Fig 4 - uploaded by Zhong-Qiu Yu
Content may be subject to copyright.
Mitochondrial autophagy is promoted in a redundant manner by Atg20, Atg24 and Atg24b. (A) Starvation-induced vacuole localization of mitomCherry was monitored in selected double and triple mutants. 20, Atg20; 24, Atg24; 24b, Atg24b; 186, Mug186. +N, nitrogen-containing medium; −N, nitrogenstarvation medium. Scale bar: 3 µm. (B) Starvation-induced processing of Sdh2-mCherry was monitored in selected double and triple mutants. Coomassie Brilliant Blue R-350 (CBB) staining of PVDF membrane after immunodetection was used to control for protein loading and blotting efficiency. The free/FL ratios were calculated as described in Fig. 1.
Source publication
Autophagy cargos include not only soluble cytosolic materials but also bulky organelles such as ER and mitochondria. In budding yeast, two proteins that contain the PX domain and the BAR domain, Atg20/Snx42 and Atg24/Snx4, are required for organelle autophagy and contribute to general autophagy in a way that can be compensated. It remains unclear w...
Contexts in source publication
Context 1
... hypothesized that Atg20, Atg24 and Atg24b function in mitochondrial autophagy in a redundant manner, similar to the way they act in ER autophagy. Thus, we examined a number of selected double and triple mutants. Consistent with the findings for ER autophagy, the vacuolar entry of the mitochondrial marker mito- mCherry was impeded in atg20? atg24? and atg24? atg24b? double mutants, but not in the atg24? mug186? double mutant and atg20? atg24b? mug186? triple mutant (Fig. 4A). Similarly, the processing of Sdh2-mCherry was defective in atg20? atg24? and atg24? atg24b? double mutants, but not the in atg24? mug186? double mutant and the atg20? atg24b? mug186? triple mutant (Fig. 4B). Taken together, these data demonstrate that, like ER autophagy, mitochondrial autophagy is promoted redundantly by Atg24, Atg20 and Atg24b. Atg24 appears to be sufficient by itself; in its absence, the mitochondrial autophagy-promoting function can be fulfilled equally well by the combined actions of Atg20 and ...
Context 2
... hypothesized that Atg20, Atg24 and Atg24b function in mitochondrial autophagy in a redundant manner, similar to the way they act in ER autophagy. Thus, we examined a number of selected double and triple mutants. Consistent with the findings for ER autophagy, the vacuolar entry of the mitochondrial marker mito- mCherry was impeded in atg20? atg24? and atg24? atg24b? double mutants, but not in the atg24? mug186? double mutant and atg20? atg24b? mug186? triple mutant (Fig. 4A). Similarly, the processing of Sdh2-mCherry was defective in atg20? atg24? and atg24? atg24b? double mutants, but not the in atg24? mug186? double mutant and the atg20? atg24b? mug186? triple mutant (Fig. 4B). Taken together, these data demonstrate that, like ER autophagy, mitochondrial autophagy is promoted redundantly by Atg24, Atg20 and Atg24b. Atg24 appears to be sufficient by itself; in its absence, the mitochondrial autophagy-promoting function can be fulfilled equally well by the combined actions of Atg20 and ...
