Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

Fengsrud, Monica; Erichsen, Egil S.; Berg, Trond; Raiborg, Camilla; Seglen, Per Ottar

doi:10.1078/0171-9335-00125

Cited by 120 publications

(99 citation statements)

References 48 publications

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“…Considering the apparent paucity of transmembrane and outer surface proteins in the autophagosomal delimiting membrane, 40 the autophagosomally enriched proteins described in the present study are likely to be associated with the inner delimiting membrane, corresponding to the cytoplasm-sequestering face of the phagophore. They would thus be in a suitable position to detect oxidized, drug-adducted, hypermethylated or otherwise denatured macromolecules either in aggregates or scattered throughout the cytoplasm, as suggested by their predominant antioxidant, drug-metabolizing, methylating and chaperoning properties.…”

Section: Discussionmentioning

confidence: 99%

“…39 Although separation of the autophagosomal delimiting membranes (corresponding roughly to phagophores) from the autophagosomal contents of sequestered cytoplasm has not yet been achieved, proteins specifically localized to the delimiting membranes should be sufficiently enriched in an autophagosomal membrane preparation to allow their detection. Autophagosomal delimiting membranes are virtually devoid of transmembrane proteins, 40 but peripheral membrane proteins appear to be present: In a previous proteomic study, using two-dimensional (2D) gel electrophoresis for protein separation and MALDI-TOF mass spectrometric tryptic peptide mass fingerprinting for protein identification, three proteins were found to be enriched in autophagosomal membranes relative to a total cytoplasmic membrane preparation (sedimentable fraction of a postnuclear supernatant, PNS). 39 In the present report, the detectability of autophagosomal membrane proteins has been greatly enhanced by the employment of more narrowly electrofocused (zoom) 2D gels, 41 improved silver staining 42 and better quantification.…”

Section: Introductionmentioning

confidence: 99%

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Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Øverbye¹,

Fengsrud²,

Seglen³

2007

Autophagy

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140

117

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Øverbye¹,

Fengsrud²,

Seglen³

2007

Autophagy

Self Cite

140

117

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“…The next change involves differentiation of the collapsed membranes into two types of subdomains, one of which forms pre-LVs and the other multilamellartype autophagosomes/autophagic vacuoles (Fengsrud et al, 2000). We define pre-LVs as reinflated (reswollen) collapsed PSV membranes that exhibit a translucent lumen and label with anti-g-TIP antibodies (Figs.…”

Section: Cryofixation and Freeze-substitution Methods Are Important Fmentioning

confidence: 99%

“…7, C and H). Based on the appearance of these multilamellar compartments exhibiting evidence of cytoplasmic degradation, we postulate that these structures correspond to multilamellar autophagosomes of animal cells (Fengsrud et al, 2000) at different stages of development. In most instances, transformation of these multilamellar autophagic vacuoles into LVs involves engulfment by expansion of the luminal space of the pre-LV domains into outer membrane sheets that surround the autophagic vacuoles (Fig.…”

Section: Vascular Cylinder Cell Developmentmentioning

confidence: 99%

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Protein Storage Vacuoles Are Transformed into Lytic Vacuoles in Root Meristematic Cells of Germinating Seedlings by Multiple, Cell Type-Specific Mechanisms

Zheng

Staehelin

2011

Plant Physiology

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We have investigated the structural events associated with vacuole biogenesis in root tip cells of tobacco (Nicotiana tabacum) seedlings preserved by high-pressure freezing and freeze-substitution techniques. Our micrographs demonstrate that the lytic vacuoles (LVs) of root tip cells are derived from protein storage vacuoles (PSVs) by cell type-specific sets of transformation events. Analysis of the vacuole transformation pathways has been aided by the phytin-dependent black osmium staining of PSV luminal contents. In epidermal and outer cortex cells, the central LVs are formed by a process involving PSV fusion, storage protein degradation, and the gradual replacement of the PSV marker protein a-tonoplast intrinsic protein (TIP) with the LV marker protein g-TIP. In contrast, in the inner cortex and vascular cylinder cells, the transformation events are more complex. During mobilization of the stored molecules, the PSV membranes collapse osmotically upon themselves, thereby squeezing the vacuolar contents into the remaining bulging vacuolar regions. The collapsed PSV membranes then differentiate into two domains: (1) vacuole "reinflation" domains that produce pre-LVs, and (2) multilamellar autophagosomal domains that are later engulfed by the pre-LVs. The multilamellar autophagosomal domains appear to originate from concentric sheets of PSV membranes that create compartments within which the cytoplasm begins to break down. Engulfment of the multilamellar autophagic vacuoles by the pre-LVs gives rise to the mature LVs. During pre-LV formation, the PSV marker a-TIP disappears and is replaced by the LV marker g-TIP. These findings demonstrate that the central LVs of root cells arise from PSVs via cell type-specific transformation pathways.

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Structural view on autophagosome formation

Noda

2023

FEBS Letters

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Autophagy is a conserved intracellular degradation system in eukaryotes, involving the sequestration of degradation targets into autophagosomes, which are subsequently delivered to lysosomes (or vacuoles in yeasts and plants) for degradation. In budding yeast, starvation‐induced autophagosome formation relies on approximately 20 core Atg proteins, grouped into six functional categories: the Atg1/ULK complex, the phosphatidylinositol‐3 kinase complex, the Atg9 transmembrane protein, the Atg2–Atg18/WIPI complex, the Atg8 lipidation system, and the Atg12–Atg5 conjugation system. Additionally, selective autophagy requires cargo receptors and other factors, including a fission factor, for specific sequestration. This review covers the 30‐year history of structural studies on core Atg proteins and factors involved in selective autophagy, examining X‐ray crystallography, NMR, and cryo‐EM techniques. The molecular mechanisms of autophagy are explored based on protein structures, and future directions in the structural biology of autophagy are discussed, considering the advancements in the era of AlphaFold.

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Ultrastructural characterization of the delimiting membranes of isolated autophagosomes and amphisomes by freeze-fracture electron microscopy

Cited by 120 publications

References 48 publications

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Proteomic Analysis of Membrane-Associated Proteins from Rat Liver Autophagosomes

Protein Storage Vacuoles Are Transformed into Lytic Vacuoles in Root Meristematic Cells of Germinating Seedlings by Multiple, Cell Type-Specific Mechanisms

Structural view on autophagosome formation

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