Atg27 tyrosine sorting motif is important for its trafficking and Atg9 localization.
TL;DR: It is suggested that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.
Abstract: During autophagy, the transmembrane protein Atg27 facilitates transport of the major autophagy membrane protein Atg9 to the preautophagosomal structure (PAS). To better understand the function of Atg27 and its relationship with Atg9, Atg27 trafficking and localization were examined. Atg27 localized to endosomes and the vacuolar membrane, in addition to previously described PAS, Golgi and Atg9-positive structures. Atg27 vacuolar membrane localization was dependent on the adaptor AP-3, which mediates direct transport from the trans-Golgi to the vacuole. The four C-terminal amino acids (YSAV) of Atg27 comprise a tyrosine sorting motif. Mutation of the YSAV abrogated Atg27 transport to the vacuolar membrane and affected its distribution in TGN/endosomal compartments, while PAS localization was normal. Also, in atg27(ΔYSAV) or AP-3 mutants, accumulation of Atg9 in the vacuolar lumen was observed upon autophagy induction. Nevertheless, PAS localization of Atg9 was normal in atg27(ΔYSAV) cells. The vacuole lumen localization of Atg9 was dependent on transport through the multivesicular body, as Atg9 accumulated in the class E compartment and vacuole membrane in atg27(ΔYSAV) vps4Δ but not in ATG27 vps4Δ cells. We suggest that Atg27 has an additional role to retain Atg9 in endosomal reservoirs that can be mobilized during autophagy.
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TL;DR: This work has shown 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.
Abstract: 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. Autophagy involves engulfment of cellular components into double-membrane vesicles called autophagosomes. The biogenesis of autophagosomes requires the cooperation of multiple proteins and lipids from various membrane sources. Our understanding of the molecular mechanisms of the initiation, growth, bending and closure of autophagosomal membranes is expanding at a rapid pace.
398 citations
TL;DR: The current understanding and potential mechanisms by which membrane trafficking participates in macroautophagy are described, with a focus on the enigma of the membrane protein Atg9, for which the proximal mechanisms determining its movement are disputable, despite its key role in autophagosome formation.
Abstract: Macroautophagy is an intracellular degradation system that involves the de novo formation of membrane structures called autophagosomes, although the detailed process by which membrane lipids are supplied during autophagosome formation is yet to be elucidated. Macroautophagy is thought to be associated with canonical membrane trafficking, but several mechanistic details are still missing. In this review, the current understanding and potential mechanisms by which membrane trafficking participates in macroautophagy are described, with a focus on the enigma of the membrane protein Atg9, for which the proximal mechanisms determining its movement are disputable, despite its key role in autophagosome formation.
56 citations
TL;DR: The yeast SNX4 sub‐family of sorting nexin containing a Bin‐Amphiphysin‐Rvs domain (SNX‐BAR) proteins, Snx4/Atg24, snx41 and Atg20/Snx42, are required for endocytic recycling and selective autophagy and it is shown that Snx 4 forms 2 functionally distinct heterodimers.
Abstract: The yeast SNX4 sub-family of SNX-BAR proteins, Snx4/Atg24, Snx41, and Atg20/Snx42, are required for endocytic recycling and selective autophagy. Here we show that Snx4 forms two functionally distinct heterodimers: Snx4–Atg20 and Snx4–Snx41. Each heterodimer coats an endosome-derived tubule that mediates retrograde sorting of distinct cargo; the v-SNARE, Snc1, is a cargo of the Snx4-Atg20 pathway, and Snx4-Snx41 mediates retrograde sorting of Atg27, an integral membrane protein implicated in selective autophagy. Live cell imaging of individual endosomes shows that Snx4 and the Vps5-Vps17 retromer SNX-BAR heterodimer operate concurrently on a maturing endosome. Consistent with this, the yeast dynamin family protein, Vps1, which was previously shown to promote fission of retromer-coated tubules, promotes fission of Snx4-Atg20 coated tubules. The results indicate that the yeast SNX-BAR proteins coat three distinct types of endosome-derived carriers that mediate endosome-to-Golgi retrograde trafficking.
53 citations
Cites background from "Atg27 tyrosine sorting motif is imp..."
...brane localization as non-punctate, continuous and circumferential overlapping signal with the vacuole membrane dye, FM4-64, as described in Segarra et al.(18)...
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...Snx4-Atg20 functions on the Snc1 v-SNARE recycling pathway to transport Snc1 to the Golgi, and Snx4-Snx41 functions to transport Atg27 to the Golgi where it is then transported via the AP3 pathway to the vacuolar membrane.(18) We speculate that Snx4-Atg20 and Snx4-Snx41 heterodimers coat distinct carriers....
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...Endosome tubule compositions were quantified as previously described (n = 100).23 To visualize vacuole membrane, yeast cell cultures were incubated with FM4-64 lipophilic dye at 32 nM at 30 C for 20 minutes in YPD, washed and resuspended in fresh medium and grown for 120 minutes to allow FM4-64 to transit to the vacuole membrane.35 We identify Atg27-ΔYSAV-2xGFP and Atg27-2xGFP vacuole membrane localization as non-punctate, continuous and circumferential overlapping signal with the vacuole membrane dye, FM4-64, as described in Segarra et al.18...
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TL;DR: The probable sources of autophagosome membrane are described and the membrane donors are described, which are still under debate and attract a great deal of interests.
Abstract: Autophagy begins with the nucleation of phagophores, which then expand to give rise to the double-membrane autophagosomes. Autophagosomes ultimately fuse with lysosomes, where the cytosolic cargoes are degraded. Accumulation of autophagosomes is a hallmark of autophagy and neurodegenerative disorders including Alzheimer's and Huntington's disease. In recent years, the sources of autophagosome membrane have attracted a great deal of interests, even so, the membrane donors for autophagosomes are still under debate. In this review, we describe the probable sources of autophagosome membrane.
