Quantitative Assay of Macroautophagy Using Pho8△60 Assay and GFP-Cleavage Assay in Yeast

Araki, Yasuhiro; Kira, Shintaro; Noda, Tetsuji

doi:10.1016/bs.mie.2016.10.027

Cited by 13 publications

(18 citation statements)

References 20 publications

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“…Pho8∆60 can be stably integrated into the genome, replacing the PHO8 gene [ 34 ]. Alternatively, plasmid-based overexpression of Pho8∆60 without deleting endogenous PHO8 has been used to measure autophagic flux [ 38 , 39 ]. This plasmid-based approach requires continuous selection for the plasmid, but avoids the assay-specific manipulation of the yeast genome, especially when using α-naphthyl phosphate as phosphatase substrate.…”

Section: End-point Measurementsmentioning

confidence: 99%

Assays to Monitor Autophagy in Saccharomyces cerevisiae

Torggler

Papinski

Kraft

2017

Cells

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Autophagy is an intracellular process responsible for the degradation and recycling of cytoplasmic components. It selectively removes harmful cellular material and enables the cell to survive starvation by mobilizing nutrients via the bulk degradation of cytoplasmic components. While research over the last decades has led to the discovery of the key factors involved in autophagy, the pathway is not yet completely understood. The first studies of autophagy on a molecular level were conducted in the yeast Saccharomyces cerevisiae. Building up on these studies, many homologs have been found in higher eukaryotes. Yeast remains a highly relevant model organism for studying autophagy, with a wide range of established methods to elucidate the molecular details of the autophagy pathway. In this review, we provide an overview of methods to study both selective and bulk autophagy, including intermediate steps in the yeast Saccharomyces cerevisiae. We compare different assays, discuss their advantages and limitations and list potential applications.

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Section: End-point Measurementsmentioning

confidence: 99%

Assays to Monitor Autophagy in Saccharomyces cerevisiae

Torggler

Papinski

Kraft

2017

Cells

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“…Because cells continue to grow when overexpressed integral-membrane proteins are shuttled to the vacuole for degradation through ER-phagy [7, 9], we hypothesized that this pathway is constitutive and does not require induction of general autophagy. Three different approaches were used to assess whether general autophagy response is induced in cells overexpressing GFP-Snc1-PEM: determining Atg8 protein level [23], assessing processing of the CVT cargo Ape1 [11], and measuring the alkaline phosphatase activity of Pho8Δ60 [24].…”

Section: Resultsmentioning

confidence: 99%

“…Although clearance of excess integral-membrane proteins during normal growth requires Atg8, and proteins needed for its lipidation, Atg5 and Atg7, Atg8 level is not increased. Second, measuring Pho8Δ60 activity is the accepted quantitative nutritional stress-induced autophagy assay [24]. During normal growth, neither overexpression of an integral-membrane protein nor its accumulation in mutants defective in its clearance, result in an induction of Pho8Δ60 activity.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Constitutive macro-ER-phagy

Lipatova

Gyurkovska²,

Zhang³

et al. 2020

Preprint

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Thirty percent of all cellular proteins are inserted into the endoplasmic reticulum (ER), which spans throughout the cytoplasm. Two well-established stress-induced pathways ensure quality control (QC) at the ER: ER-phagy and ER-associated degradation (ERAD), which shuttle cargo for degradation to the lysosome and proteasome, respectively. In contrast, not much is known about constitutive ER-phagy. We have previously reported that excess of integral-membrane proteins is delivered from the ER to the lysosome via autophagy during normal growth of yeast cells. Here, we characterize this pathway as constitutive ER-phagy. Constitutive and stress-induced ER-phagy share the basic macro-autophagy machinery including the conserved Atgs and Ypt1 GTPase. However, induction of stress-induced autophagy is not needed for constitutive ER-phagy to occur. Moreover, the selective receptors needed for starvation- induced ER-phagy, Atg39 and Atg40, are not required for constitutive ER-phagy and neither these receptors nor their cargos are delivered through it to the vacuole. As for ERAD, while constitutive ER-phagy recognizes cargo different from that recognized by ERAD, these two ER-QC pathways can partially substitute for each other. Because accumulation of membrane proteins is associated with disease, and constitutive ER-phagy players are conserved from yeast to mammalian cells, this process could be critical for human health.

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“…In yeast cells, Atg17/FIP200, Atg31, and Atg29 proteins form a stable complex independent of nutritional status. Atg1, Atg11, and Atg13 form polymer complex with Atg17-Atg31-Atg29 as a platform for the recruitment of other ATG proteins (Araki et al, 2017). As shown in Supplementary Figures S1A,B, under nitrogen starvation and glucose starvation, Atg17 protein appears as puncta in the fzo1 and ugo1 yeast strain, indicating that mitochondrial fusion machinery is not involved in PAS formation.…”

Section: Mitochondrial Fusion Machinerymentioning

confidence: 96%

“…Next, to investigate autophagy level induced by energy deficiency, we compared autophagy under SD-N and SD-G by ALP assay (Araki et al, 2017). As shown in Figure 5A, the ALP activity under nitrogen starvation is about two times higher than that of under glucose starvation, indicating the intensity of autophagy induced by glucose starvation is weaker than that by nitrogen starvation.…”

Section: Glucose Starvation Does Not Induce Mitophagymentioning

confidence: 99%

Mitochondrial Fusion Machinery Specifically Involved in Energy Deprivation-Induced Autophagy

Yao

Kai

et al. 2020

Front. Cell Dev. Biol.

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Mitochondria are highly dynamic organelles, which can form a network in cells through fusion, fission, and tubulation. Its morphology is closely related to the function of mitochondria. The damaged mitochondria can be removed by mitophagy. However, the relationship between mitochondrial morphology and non-selective autophagy is not fully understood. We found that mitochondrial fusion machinery, not fission or tubulation machinery, is essential for energy deprivation-induced autophagy. In response to glucose starvation, deletion of mitochondrial fusion proteins severely impaired the association of Atg1/ULK1 with Atg13, and then affected the recruitment of Atg1 and other autophagic proteins to PAS (phagophore assembly site). Furthermore, the deletion of fusion proteins blocks mitochondrial respiration, the binding of Snf1-Mec1, the phosphorylation of Mec1 by Snf1, and the dissociation of Mec1 from mitochondria under prolonged starvation. We propose that mitochondrial fusion machinery regulates energy deprivation-induced autophagy through maintaining mitochondrial respiration.

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Quantitative Assay of Macroautophagy Using Pho8△60 Assay and GFP-Cleavage Assay in Yeast

Cited by 13 publications

References 20 publications

Assays to Monitor Autophagy in Saccharomyces cerevisiae

Assays to Monitor Autophagy in Saccharomyces cerevisiae

Characterization of Constitutive macro-ER-phagy

Mitochondrial Fusion Machinery Specifically Involved in Energy Deprivation-Induced Autophagy

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