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Tor-mediated induction of autophagy via an Apg1 protein kinase complex.

Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M, Ohsumi Y - J. Cell Biol. (2000)

Bottom Line: In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1.This Apg1-Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions.Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.

ABSTRACT
Autophagy is a membrane trafficking to vacuole/lysosome induced by nutrient starvation. In Saccharomyces cerevisiae, Tor protein, a phosphatidylinositol kinase-related kinase, is involved in the repression of autophagy induction by a largely unknown mechanism. Here, we show that the protein kinase activity of Apg1 is enhanced by starvation or rapamycin treatment. In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1. This Apg1-Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions. Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

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Apg1–Apg13 association is required for autophagy, but not for the Cvt pathway. A, Apg1-binding site of Apg13. Interaction of the indicated Apg13 fragments (prey) with Apg1 (bait) in the yeast two-hybrid system. Binding activity was estimated by β-galactosidase activity (Kaiser et al. 1994, shown as means and errors calculated from three independent experiments). B, Apg1–Apg13 association is required specifically for autophagy. Wild-type cells (TN125) or apg13Δ cells (YYK130) harboring the indicated APG13 constructs grown in YEPD were analyzed by anti-API immunoblot (top). An ALP assay was performed after 4 h SD(−N) incubation (bottom). C, Apg1 kinase assay was performed using the above APG13 transformants before or after rapamycin (0.2 μg/ml, 1 h) treatment.
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Figure 4: Apg1–Apg13 association is required for autophagy, but not for the Cvt pathway. A, Apg1-binding site of Apg13. Interaction of the indicated Apg13 fragments (prey) with Apg1 (bait) in the yeast two-hybrid system. Binding activity was estimated by β-galactosidase activity (Kaiser et al. 1994, shown as means and errors calculated from three independent experiments). B, Apg1–Apg13 association is required specifically for autophagy. Wild-type cells (TN125) or apg13Δ cells (YYK130) harboring the indicated APG13 constructs grown in YEPD were analyzed by anti-API immunoblot (top). An ALP assay was performed after 4 h SD(−N) incubation (bottom). C, Apg1 kinase assay was performed using the above APG13 transformants before or after rapamycin (0.2 μg/ml, 1 h) treatment.

Mentions: From these results, we hypothesized that Apg13 binding to Apg1 is required for the induction of autophagy, but not for the Cvt pathway. Therefore, we examined autophagy using apg13 mutants. The two-hybrid assay was used to determine the Apg1-binding site on Apg13, using various APG13 open reading frame fragments as prey. A central 89-amino acid region (432–520) was found to be responsible for Apg1 binding (Fig. 4 A). We also isolated a COOH-terminal truncated form of Apg13, Apg13(1–448), whose Ser449 was mutated to a stop codon using in vitro mutagenesis (Kaiser et al. 1994). This mutant has a mutation within the putative Apg1-binding site, and is unable to associate with Apg1, as confirmed by the two-hybrid assay and coimmunoprecipitation (Fig. 4 A, and data not shown). This result indicates a role for a domain of Apg13 around amino acid 448 in binding Apg1. We tested several COOH-terminal truncated Apg13 mutants including Apg13(1–448) for autophagy activity. Apg13(1–448) did not rescue the autophagic defect at all (Fig. 4 B, bottom), presumably due to the absence of the Apg1-binding domain. Another COOH-terminal truncated form, Apg13 (1–568), which contains the entire putative Apg1-binding domain, displayed partial, but significant, autophagic activity, when compared with Apg13(1–448). Apg13(1–568) was less competent for autophagic import than full-length Apg13, suggesting that additional sequences downstream of the Apg1 binding site are important for maximal activity. On the other hand, Apg13(1–448) partially, but significantly, rescued vacuolar-targeting of precursor API (Fig. 4 B, top), suggesting that this truncated protein is still functional for the Cvt pathway. Apg1 activity in the apg13Δ mutant was partially restored in the presence of either Apg13(1–448) or Apg13(1–568), and was fully recovered in the presence of the whole APG13 construct (Fig. 4 C). The kinase activity of the Apg13(1–568) transformant was clearly higher than that of the Apg13(1–448) transformant. These results confirmed the hypothesis that the Apg1–Apg13 association and subsequent activation of Apg1 are required for autophagy induction in response to starvation.


Tor-mediated induction of autophagy via an Apg1 protein kinase complex.

