scholarly journals Determining Atg protein stoichiometry at the phagophore assembly site by fluorescence microscopy

Autophagy ◽  
2010 ◽  
Vol 6 (1) ◽  
pp. 144-147 ◽  
Author(s):  
Jiefei Geng ◽  
Daniel J. Klionsky
Keyword(s):  
Fluorescence Microscopy ◽  
Phagophore Assembly Site ◽  
Assembly Site

scholarly journals Quantitative analysis of autophagy-related protein stoichiometry by fluorescence microscopy

2008 ◽  
Vol 182 (1) ◽  
pp. 129-140 ◽  
Author(s):  
Jiefei Geng ◽  
Misuzu Baba ◽  
Usha Nair ◽  
Daniel J. Klionsky
Keyword(s):  
Quantitative Analysis ◽  
Fluorescence Microscopy ◽  
Periodic Change ◽  
Related Protein ◽  
Atg Proteins ◽  
Autophagy Induction ◽  
Phagophore Assembly Site ◽  
Insight Into ◽  
Assembly Site

In yeast, ∼31 autophagy-related (Atg) proteins have been identified. Most of them reside at the phagophore assembly site (PAS), although the function of the PAS mostly remains unclear. One reason for the latter is the lack of stoichiometric information regarding the Atg proteins at this site. We report the application of fluorescence microscopy to study the amount of Atg proteins at the PAS. We find that an increase in the amount of Atg11 at the PAS enhances the recruitment of Atg8 and Atg9 to this site and facilitates the formation of more cytoplasm-to-vacuole targeting vesicles. In response to autophagy induction, the amount of most Atg proteins remains unchanged at the PAS, whereas we see an enhanced recruitment of Atg8 and 9 at this site. During autophagy, the amount of Atg8 at the PAS showed a periodic change, indicating the formation of autophagosomes. Application of this method and further analysis will provide more insight into the functions of Atg proteins.

scholarly journals PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex

2019 ◽  
Vol 116 (5) ◽  
pp. 1613-1620 ◽  
Author(s):  
Gonen Memisoglu ◽  
Vinay V. Eapen ◽  
Ying Yang ◽  
Daniel J. Klionsky ◽  
James E. Haber
Keyword(s):  
Kinase Activity ◽  
Budding Yeast ◽  
Nutrient Starvation ◽  
Phosphorylation Sites ◽  
Phagophore Assembly Site ◽  
Assembly Site

Macroautophagy is orchestrated by the Atg1-Atg13 complex in budding yeast. Under nutrient-rich conditions, Atg13 is maintained in a hyperphosphorylated state by the TORC1 kinase. After nutrient starvation, Atg13 is dephosphorylated, triggering Atg1 kinase activity and macroautophagy induction. The phosphatases that dephosphorylate Atg13 remain uncharacterized. Here, we show that two redundant PP2C phosphatases, Ptc2 and Ptc3, regulate macroautophagy by dephosphorylating Atg13 and Atg1. In the absence of these phosphatases, starvation-induced macroautophagy and the cytoplasm-to-vacuole targeting pathway are inhibited, and the recruitment of the essential autophagy machinery to the phagophore assembly site is impaired. Expressing a genomic ATG13-8SA allele lacking key TORC1 phosphorylation sites partially bypasses the macroautophagy defect in ptc2Δ ptc3Δ strains. Moreover, Ptc2 and Ptc3 interact with the Atg1-Atg13 complex. Taken together, these results suggest that PP2C-type phosphatases promote macroautophagy by regulating the Atg1 complex.

scholarly journals Vacuolar protein Tag1 and Atg1–Atg13 regulate autophagy termination during persistent starvation in S. cerevisiae

2021 ◽  
Vol 134 (4) ◽  
pp. jcs253682
Author(s):  
Shintaro Kira ◽  
Masafumi Noguchi ◽  
Yasuhiro Araki ◽  
Yu Oikawa ◽  
Tamotsu Yoshimori ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Membrane Protein ◽  
Nitrogen Starvation ◽  
Genetic Screen ◽  
Vacuolar Membrane ◽  
Phagophore Assembly Site ◽  
Vacuolar Protein ◽  
Assembly Site ◽  
Starvation Conditions

ABSTRACTUnder starvation conditions, cells degrade their own components via autophagy in order to provide sufficient nutrients to ensure their survival. However, even if starvation persists, the cell is not completely degraded through autophagy, implying the existence of some kind of termination mechanism. In the yeast Saccharomyces cerevisiae, autophagy is terminated after 10–12 h of nitrogen starvation. In this study, we found that termination is mediated by re-phosphorylation of Atg13 by the Atg1 protein kinase, which is also affected by PP2C phosphatases, and the eventual dispersion of the pre-autophagosomal structure, also known as the phagophore assembly site (PAS). In a genetic screen, we identified an uncharacterized vacuolar membrane protein, Tag1, as a factor responsible for the termination of autophagy. Re-phosphorylation of Atg13 and eventual PAS dispersal were defective in the Δtag1 mutant. The vacuolar luminal domain of Tag1 and autophagic progression are important for the behaviors of Tag1. Together, our findings reveal the mechanism and factors responsible for termination of autophagy in yeast.

