Trends Cell Biol ; 32(7): 574-584, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35067425

RESUMO

Eukaryotic cells have evolved different modes of autophagy, including macroautophagy and microautophagy, to deliver their own components to lysosomes or vacuoles for degradation. While an increasing body of research has established that autophagy plays pivotal roles for the maintenance and regulation of various cellular constituents, recent studies have begun to reveal that parts of the nucleus, for example, nucleus-derived vesicles and nuclear proteins, also become targets of autophagic degradation in different physiological or pathological contexts, including nutrient deprivation, defective nuclear pore complex (NPC) assembly, DNA damage, cellular senescence, and oncogenic insults. Here, we overview our current knowledge on the mechanisms and physiological roles of these 'nucleophagy' pathways and discuss their possible interplays and remaining issues.


Assuntos
Autofagia , Núcleo Celular , Autofagia/fisiologia , Núcleo Celular/metabolismo , Humanos , Lisossomos/metabolismo , Proteínas Nucleares/metabolismo
10.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35061008

RESUMO

In selective autophagy of the nucleus (hereafter nucleophagy), nucleus-derived double-membrane vesicles (NDVs) are formed, sequestered within autophagosomes, and delivered to lysosomes or vacuoles for degradation. In Saccharomyces cerevisiae, the nuclear envelope (NE) protein Atg39 acts as a nucleophagy receptor, which interacts with Atg8 to target NDVs to the forming autophagosomal membranes. In this study, we revealed that Atg39 is anchored to the outer nuclear membrane via its transmembrane domain and also associated with the inner nuclear membrane via membrane-binding amphipathic helices (APHs) in its perinuclear space region, thereby linking these membranes. We also revealed that autophagosome formation-coupled Atg39 crowding causes the NE to protrude toward the cytoplasm, and the tips of the protrusions are pinched off to generate NDVs. The APHs of Atg39 are crucial for Atg39 crowding in the NE and subsequent NE protrusion. These findings suggest that the nucleophagy receptor Atg39 plays pivotal roles in NE deformation during the generation of NDVs to be degraded by nucleophagy.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Membrana Nuclear/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/metabolismo
11.
Nat Struct Mol Biol ; 28(7): 583-593, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34239122

RESUMO

Autophagosome biogenesis is an essential feature of autophagy. Lipidation of Atg8 plays a critical role in this process. Previous in vitro studies identified membrane tethering and hemi-fusion/fusion activities of Atg8, yet definitive roles in autophagosome biogenesis remained controversial. Here, we studied the effect of Atg8 lipidation on membrane structure. Lipidation of Saccharomyces cerevisiae Atg8 on nonspherical giant vesicles induced dramatic vesicle deformation into a sphere with an out-bud. Solution NMR spectroscopy of Atg8 lipidated on nanodiscs identified two aromatic membrane-facing residues that mediate membrane-area expansion and fragmentation of giant vesicles in vitro. These residues also contribute to the in vivo maintenance of fragmented vacuolar morphology under stress in fission yeast, a moonlighting function of Atg8. Furthermore, these aromatic residues are crucial for the formation of a sufficient number of autophagosomes and regulate autophagosome size. Together, these data demonstrate that Atg8 can cause membrane perturbations that underlie efficient autophagosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Membrana Celular/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Nanoestruturas , Ressonância Magnética Nuclear Biomolecular , Fosfatidiletanolaminas/química , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo
13.
Autophagy ; 16(12): 2299-2300, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33043769

RESUMO

Reticulophagy (or ER-phagy) is a type of selective autophagy that targets the endoplasmic reticulum (ER). In the process of reticulophagy, part of the ER is fragmented and packed within autophagosomes. However, the underlying mechanism that induces this local remodeling of ER subdomains was poorly understood. Our recent study showed that in the budding yeast Saccharomyces cerevisiae the reticulophagy receptor Atg40 plays an important role in ER remodeling beyond its role as a tether between the ER and the phagophore [1]. Atg40 has an ability to generate positive membrane curvature through the reticulon-like domain and locally forms a super assemblage though its binding to Atg8 at ER-phagophore contacts. These Atg40 assemblages cause folding of the ER subdomains to allow them to be efficiently packed into autophagosomes. Furthermore, our structural analysis identified an evolutionarily conserved short helix that assists strong Atg8-binding of reticulophagy receptors.


Assuntos
Autofagia , Proteínas de Saccharomyces cerevisiae , Autofagossomos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Retículo Endoplasmático , Receptores Citoplasmáticos e Nucleares , Saccharomyces cerevisiae
14.
Nat Struct Mol Biol ; 27(12): 1185-1193, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33106658

RESUMO

The molecular function of Atg9, the sole transmembrane protein in the autophagosome-forming machinery, remains unknown. Atg9 colocalizes with Atg2 at the expanding edge of the isolation membrane (IM), where Atg2 receives phospholipids from the endoplasmic reticulum (ER). Here we report that yeast and human Atg9 are lipid scramblases that translocate phospholipids between outer and inner leaflets of liposomes in vitro. Cryo-EM of fission yeast Atg9 reveals a homotrimer, with two connected pores forming a path between the two membrane leaflets: one pore, located at a protomer, opens laterally to the cytoplasmic leaflet; the other, at the trimer center, traverses the membrane vertically. Mutation of residues lining the pores impaired IM expansion and autophagy activity in yeast and abolished Atg9's ability to transport phospholipids between liposome leaflets. These results suggest that phospholipids delivered by Atg2 are translocated from the cytoplasmic to the luminal leaflet by Atg9, thereby driving autophagosomal membrane expansion.