Kamada Y, Funakoshi T, Shintani T, Nagano K, Ohsumi M, Ohsumi Y - J. Cell Biol. (2000)

Apg1–Apg13 association is required for autophagy, but not for the Cvt pathway. A, Apg1-binding site of Apg13. Interaction of the indicated Apg13 fragments (prey) with Apg1 (bait) in the yeast two-hybrid system. Binding activity was estimated by β-galactosidase activity (Kaiser et al. 1994, shown as means and errors calculated from three independent experiments). B, Apg1–Apg13 association is required specifically for autophagy. Wild-type cells (TN125) or apg13Δ cells (YYK130) harboring the indicated APG13 constructs grown in YEPD were analyzed by anti-API immunoblot (top). An ALP assay was performed after 4 h SD(−N) incubation (bottom). C, Apg1 kinase assay was performed using the above APG13 transformants before or after rapamycin (0.2 μg/ml, 1 h) treatment.
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Figure 4: Apg1–Apg13 association is required for autophagy, but not for the Cvt pathway. A, Apg1-binding site of Apg13. Interaction of the indicated Apg13 fragments (prey) with Apg1 (bait) in the yeast two-hybrid system. Binding activity was estimated by β-galactosidase activity (Kaiser et al. 1994, shown as means and errors calculated from three independent experiments). B, Apg1–Apg13 association is required specifically for autophagy. Wild-type cells (TN125) or apg13Δ cells (YYK130) harboring the indicated APG13 constructs grown in YEPD were analyzed by anti-API immunoblot (top). An ALP assay was performed after 4 h SD(−N) incubation (bottom). C, Apg1 kinase assay was performed using the above APG13 transformants before or after rapamycin (0.2 μg/ml, 1 h) treatment.
Mentions: From these results, we hypothesized that Apg13 binding to Apg1 is required for the induction of autophagy, but not for the Cvt pathway. Therefore, we examined autophagy using apg13 mutants. The two-hybrid assay was used to determine the Apg1-binding site on Apg13, using various APG13 open reading frame fragments as prey. A central 89-amino acid region (432–520) was found to be responsible for Apg1 binding (Fig. 4 A). We also isolated a COOH-terminal truncated form of Apg13, Apg13(1–448), whose Ser449 was mutated to a stop codon using in vitro mutagenesis (Kaiser et al. 1994). This mutant has a mutation within the putative Apg1-binding site, and is unable to associate with Apg1, as confirmed by the two-hybrid assay and coimmunoprecipitation (Fig. 4 A, and data not shown). This result indicates a role for a domain of Apg13 around amino acid 448 in binding Apg1. We tested several COOH-terminal truncated Apg13 mutants including Apg13(1–448) for autophagy activity. Apg13(1–448) did not rescue the autophagic defect at all (Fig. 4 B, bottom), presumably due to the absence of the Apg1-binding domain. Another COOH-terminal truncated form, Apg13 (1–568), which contains the entire putative Apg1-binding domain, displayed partial, but significant, autophagic activity, when compared with Apg13(1–448). Apg13(1–568) was less competent for autophagic import than full-length Apg13, suggesting that additional sequences downstream of the Apg1 binding site are important for maximal activity. On the other hand, Apg13(1–448) partially, but significantly, rescued vacuolar-targeting of precursor API (Fig. 4 B, top), suggesting that this truncated protein is still functional for the Cvt pathway. Apg1 activity in the apg13Δ mutant was partially restored in the presence of either Apg13(1–448) or Apg13(1–568), and was fully recovered in the presence of the whole APG13 construct (Fig. 4 C). The kinase activity of the Apg13(1–568) transformant was clearly higher than that of the Apg13(1–448) transformant. These results confirmed the hypothesis that the Apg1–Apg13 association and subsequent activation of Apg1 are required for autophagy induction in response to starvation.

Bottom Line: In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1.This Apg1-Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions.Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.

ABSTRACT
Autophagy is a membrane trafficking to vacuole/lysosome induced by nutrient starvation. In Saccharomyces cerevisiae, Tor protein, a phosphatidylinositol kinase-related kinase, is involved in the repression of autophagy induction by a largely unknown mechanism. Here, we show that the protein kinase activity of Apg1 is enhanced by starvation or rapamycin treatment. In addition, we have also found that Apg13, which binds to and activates Apg1, is hyperphosphorylated in a Tor-dependent manner, reducing its affinity to Apg1. This Apg1-Apg13 association is required for autophagy, but not for the cytoplasm-to-vacuole targeting (Cvt) pathway, another vesicular transport mechanism in which factors essential for autophagy (Apg proteins) are also employed under vegetative growth conditions. Finally, other Apg1-associating proteins, such as Apg17 and Cvt9, are shown to function specifically in autophagy or the Cvt pathway, respectively, suggesting that the Apg1 complex plays an important role in switching between two distinct vesicular transport systems in a nutrient-dependent manner.

Show MeSH
Related in: MedlinePlus