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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: 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.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-associating proteins are required for Apg1 activation. A, Wild-type (TN125, lane 1), apg1Δ (YYK126, lane 2), apg13Δ (YYK130, lane 3), and apg17Δ (YYK121, lane 4) cells were analyzed with anti-API blot (top) and ALP assay (bottom). B, Wild-type (KA311A), apg13Δ (YYK119), apg17Δ (YYK111), and cvt9Δ (YYK107) cells expressing HAApg1 were treated with rapamycin (0.2 μg/ml, 1 h) and analyzed by kinase assay.
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Figure 2: Apg1-associating proteins are required for Apg1 activation. A, Wild-type (TN125, lane 1), apg1Δ (YYK126, lane 2), apg13Δ (YYK130, lane 3), and apg17Δ (YYK121, lane 4) cells were analyzed with anti-API blot (top) and ALP assay (bottom). B, Wild-type (KA311A), apg13Δ (YYK119), apg17Δ (YYK111), and cvt9Δ (YYK107) cells expressing HAApg1 were treated with rapamycin (0.2 μg/ml, 1 h) and analyzed by kinase assay.

Mentions: Next, we performed a two-hybrid screening with APG1 as bait to identify Apg1-associating proteins, which may regulate Apg1 activity. The following three genes were obtained from the screen: APG13 (Funakoshi et al. 1997) and two novel genes, which were subsequently found to be essential for either autophagy or the Cvt pathway, or both. One gene, designated as APG17 (YLR423c), was essential for only autophagy and was not required for the Cvt pathway (Fig. 2 A). The other, CVT9 (Harding et al. 1996; D.J. Klionsky, personal communication), was required for the Cvt pathway, but not for autophagy. Among the 16 APG genes discovered so far, APG17 is the first one identified whose function is restricted to autophagy. It is interesting to note that Apg1 binds to proteins whose function is specific to either autophagy (Apg17) or the Cvt pathway (Cvt9). Overexpression of APG1 in an apg13Δ mutant partially rescues the autophagy defect (Funakoshi et al. 1997). Similarly, the apg17Δ mutant was also rescued by overexpression of APG1 (data not shown), indicating that these three genes interact functionally. We tested whether these Apg1-associated proteins are involved in Apg1 activation. As shown in Fig. 2 B, deletion of each gene affected Apg1 activation by rapamycin treatment. In the apg13Δ mutant, attenuated Apg1 activity was observed. In rapamycin-treated apg17Δ cells, Apg1 activity also was found to be largely impaired (∼20% of the wild-type). On the other hand, deletion of CVT9, which is not needed for autophagy, resulted in rapamycin-induced activation of Apg1 to 50% of wild-type. The effects of deleting APG13 and APG17 on Apg1 activity are not the result of a general autophagy defect, because deletion of other APG genes, such as APG5 (Mizushima et al. 1998), does not affect the activation of Apg1 (data not shown). These results indicate that the activated state of Apg1 is required for autophagy induction, and that Apg13 and Apg17 play a key role in the activation of Apg1 in response to Tor inhibition.


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-associating proteins are required for Apg1 activation. A, Wild-type (TN125, lane 1), apg1Δ (YYK126, lane 2), apg13Δ (YYK130, lane 3), and apg17Δ (YYK121, lane 4) cells were analyzed with anti-API blot (top) and ALP assay (bottom). B, Wild-type (KA311A), apg13Δ (YYK119), apg17Δ (YYK111), and cvt9Δ (YYK107) cells expressing HAApg1 were treated with rapamycin (0.2 μg/ml, 1 h) and analyzed by kinase assay.
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Figure 2: Apg1-associating proteins are required for Apg1 activation. A, Wild-type (TN125, lane 1), apg1Δ (YYK126, lane 2), apg13Δ (YYK130, lane 3), and apg17Δ (YYK121, lane 4) cells were analyzed with anti-API blot (top) and ALP assay (bottom). B, Wild-type (KA311A), apg13Δ (YYK119), apg17Δ (YYK111), and cvt9Δ (YYK107) cells expressing HAApg1 were treated with rapamycin (0.2 μg/ml, 1 h) and analyzed by kinase assay.
Mentions: Next, we performed a two-hybrid screening with APG1 as bait to identify Apg1-associating proteins, which may regulate Apg1 activity. The following three genes were obtained from the screen: APG13 (Funakoshi et al. 1997) and two novel genes, which were subsequently found to be essential for either autophagy or the Cvt pathway, or both. One gene, designated as APG17 (YLR423c), was essential for only autophagy and was not required for the Cvt pathway (Fig. 2 A). The other, CVT9 (Harding et al. 1996; D.J. Klionsky, personal communication), was required for the Cvt pathway, but not for autophagy. Among the 16 APG genes discovered so far, APG17 is the first one identified whose function is restricted to autophagy. It is interesting to note that Apg1 binds to proteins whose function is specific to either autophagy (Apg17) or the Cvt pathway (Cvt9). Overexpression of APG1 in an apg13Δ mutant partially rescues the autophagy defect (Funakoshi et al. 1997). Similarly, the apg17Δ mutant was also rescued by overexpression of APG1 (data not shown), indicating that these three genes interact functionally. We tested whether these Apg1-associated proteins are involved in Apg1 activation. As shown in Fig. 2 B, deletion of each gene affected Apg1 activation by rapamycin treatment. In the apg13Δ mutant, attenuated Apg1 activity was observed. In rapamycin-treated apg17Δ cells, Apg1 activity also was found to be largely impaired (∼20% of the wild-type). On the other hand, deletion of CVT9, which is not needed for autophagy, resulted in rapamycin-induced activation of Apg1 to 50% of wild-type. The effects of deleting APG13 and APG17 on Apg1 activity are not the result of a general autophagy defect, because deletion of other APG genes, such as APG5 (Mizushima et al. 1998), does not affect the activation of Apg1 (data not shown). These results indicate that the activated state of Apg1 is required for autophagy induction, and that Apg13 and Apg17 play a key role in the activation of Apg1 in response to Tor inhibition.

Bottom Line: 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.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