Context 3
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Context 4
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Context 5
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Context 6
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Context 7
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Context 8
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Context 9
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Context 10
... Atg20 and Mug186, suggesting that Atg24 can engage only one interaction partner at a time. Similarly, no interaction was found between Atg24 and Atg24b, despite both of them being able to interact with Atg20. In addition to the hetero-interactions, the coimmunoprecipitation analysis also revealed that Atg24 can engage in a homotypic interaction (Fig. 5C). Given the results of the mutant-phenotype analysis, we speculate that the functional forms of these proteins are the oligomers (including dimers), and either the homo-oligomer of Atg24 or the hetero-oligomer of Atg20 and Atg24b is sufficient to promote organelle autophagy (Fig. 5F). No other Atg proteins were found in our AP-MS analysis. Because budding yeast Atg20 and Atg24 interact with Atg17 ( Nice et al., 2002), we used a yeast two-hybrid (Y2H) assay to examine whether fission yeast Atg20, Atg24 and Atg24b interact with Atg17 or with several other Atg proteins that could act together with Atg17, including Atg1, Atg11, Atg13 and Atg101. The results were all negative (Fig. S3C). To further understand the functional relevance of the hetero- interactions between Atg20-family and Atg24-family proteins, we examined whether their subcellular localizations were affected by each other. The only interactions we have observed for Atg24b and Mug186 are their hetero-interactions with Atg20 and Atg24, respectively. Supporting the importance of these interactions, puncta formation by Atg24b was completely abolished in atg20? cells (Fig. S4A), and puncta formation by Mug186 was completely abolished in atg24? cells (Fig. S4B). Atg20 can interact with either Atg24 or Atg24b. Under nutrient-rich conditions, Atg20 no longer formed puncta in atg24? cells (Fig. S4C), consistent with the fact that Atg24 but not Atg24b forms puncta under such conditions. The PAS localization of Atg20 in starved cells remained largely normal in atg24? or atg24b? single mutants (Fig. S4C). In contrast, in the atg24? atg24b? double mutant, Atg8-colocalizing Atg20 puncta were severely diminished (Fig. S4C). Thus, the PAS localization of Atg20 redundantly depends on its two interaction partners Atg24 and Atg24b. Atg24 can engage not only in hetero-interactions with Atg20 and Mug186, but also in a homo-interaction with itself. Deleting mug186 strongly reduced the numbers of puncta formed by Atg24 but did not affect the PAS localization of Atg24, and this deletion actually made it easier to observe the PAS localization of Atg24 than it was to observe in the wild-type cells (Fig. S4D). Deleting atg20 either in wild-type or mug186? background did not obviously alter the localization pattern of Atg24 (Fig. S4D), consistent with the model that Atg24 homo-oligomers can function in the absence of both Atg20 and Mug186. The results of these analyses are summarized in Fig. ...
Citations
... We cultured the cells in Edinburgh Minimal Medium containing 2 % glucose and five supplements (adenine, histidine, leucine, lysine, and uracil) (referred to as EMM5S). To visualize mitochondria, Sdh2, a protein localizing within the mitochondrial matrix [39], was tagged with a green fluorescent protein (GFP). Microscopy revealed that mitochondria were tubular in WT cells but became fragmented in rpl2702Δ cells ( Fig. 1A and B). ...
Ribosomes mediate protein synthesis, which is one of the most energy-demanding activities within the cell, and mitochondria are one of the main sources generating energy. How mitochondrial morphology and functions are adjusted to cope with ribosomal defects, which can impair protein synthesis and affect cell viability, is poorly understood. Here, we used the fission yeast Schizosaccharomyces Pombe as a model organism to investigate the interplay between ribosome and mitochondria. We found that a ribosomal insult, caused by the absence of Rpl2702, activates a signaling pathway involving Sty1/MAPK and mTOR to modulate mitochondrial morphology and functions. Specifically, we demonstrated that Sty1/MAPK induces mitochondrial fragmentation in a mTOR-independent manner while both Sty1/MAPK and mTOR increases the levels of mitochondrial membrane potential and mitochondrial reactive oxygen species (mROS). Moreover, we demonstrated that Sty1/MAPK acts upstream of Tor1/TORC2 and Tor1/TORC2 and is required to activate Tor2/TORC1. The enhancements of mitochondrial membrane potential and mROS function to promote proliferation of cells bearing ribosomal defects. Hence, our study reveals a previously uncharacterized Sty1/MAPK-mTOR signaling axis that regulates mitochondrial morphology and functions in response to ribosomal insults and provides new insights into the molecular and physiological adaptations of cells to impaired protein synthesis.
... Atg24 (also termed Snx4) is required for autophagy of large cargoes such as organelles, ribosomes, and proteasomes. [44][45][46][47] In atg24D cells, the degradation of Gsy2 was clearly impaired, while that of Pgk1 was only slightly decreased ( Figures 2D and 2E). Together, these results show that prolonged starvation-induced glycophagy does not require Atg11 but does require Atg24. ...
The mechanisms governing autophagy of proteins and organelles have been well studied, but how other cytoplasmic components such as RNA and polysaccharides are degraded remains largely unknown. In this study, we examine autophagy of glycogen, a storage form of glucose. We find that cells accumulate glycogen in the cytoplasm during nitrogen starvation and that this carbohydrate is rarely observed within autophagosomes and autophagic bodies. However, sequestration of glycogen by autophagy is observed following prolonged nitrogen starvation. We identify a yet-uncharacterized open reading frame, Yil024c (herein Atg45), as encoding a cytosolic receptor protein that mediates autophagy of glycogen (glycophagy). Furthermore, we show that, during sporulation, Atg45 is highly expressed and is associated with an increase in glycophagy. Our results suggest that cells regulate glycophagic activity by controlling the expression level of Atg45.