50 citations
Cites background from "Atg27 tyrosine sorting motif is imp..."
...— ATG27 [20] transmembrane protein retrieval of Atg9 from the vacuole...
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TL;DR: Advances regarding retrograde trafficking and recycling pathways are discussed, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.
Abstract: The endosomal system functions as a network of protein and lipid sorting stations that receives molecules from endocytic and secretory pathways and directs them to the lysosome for degradation, or exports them from the endosome via retrograde trafficking or plasma membrane recycling pathways. Retrograde trafficking pathways describe endosome-to-Golgi transport while plasma membrane recycling pathways describe trafficking routes that return endocytosed molecules to the plasma membrane. These pathways are crucial for lysosome biogenesis, nutrient acquisition and homeostasis and for the physiological functions of many types of specialized cells. Retrograde and recycling sorting machineries of eukaryotic cells were identified chiefly through genetic screens using the budding yeast Saccharomyces cerevisiae system and discovered to be highly conserved in structures and functions. In this review, we discuss advances regarding retrograde trafficking and recycling pathways, including new discoveries that challenge existing ideas about the organization of the endosomal system, as well as how these pathways intersect with cellular homeostasis pathways.
47 citations
Cites background from "Atg27 tyrosine sorting motif is imp..."
...is broadly distributed throughout organelles of the endovacuolar system, including the vacuole membrane.(82) Autophagy induction results...
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References
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TL;DR: A new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications that should further facilitate the rapid analysis of gene function in S. cerevisiae.
Abstract: An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications Using as selectable marker the S cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5 + or Escherichia coli kan r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging) Because of the modular nature of the plasmids, they allow eYcient and economical use of a small number of PCR primers for a wide variety of gene manipulations Thus, these plasmids should further facilitate the rapid analysis of gene function in S cerevisiae ? 1998 John Wiley & Sons, Ltd
5,301 citations
"Atg27 tyrosine sorting motif is imp..." refers methods in this paper
...Atg27 localizes to the vacuolar membrane via the AP-3 pathway To examine Atg27’s localization and trafficking the coding sequence for GFP was inserted at the 3′ end of the ATG27 open reading frame (18)....
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...Unless otherwise indicated, the Longtine method was used for generating the fluorescently tagged reporters and deletion mutants (18)....
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TL;DR: This protocol provides a guide to interpreting the output of structure prediction servers in general and one such tool in particular, the protein homology/analogy recognition engine (Phyre), which can reliably detect up to twice as many remote homologies as standard sequence-profile searching.
Abstract: Determining the structure and function of a novel protein is a cornerstone of many aspects of modern biology. Over the past decades, a number of computational tools for structure prediction have been developed. It is critical that the biological community is aware of such tools and is able to interpret their results in an informed way. This protocol provides a guide to interpreting the output of structure prediction servers in general and one such tool in particular, the protein homology/analogy recognition engine (Phyre). New profile–profile matching algorithms have improved structure prediction considerably in recent years. Although the performance of Phyre is typical of many structure prediction systems using such algorithms, all these systems can reliably detect up to twice as many remote homologies as standard sequence-profile searching. Phyre is widely used by the biological community, with >150 submissions per day, and provides a simple interface to results. Phyre takes 30 min to predict the structure of a 250-residue protein.
4,403 citations
"Atg27 tyrosine sorting motif is imp..." refers background in this paper
...Of interest, the large Atg27 luminal region is related to the mannose 6-phosphate receptor homology (MRH) domain family of proteins (42,43)....
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TL;DR: This volume and its companion, Volume 350, are specifically designed to meet the needs of graduate students and postdoctoral students as well as researchers, by providing all the up-to-date methods necessary to study genes in yeast.
Abstract: This volume and its companion, Volume 350, are specifically designed to meet the needs of graduate students and postdoctoral students as well as researchers, by providing all the up-to-date methods necessary to study genes in yeast. Procedures are included that enable newcomers to set up a yeast laboratory and to master basic manipulations. Relevant background and reference information given for procedures can be used as a guide to developing protocols in a number of disciplines. Specific topics addressed in this book include cytology, biochemistry, cell fractionation, and cell biology.
2,659 citations
TL;DR: The molecular mechanism of autophagosome formation is described with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagous membrane membrane.
Abstract: Macroautophagy is mediated by a unique organelle, the autophagosome, which encloses a portion of cytoplasm for delivery to the lysosome. Autophagosome formation is dynamically regulated by starvation and other stresses and involves complicated membrane reorganization. Since the discovery of yeast Atg-related proteins, autophagosome formation has been dissected at the molecular level. In this review we describe the molecular mechanism of autophagosome formation with particular focus on the function of Atg proteins and the long-standing discussion regarding the origin of the autophagosome membrane.
2,522 citations
"Atg27 tyrosine sorting motif is imp..." refers background in this paper
...Although the membrane trafficking events underlying autophagy and the source of membrane for autophagosome formation are of great interest, they are still not completely understood (1,4)....
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TL;DR: A core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process.
Abstract: Eukaryotic cells employ autophagy to degrade damaged or obsolete organelles and proteins. Central to this process is the formation of autophagosomes, double-membrane vesicles responsible for delivering cytoplasmic material to lysosomes. In the past decade many autophagy-related genes, ATG, have been identified that are required for selective and/or nonselective autophagic functions. In all types of autophagy, a core molecular machinery has a critical role in forming sequestering vesicles, the autophagosome, which is the hallmark morphological feature of this dynamic process. Additional components allow autophagy to adapt to the changing needs of the cell.
2,057 citations