scholarly journals An Atg9-containing compartment that functions in the early steps of autophagosome biogenesis

2010 ◽  
Vol 190 (6) ◽  
pp. 1005-1022 ◽  
Author(s):  
Muriel Mari ◽  
Janice Griffith ◽  
Ester Rieter ◽  
Lakshmi Krishnappa ◽  
Daniel J. Klionsky ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
De Novo ◽  
En Bloc ◽  
Genetic Analyses ◽  
Double Membrane ◽  
Precise Nature ◽  
Phagophore Assembly Site ◽  
Assembly Site ◽  
Key Questions ◽  
Phosphatidylinositol 3

Eukaryotes use the process of autophagy, in which structures targeted for lysosomal/vacuolar degradation are sequestered into double-membrane autophagosomes, in numerous physiological and pathological situations. The key questions in the field relate to the origin of the membranes as well as the precise nature of the rearrangements that lead to the formation of autophagosomes. We found that yeast Atg9 concentrates in a novel compartment comprising clusters of vesicles and tubules, which are derived from the secretory pathway and are often adjacent to mitochondria. We show that these clusters translocate en bloc next to the vacuole to form the phagophore assembly site (PAS), where they become the autophagosome precursor, the phagophore. In addition, genetic analyses indicate that Atg1, Atg13, and phosphatidylinositol-3-phosphate are involved in the further rearrangement of these initial membranes. Thus, our data reveal that the Atg9-positive compartments are important for the de novo formation of the PAS and the sequestering vesicle that are the hallmarks of autophagy.

scholarly journals Spatial control of avidity regulates initiation and progression of selective autophagy

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David M. Hollenstein ◽  
Mariya Licheva ◽  
Nicole Konradi ◽  
David Schweida ◽  
Hector Mancilla ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Membrane ◽  
Vacuolar Membrane ◽  
Phosphatidylinositol 3 Kinase ◽  
Autophagosome Formation ◽  
Spatial Control ◽  
Selective Autophagy ◽  
Phagophore Assembly Site ◽  
Assembly Site ◽  
Phosphatidylinositol 3

AbstractAutophagosomes form at the endoplasmic reticulum in mammals, and between the vacuole and the endoplasmic reticulum in yeast. However, the roles of these sites and the mechanisms regulating autophagosome formation are incompletely understood. Vac8 is required for autophagy and recruits the Atg1 kinase complex to the vacuole. Here we show that Vac8 acts as a central hub to nucleate the phagophore assembly site at the vacuolar membrane during selective autophagy. Vac8 directly recruits the cargo complex via the Atg11 scaffold. In addition, Vac8 recruits the phosphatidylinositol 3-kinase complex independently of autophagy. Cargo-dependent clustering and Vac8-dependent sequestering of these early autophagy factors, along with local Atg1 activation, promote phagophore assembly site assembly at the vacuole. Importantly, ectopic Vac8 redirects autophagosome formation to the nuclear membrane, indicating that the vacuolar membrane is not specifically required. We propose that multiple avidity-driven interactions drive the initiation and progression of selective autophagy.

scholarly journals Harpooning the Cvt complex to the phagophore assembly site

Autophagy ◽  
2008 ◽  
Vol 4 (7) ◽  
pp. 914-916 ◽  
Author(s):  
Iryna Monastyrska ◽  
Fulvio Reggiori ◽  
Daniel J. Klionsky
Keyword(s):  
Phagophore Assembly Site ◽  
Assembly Site

scholarly journals Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Autophagy ◽  
2016 ◽  
Vol 12 (4) ◽  
pp. 648-658 ◽  
Author(s):  
Yuchen Feng ◽  
Steven K. Backues ◽  
Misuzu Baba ◽  
Jin-Mi Heo ◽  
J. Wade Harper ◽  
...  
Keyword(s):  
Autophagosome Formation ◽  
Phagophore Assembly Site ◽  
Assembly Site

scholarly journals Mammalian BCAS3 and C16orf70 associate with the phagophore assembly site in response to selective and non-selective autophagy

Autophagy ◽  
2021 ◽  
pp. 1-26
Author(s):  
Waka Kojima ◽  
Koji Yamano ◽  
Hidetaka Kosako ◽  
Kenichiro Imai ◽  
Reika Kikuchi ◽  
...  
Keyword(s):  
Selective Autophagy ◽  
Phagophore Assembly Site ◽  
Assembly Site

scholarly journals Solution Structure of the Atg1 Complex: Implications for the Architecture of the Phagophore Assembly Site

Structure ◽  
2015 ◽  
Vol 23 (5) ◽  
pp. 809-818 ◽  
Author(s):  
Jürgen Köfinger ◽  
Michael J. Ragusa ◽  
Il-Hyung Lee ◽  
Gerhard Hummer ◽  
James H. Hurley
Keyword(s):  
Solution Structure ◽  
Phagophore Assembly Site ◽  
Assembly Site
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