... Silencing of the ATG32 gene results in the retardation of protein recruitment during autophagy that impaired mitochondrial degeneration and is not involved in the degradation of other forms of autophagy pexophagy or nucleophagy [99,106]. Whereas in fission yeast upon nitrogen starvation autophagy of mitochondria and ER is induced by the interaction of three proteins (ATG20, ATG24, and ATG24b) as in plants and animals, ATG8-interacting motifs act as receptor proteins during mitophagy [10,40]. In fission yeast, ATG43 localized to the mitochondrial outer membrane function as a receptor during mitophagy and interacts with a ubiquitinlike ATG8 protein. ...
... Moreover, during the process of autophagy ATG11 after binding with the Cvt activates dimerization that results in the regulation of other ATG components like ATG1 and ATG17; for the transportation of ATG9 anterograde and ATG19-prAPE1 complex from mitochondria to PAS; in the expansion of cell life [28][29][30]. ATG24 with other ATG-proteins (ATG20, ATG24, and ATG24b) is required for organelle autophagy (ER and mitochondria) and general autophagy when cells are subjected to nitrogen starvation [40]. MoATG24 plays a specific role in the direct recruitment of mitochondria to autophagy bodies and in stress response and was not involved in pexophagy and macroautophagy [109]. ...
... In contrast to the severe ER-phagy and nucleophagy defects of yep1Δ cells, bulk autophagy in yep1Δ cells was largely normal as indicated by the processing of CFP-Atg8 (Fig 1J). In addition, another readout of bulk autophagy, the processing of fluorescent protein-tagged cytosolic protein Tdh1 (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)) [43], was also largely unaffected in yep1Δ cells (S1E Fig). Consistent with the lack of bulk autophagy defects, transmission electron microscopy (TEM) analysis showed that autophagosome accumulation in the fsc1Δ mutant, which is defective in autophagosome-vacuole fusion [44], was not notably affected by the deletion of yep1 (S1F Fig). ...
... Thin sections were examined using an FEI Tecnai G2 Spirit electron microscope equipped with a Gatan 895 4k×4k CCD camera. The diameters of the ring-shaped structures were determined using the method previously used for measuring the sizes of autophagosomes or autophagic bodies [43,76]. P values were calculated using Welch's t test. ...
... Yop1 (133-182) appears in Fig 4I. Yop1 and Yop1 [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] appear in (G). Yop1 ...
Selective macroautophagy of the endoplasmic reticulum (ER) and the nucleus, known as ER-phagy and nucleophagy, respectively, are processes whose mechanisms remain inadequately understood. Through an imaging-based screen, we find that in the fission yeast Schizosaccharomyces pombe , Yep1 (also known as Hva22 or Rop1), the ortholog of human REEP1-4, is essential for ER-phagy and nucleophagy but not for bulk autophagy. In the absence of Yep1, the initial phase of ER-phagy and nucleophagy proceeds normally, with the ER-phagy/nucleophagy receptor Epr1 coassembling with Atg8. However, ER-phagy/nucleophagy cargos fail to reach the vacuole. Instead, nucleus- and cortical-ER-derived membrane structures not enclosed within autophagosomes accumulate in the cytoplasm. Intriguingly, the outer membranes of nucleus-derived structures remain continuous with the nuclear envelope-ER network, suggesting a possible outer membrane fission defect during cargo separation from source compartments. We find that the ER-phagy role of Yep1 relies on its abilities to self-interact and shape membranes and requires its C-terminal amphipathic helices. Moreover, we show that human REEP1-4 and budding yeast Atg40 can functionally substitute for Yep1 in ER-phagy, and Atg40 is a divergent ortholog of Yep1 and REEP1-4. Our findings uncover an unexpected mechanism governing the autophagosomal enclosure of ER-phagy/nucleophagy cargos and shed new light on the functions and evolution of REEP family proteins.
... By contrast, the size and number of autophagic bodies did not differ largely between wild-type and atg24Δ cells ( Supplementary Fig. 2d, e), providing important insight into the mechanism underlying autophagosome biogenesis; the final size of the autophagosome and the overall efficiency of autophagosome formation can be determined independently of the opening size of the expanding IM. However, in the fission yeast Schizosaccharomyces pombe, cells lacking Atg20 and Atg24 homologs accumulated autophagosomes smaller than those of control cells 25 . Therefore, multiple mechanisms may govern autophagosome morphogenesis, and these mechanisms may vary among organisms. ...
