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Phosphorylation of Atg31 is required for autophagy.

Feng W, Wu T, Dan X, Chen Y, Li L, Chen S, Miao D, Deng H, Gong X, Yu L - Protein Cell (2015)

Bottom Line: S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment.Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS.Our data demonstrate the role of phosphorylation of Atg31 in autophagy.

View Article: PubMed Central - PubMed

Affiliation: PTN Program, College of Life Science, Peking University, Beijing, 100871, China.

ABSTRACT
Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.

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Impairment of Atg9 recycling in the S174A mutant. (A) Wild-type or S174A mutant cells expressing Atg9-GFP were transferred to nitrogen starvation for 0 h or 2 h and observed by confocal microscopy. Scale bar, 2 µm. (B) Cells from (A) were assessed for the number and intensity of Atg9 puncta. (C) Wild-type or S174 mutant cells expressing Atg9-GFP and Cherry red-Atg8 were transferred to SD-N medium for 2 h and observed by confocal microscopy. Scale bar, 2 µm. (D) Cells from (C) were assessed for co-localization between Atg9 and Atg8. 100 cells were assessed in a blind fashion and quantified. Error bars indicate standard deviation (s.d.) (n = 3)
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Fig4: Impairment of Atg9 recycling in the S174A mutant. (A) Wild-type or S174A mutant cells expressing Atg9-GFP were transferred to nitrogen starvation for 0 h or 2 h and observed by confocal microscopy. Scale bar, 2 µm. (B) Cells from (A) were assessed for the number and intensity of Atg9 puncta. (C) Wild-type or S174 mutant cells expressing Atg9-GFP and Cherry red-Atg8 were transferred to SD-N medium for 2 h and observed by confocal microscopy. Scale bar, 2 µm. (D) Cells from (C) were assessed for co-localization between Atg9 and Atg8. 100 cells were assessed in a blind fashion and quantified. Error bars indicate standard deviation (s.d.) (n = 3)

Mentions: Atg9 is a multi-pass transmembrane protein that plays a key role in autophagosome formation. Atg9-positive vesicles are highly mobile structures in the cytoplasm (Yamamoto et al., 2012) that recycle Atg9 and other molecular from the PAS to the cytoplasmic pool (Reggiori et al., 2004). We found that the number of Atg9 puncta is reduced in the S174A mutant. Furthermore, the fluorescence intensity of the Atg9 puncta is dramatically enhanced, indicating that the dynamic recycling of Atg9 between different pools is impaired and Atg9 accumulates in these puncta (Fig. 4A and 4B). Since Atg9 puncta in S174A mutants are co-localized with the PAS marker Atg8 (Fig. 4C and 4D), we conclude the recycling of Atg9 between the PAS and the cytoplasmic pool is impaired.Figure 4


Phosphorylation of Atg31 is required for autophagy.

Feng W, Wu T, Dan X, Chen Y, Li L, Chen S, Miao D, Deng H, Gong X, Yu L - Protein Cell (2015)

Impairment of Atg9 recycling in the S174A mutant. (A) Wild-type or S174A mutant cells expressing Atg9-GFP were transferred to nitrogen starvation for 0 h or 2 h and observed by confocal microscopy. Scale bar, 2 µm. (B) Cells from (A) were assessed for the number and intensity of Atg9 puncta. (C) Wild-type or S174 mutant cells expressing Atg9-GFP and Cherry red-Atg8 were transferred to SD-N medium for 2 h and observed by confocal microscopy. Scale bar, 2 µm. (D) Cells from (C) were assessed for co-localization between Atg9 and Atg8. 100 cells were assessed in a blind fashion and quantified. Error bars indicate standard deviation (s.d.) (n = 3)
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Related In: Results  -  Collection

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Fig4: Impairment of Atg9 recycling in the S174A mutant. (A) Wild-type or S174A mutant cells expressing Atg9-GFP were transferred to nitrogen starvation for 0 h or 2 h and observed by confocal microscopy. Scale bar, 2 µm. (B) Cells from (A) were assessed for the number and intensity of Atg9 puncta. (C) Wild-type or S174 mutant cells expressing Atg9-GFP and Cherry red-Atg8 were transferred to SD-N medium for 2 h and observed by confocal microscopy. Scale bar, 2 µm. (D) Cells from (C) were assessed for co-localization between Atg9 and Atg8. 100 cells were assessed in a blind fashion and quantified. Error bars indicate standard deviation (s.d.) (n = 3)
Mentions: Atg9 is a multi-pass transmembrane protein that plays a key role in autophagosome formation. Atg9-positive vesicles are highly mobile structures in the cytoplasm (Yamamoto et al., 2012) that recycle Atg9 and other molecular from the PAS to the cytoplasmic pool (Reggiori et al., 2004). We found that the number of Atg9 puncta is reduced in the S174A mutant. Furthermore, the fluorescence intensity of the Atg9 puncta is dramatically enhanced, indicating that the dynamic recycling of Atg9 between different pools is impaired and Atg9 accumulates in these puncta (Fig. 4A and 4B). Since Atg9 puncta in S174A mutants are co-localized with the PAS marker Atg8 (Fig. 4C and 4D), we conclude the recycling of Atg9 between the PAS and the cytoplasmic pool is impaired.Figure 4

Bottom Line: S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment.Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS.Our data demonstrate the role of phosphorylation of Atg31 in autophagy.

View Article: PubMed Central - PubMed

Affiliation: PTN Program, College of Life Science, Peking University, Beijing, 100871, China.

ABSTRACT
Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.

Show MeSH
Related in: MedlinePlus