Assuntos
Autofagossomos/química , Proteínas Relacionadas à Autofagia/química , Proteínas de Membrana/química , Fosfolipídeos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Transporte Vesicular/química , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Sítios de Ligação , Transporte Biológico , Microscopia Crioeletrônica , Expressão Gênica , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfolipídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteolipídeos/química , Proteolipídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína Vermelha Fluorescente
15.
J Biol Chem ; 295(48): 16292-16298, 2020 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-32958557

RESUMO

In macroautophagy (hereafter autophagy), cytoplasmic molecules and organelles are randomly or selectively sequestered within double-membrane vesicles called autophagosomes and delivered to lysosomes or vacuoles for degradation. In selective autophagy, the specificity of degradation targets is determined by autophagy receptors. In the budding yeast Saccharomyces cerevisiae, autophagy receptors interact with specific targets and Atg11, resulting in the recruitment of a protein complex that initiates autophagosome formation. Previous studies have revealed that autophagy receptors are regulated by posttranslational modifications. In selective autophagy of peroxisomes (pexophagy), the receptor Atg36 localizes to peroxisomes by binding to the peroxisomal membrane protein Pex3. We previously reported that Atg36 is phosphorylated by Hrr25 (casein kinase 1δ), increasing the Atg36-Atg11 interaction and thereby stimulating pexophagy initiation. However, the regulatory mechanisms underlying Atg36 phosphorylation are unknown. Here, we show that Atg36 phosphorylation is abolished in cells lacking Pex3 or expressing a Pex3 mutant defective in the interaction with Atg36, suggesting that the interaction with Pex3 is essential for the Hrr25-mediated phosphorylation of Atg36. Using recombinant proteins, we further demonstrated that Pex3 directly promotes Atg36 phosphorylation by Hrr25. A co-immunoprecipitation analysis revealed that the interaction of Atg36 with Hrr25 depends on Pex3. These results suggest that Pex3 increases the Atg36-Hrr25 interaction and thereby stimulates Atg36 phosphorylation on the peroxisomal membrane. In addition, we found that Pex3 binding protects Atg36 from proteasomal degradation. Thus, Pex3 confines Atg36 activity to the peroxisome by enhancing its phosphorylation and stability on this organelle.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Caseína Quinase I/metabolismo , Proteínas de Membrana/metabolismo , Peroxinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas à Autofagia/genética , Caseína Quinase I/genética , Proteínas de Membrana/genética , Peroxinas/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
16.
Nat Commun ; 11(1): 3306, 2020 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-32620754

RESUMO

The endoplasmic reticulum (ER) is selectively degraded by autophagy (ER-phagy) through proteins called ER-phagy receptors. In Saccharomyces cerevisiae, Atg40 acts as an ER-phagy receptor to sequester ER fragments into autophagosomes by binding Atg8 on forming autophagosomal membranes. During ER-phagy, parts of the ER are morphologically rearranged, fragmented, and loaded into autophagosomes, but the mechanism remains poorly understood. Here we find that Atg40 molecules assemble in the ER membrane concurrently with autophagosome formation via multivalent interaction with Atg8. Atg8-mediated super-assembly of Atg40 generates highly-curved ER regions, depending on its reticulon-like domain, and supports packing of these regions into autophagosomes. Moreover, tight binding of Atg40 to Atg8 is achieved by a short helix C-terminal to the Atg8-family interacting motif, and this feature is also observed for mammalian ER-phagy receptors. Thus, this study significantly advances our understanding of the mechanisms of ER-phagy and also provides insights into organelle fragmentation in selective autophagy of other organelles.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia/química , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/química , Proteínas Relacionadas à Autofagia/genética , Sítios de Ligação/genética , Estresse do Retículo Endoplasmático/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência , Mutação , Ligação Proteica , Domínios Proteicos , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
17.
J Cell Biol ; 219(7)2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32453403

RESUMO

The mechanisms underlying turnover of the nuclear pore complex (NPC) and the component nucleoporins (Nups) are still poorly understood. In this study, we found that the budding yeast Saccharomyces cerevisiae triggers NPC degradation by autophagy upon the inactivation of Tor kinase complex 1. This degradation largely depends on the selective autophagy-specific factor Atg11 and the autophagy receptor-binding ability of Atg8, suggesting that the NPC is degraded via receptor-dependent selective autophagy. Immunoelectron microscopy revealed that NPCs embedded in nuclear envelope-derived double-membrane vesicles are sequestered within autophagosomes. At least two pathways are involved in NPC degradation: Atg39-dependent nucleophagy (selective autophagy of the nucleus) and a pathway involving an unknown receptor. In addition, we found the interaction between Nup159 and Atg8 via the Atg8-family interacting motif is important for degradation of this nucleoporin not assembled into the NPC. Thus, this study provides the first evidence for autophagic degradation of the NPC and Nups, which we term "NPC-phagy" and "nucleoporinophagy."