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.
... It was apparent that in yta4Δ cells, the mitochondrial number increased, whereas the number of mitochondrial branches and junctions decreased significantly, suggesting that the absence of Yta4 causes mitochondrial fragmentation and reduces the complexity of the mitochondrial network ( Fig 1B). For ease of observation by live-cell microscopy, we tagged Sdh2, a mitochondrial matrix protein [27], at its own locus with mCherry in WT and yta4Δ cells or yta4Δ cells expressing Yta4-13Myc from its own promoter. As shown in Fig 1C and 1D, the absence of Yta4 similarly caused mitochondrial fragmentation in these Sdh2-mCherry-expressing cells, while ectopic expression of Yta4-13Myc in yta4Δ cells restored mitochondria to wild-type-like tubular morphology (Fig 1C). ...
Mitochondria are in a constant balance of fusion and fission. Excessive fission or deficient fusion leads to mitochondrial fragmentation, causing mitochondrial dysfunction and physiological disorders. How the cell prevents excessive fission of mitochondria is not well understood. Here, we report that the fission yeast AAA-ATPase Yta4, which is the homolog of budding yeast Msp1 responsible for clearing mistargeted tail-anchored (TA) proteins on mitochondria, plays a critical role in preventing excessive mitochondrial fission. The absence of Yta4 leads to mild mitochondrial fragmentation in a Dnm1-dependent manner but severe mitochondrial fragmentation upon induction of mitochondrial depolarization. Overexpression of Yta4 delocalizes the receptor proteins of Dnm1, i.e., Fis1 (a TA protein) and Mdv1 (the bridging protein between Fis1 and Dnm1), from mitochondria and reduces the localization of Dnm1 to mitochondria. The effect of Yta4 overexpression on Fis1 and Mdv1, but not Dnm1, depends on the ATPase and translocase activities of Yta4. Moreover, Yta4 interacts with Dnm1, Mdv1, and Fis1. In addition, Yta4 competes with Dnm1 for binding Mdv1 and decreases the affinity of Dnm1 for GTP and inhibits Dnm1 assembly in vitro. These findings suggest a model, in which Yta4 inhibits mitochondrial fission by inhibiting the function of the mitochondrial divisome composed of Fis1, Mdv1, and Dnm1. Therefore, the present work reveals an uncharacterized molecular mechanism underlying the inhibition of mitochondrial fission.
... Autophagy is a conserved proteolytic pathway from yeast to mammals; many Atg proteins have been identified in studies using S. cerevisiae, and similar proteins have been confirmed in S. pombe [43,44]. Atg11 is required for selective autophagy in S. cerevisiae but for general autophagy in S. pombe [45]. Atg11 but not Atg13, Atg17, or Atg101 in S. pombe is required for the kinase activity of Atg1 [46], suggesting that Atg protein functions may differ slightly among yeast species. ...
Cyclins are degraded by the anaphase-promoting complex/cyclosome (APC/C)-mediated proteasome in normal mitosis. We showed that Cdc13 (cyclin B) is also degraded by macroautophagy/autophagy in sulfur-deficient fission yeast. Sulfur depletion causes G2 cell cycle arrest and reduces cell size; however, the associated mechanisms are unknown. We found that autophagy is required for the degradation of Cdc13, which is associated with cell cycle arrest and reduced cell size, by examining cell morphology under sulfur depletion. The analysis of the Cdc13-GFP fusion protein supported the conclusion that Cdc13 is degraded by autophagy. Moreover, we showed that sulfur depletion results in the inactivation of target of rapamycin complex 1 (TORC1) activity via Ecl1-family proteins. Our data indicate that the cyclin is degraded by two different systems: APC/C-mediated proteasome and autophagy. The latter is induced under nutrient-depleted situations. This switch in degradation systems will contribute to appropriate cell cycle arrest when resources are depleted.
Abbreviations: APC, anaphase-promoting complex; CDK, cyclin-dependent kinase; DB, destruction box; EMM, Edinburgh minimal medium; GFP, green fluorescent protein; PCR, polymerase chain reaction; TOR, target of rapamycin; UPS, ubiquitin-proteasome system
... Most of the key components required in autophagy have been identified in budding yeast, including the receptor on the mitochondrial outer membrane (Atg32) recognized by the autophagy machinery [21]. Recently, two studies have also revealed the identity of specific components of the mitophagy pathway in fission yeast [22,23]. ...
... In S. pombe, three Atg proteins, Atg20, Atg24, and Atg24b, have also been implicated in the degradation of mitochondria by autophagy [22]. Comparable to the atg43-1 mutant (Fig. 6D, E), double deletion of these Atg proteins led to impaired mitophagy (Additional file 8: Fig. S5B) and decreased survival (Additional file 8. Fig. S5C) during chronological aging after growth in low glucose media. ...
Background
In many organisms, aging is characterized by a loss of mitochondrial homeostasis. Multiple factors such as respiratory metabolism, mitochondrial fusion/fission, or mitophagy have been linked to cell longevity, but the exact impact of each one on the aging process is still unclear.
Results
Using the deletion mutant collection of the fission yeast Schizosaccharomyces pombe , we have developed a genome-wide screening for mutants with altered chronological lifespan. We have identified four mutants associated with proteolysis at the mitochondria that exhibit opposite effects on longevity. The analysis of the respiratory activity of these mutants revealed a positive correlation between increased respiration rate and prolonged lifespan. We also found that the phenotype of the long-lived protease mutants could not be explained by impaired mitochondrial fusion/fission activities, but it was dependent on mitophagy induction. The anti-aging role of mitophagy was supported by the effect of a mutant defective in degradation of mitochondria, which shortened lifespan of the long-lived mutants.
Conclusions
Our characterization of the mitochondrial protease mutants demonstrates that mitophagy sustains the lifespan extension of long-lived mutants displaying a higher respiration potential.
... Fission yeast autophagy factors were first identified by their homology to previously known budding yeast ATG genes [19,20] and later also by unbiased genetic screens that revealed additional autophagy factors that cannot be easily identified by homology search [14,21]. Molecular characterization of these factors in the last decade has led to rich mechanistic insights [13][14][15]17,18,[21][22][23][24][25]. In addition, structural biology analyses of S. pombe autophagy proteins have also shed new light on the molecular machinery of autophagy [12,26,27]. ...
... Targeting cytosolic hydrolases to the vacuole [17,18] Atg19 NBR1 Atg20 Atg20 family protein promoting ER and mitochondrial autophagy [22] Atg20 − Atg24 and Atg24b Atg24 family proteins promoting ER and mitochondrial autophagy [22] Atg24 − Epr1 ER-phagy receptor [25] − − ...
... Targeting cytosolic hydrolases to the vacuole [17,18] Atg19 NBR1 Atg20 Atg20 family protein promoting ER and mitochondrial autophagy [22] Atg20 − Atg24 and Atg24b Atg24 family proteins promoting ER and mitochondrial autophagy [22] Atg24 − Epr1 ER-phagy receptor [25] − − ...
Autophagy is a conserved process that delivers cytoplasmic components to the vacuole/lysosome. It plays important roles in maintaining cellular homeostasis and conferring stress resistance. In the fission yeast Schizosaccharomyces pombe, autophagy is important for cell survival under nutrient depletion and ER stress conditions. Experimental analyses of fission yeast autophagy machinery in the last 10 years have unveiled both similarities and differences in autophagosome biogenesis mechanisms between fission yeast and other model eukaryotes for autophagy research, in particular, the budding yeast Saccharomyces cerevisiae. More recently, selective autophagy pathways that deliver hydrolytic enzymes, the ER, and mitochondria to the vacuole have been discovered in fission yeast, yielding novel insights into how cargo selectivity can be achieved in autophagy. Here, we review the progress made in understanding the autophagy machinery in fission yeast.
... Thus, the Atg11 requirement cannot be used as evidence to assess whether a cargo is selectively targeted by autophagy in S. pombe. As in S. cerevisiae, nitrogen starvation induced autophagic elimination of mitochondria in this fission yeast, but the molecular mechanism underlying this degradation, and whether this pathway is selective to mitochondria, remains uncertain [93,94]. In their recent work, Fukuda et al. (2020) identified the protein Atg43 as a new mitophagy receptor in S. pombe [95]. ...
Mitophagy, the selective degradation of mitochondria by autophagy, is one of the most important mechanisms of mitochondrial quality control, and its proper functioning is essential for cellular homeostasis. In this review, we describe the most important milestones achieved during almost 2 decades of research on yeasts, which shed light on the molecular mechanisms, regulation, and role of the Atg32 receptor in this process. We analyze the role of ROS in mitophagy and discuss the physiological roles of mitophagy in unicellular organisms, such as yeast; these roles are very different from those in mammals. Additionally, we discuss some of the different tools available for studying mitophagy.