Assuntos
Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Autofagia/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagia/efeitos dos fármacos , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Regulação Fúngica da Expressão Gênica , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Microscopia Imunoeletrônica , Poro Nuclear/efeitos dos fármacos , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteólise/efeitos dos fármacos , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Proteínas de Transporte Vesicular/metabolismo
18.
Nat Rev Mol Cell Biol ; 21(8): 439-458, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32372019

RESUMO

Autophagosomes are double-membrane vesicles newly formed during autophagy to engulf a wide range of intracellular material and transport this autophagic cargo to lysosomes (or vacuoles in yeasts and plants) for subsequent degradation. Autophagosome biogenesis responds to a plethora of signals and involves unique and dynamic membrane processes. Autophagy is an important cellular mechanism allowing the cell to meet various demands, and its disruption compromises homeostasis and leads to various diseases, including metabolic disorders, neurodegeneration and cancer. Thus, not surprisingly, the elucidation of the molecular mechanisms governing autophagosome biogenesis has attracted considerable interest. Key molecules and organelles involved in autophagosome biogenesis, including autophagy-related (ATG) proteins and the endoplasmic reticulum, have been discovered, and their roles and relationships have been investigated intensely. However, several fundamental questions, such as what supplies membranes/lipids to build the autophagosome and how the membrane nucleates, expands, bends into a spherical shape and finally closes, have proven difficult to address. Nonetheless, owing to recent studies with new approaches and technologies, we have begun to unveil the mechanisms underlying these processes on a molecular level. We now know that autophagosome biogenesis is a highly complex process, in which multiple proteins and lipids from various membrane sources, supported by the formation of membrane contact sites, cooperate with biophysical phenomena, including membrane shaping and liquid-liquid phase separation, to ensure seamless segregation of the autophagic cargo. Together, these studies pave the way to obtaining a holistic view of autophagosome biogenesis.


Assuntos
Autofagossomos/metabolismo , Autofagossomos/fisiologia , Animais , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Lisossomos/metabolismo , Macroautofagia , Transporte Proteico
19.
Artigo em Inglês | MEDLINE | ID: mdl-31932525

RESUMO

Autophagy is an intracellular degradation system that is present in most eukaryotes. In the process of autophagy, double membrane vesicles called autophagosomes sequester a wide variety of cellular constituents and deliver them to lytic organelles: lysosomes in mammals and vacuoles in yeast and plants. Although autophagy used to be considered a non-selective process in its target sequestration into autophagosomes, recent studies have revealed that autophagosomes can also selectively sequester certain proteins and organelles that have become unnecessary or harmful for the cell. We recently discovered that the endoplasmic reticulum (ER) is degraded by autophagy in a selective manner in the budding yeast Saccharomyces cerevisiae, and identified "receptor proteins" that play pivotal roles in this "ER-phagy" pathway. Moreover, several ER-phagy receptors in mammalian cells have also been reported. This report provides an overview of our current knowledge on ER-phagy and discuss their mechanisms, physiological roles, and possible links to human diseases.


Assuntos
Autofagossomos/metabolismo , Autofagia , Retículo Endoplasmático/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Estresse do Retículo Endoplasmático , Humanos , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Cell Struct Funct ; 45(1): 1-8, 2020 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-31787665

RESUMO

The polytopic plasma membrane protein Rim21 senses both the elevation of ambient pH and alterations in plasma membrane lipid asymmetry in the Rim101 pathway in budding yeast. Rim21 is known to undergo N-glycosylation, but the site and function of the glycosylation modification is not known. Using a systematic mutation analysis, we found that Rim21 is N-glycosylated at an unconventional motif located in the N-terminal extracellular region. The Rim21 mutant protein that failed to receive N-glycosylation showed prolonged protein lifetime compared to that of WT Rim21 protein. Although both the WT and mutant Rim21 localized to the plasma membrane, they exhibited different biochemical fractionation profiles. The mutant Rim21, but not WT Rim21, was mainly fractionated into the heavy membrane fraction. Further, compared to WT Rim21, mutant Rim21 was more easily solubilized with digitonin but was conversely more resistant to solubilization with Triton X-100. Despite these different biochemical properties from WT Rim21, mutant Rim21 protein could still activate the Rim101 pathway in response to external alkalization. Collectively, N-glycosylation of Rim21 is not indispensable for its activity as a sensor protein, but modulates the residence of Rim21 protein to some microdomains within the plasma membrane with distinct lipid conditions, thereby affecting its turnover.Key words: plasma membrane, lipid asymmetry, N-linked glycosylation, microdomain, Saccharomyces cerevisiae.


Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Membrana Celular/genética , Glicosilação , Saccharomyces cerevisiae/metabolismo
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