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Structure of the novel C-terminal domain of vacuolar protein sorting 30/autophagy-related protein 6 and its specific role in autophagy.

Noda NN, Kobayashi T, Adachi W, Fujioka Y, Ohsumi Y, Inagaki F - J. Biol. Chem. (2012)

Bottom Line: Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain.On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting.These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry, Tokyo, Tokyo 141-0021, Japan. nn@bikaken.or.jp

ABSTRACT
Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

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Analysis of Vps30 interaction with Atg14 and Vps38. Coimmunoprecipitation experiments were performed as described under “Experimental Procedures.” Protein bands for Vps30, Atg14, Vps34, and Vps38 were detected using anti-Myc, anti-HA, anti-Vps34, and anti-HA antibodies, respectively. The samples used for the upper three panels were prepared from vps30Δ yeast cells (KVY135) expressing Vps30 mutants and Atg14-HA-GFP, whereas those used for the bottom panel were prepared from KVY135 cells expressing Vps30 mutants and Vps38-HA-GFP. Asterisks indicate degraded products of Vps30.
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Figure 4: Analysis of Vps30 interaction with Atg14 and Vps38. Coimmunoprecipitation experiments were performed as described under “Experimental Procedures.” Protein bands for Vps30, Atg14, Vps34, and Vps38 were detected using anti-Myc, anti-HA, anti-Vps34, and anti-HA antibodies, respectively. The samples used for the upper three panels were prepared from vps30Δ yeast cells (KVY135) expressing Vps30 mutants and Atg14-HA-GFP, whereas those used for the bottom panel were prepared from KVY135 cells expressing Vps30 mutants and Vps38-HA-GFP. Asterisks indicate degraded products of Vps30.

Mentions: Vps30 interacts directly with Atg14 and forms the autophagy-specific PI 3-kinase complex I. Because Vps30BARA has been shown as indispensible for autophagy, we next examined whether Vps30BARA is responsible for the interaction with Atg14 by coimmunoprecipitation experiments. Myc-tagged Vps30WT and three truncated forms of Vps30, Vps30ΔBARA, Vps30BARA, and Vps30CCD+BARA, were co-expressed with Atg14 fused to a 3× hemagglutinin (HA) tag and green fluorescent protein (GFP; Atg14-HA-GFP) in vps30Δ cells, and Myc-tagged Vps30s were pulled down with anti-Myc antibody and protein G-Sepharose beads. As shown in Fig. 4, Vps30WT, Vps30ΔBARA, and Vps30CCD+BARA but not Vps30BARA interacted with Atg14. These results suggest that Vps30BARA is dispensable for the interaction with Atg14. Similarly, immunoprecipitation experiments using vps30Δ cells co-expressing truncated mutants of Vps30 and Vps38-HA-GFP showed that Vps30BARA is dispensable for the interaction with Vps38. Vps34 was coimmunoprecipitated with Vps30WT and truncated forms of Vps30 except for Vps30BARA, which is consistent with the previous report that Vps30 interacts with Vps34 through either Atg14 or Vps38 (5).


Structure of the novel C-terminal domain of vacuolar protein sorting 30/autophagy-related protein 6 and its specific role in autophagy.

Noda NN, Kobayashi T, Adachi W, Fujioka Y, Ohsumi Y, Inagaki F - J. Biol. Chem. (2012)

Analysis of Vps30 interaction with Atg14 and Vps38. Coimmunoprecipitation experiments were performed as described under “Experimental Procedures.” Protein bands for Vps30, Atg14, Vps34, and Vps38 were detected using anti-Myc, anti-HA, anti-Vps34, and anti-HA antibodies, respectively. The samples used for the upper three panels were prepared from vps30Δ yeast cells (KVY135) expressing Vps30 mutants and Atg14-HA-GFP, whereas those used for the bottom panel were prepared from KVY135 cells expressing Vps30 mutants and Vps38-HA-GFP. Asterisks indicate degraded products of Vps30.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351336&req=5

Figure 4: Analysis of Vps30 interaction with Atg14 and Vps38. Coimmunoprecipitation experiments were performed as described under “Experimental Procedures.” Protein bands for Vps30, Atg14, Vps34, and Vps38 were detected using anti-Myc, anti-HA, anti-Vps34, and anti-HA antibodies, respectively. The samples used for the upper three panels were prepared from vps30Δ yeast cells (KVY135) expressing Vps30 mutants and Atg14-HA-GFP, whereas those used for the bottom panel were prepared from KVY135 cells expressing Vps30 mutants and Vps38-HA-GFP. Asterisks indicate degraded products of Vps30.
Mentions: Vps30 interacts directly with Atg14 and forms the autophagy-specific PI 3-kinase complex I. Because Vps30BARA has been shown as indispensible for autophagy, we next examined whether Vps30BARA is responsible for the interaction with Atg14 by coimmunoprecipitation experiments. Myc-tagged Vps30WT and three truncated forms of Vps30, Vps30ΔBARA, Vps30BARA, and Vps30CCD+BARA, were co-expressed with Atg14 fused to a 3× hemagglutinin (HA) tag and green fluorescent protein (GFP; Atg14-HA-GFP) in vps30Δ cells, and Myc-tagged Vps30s were pulled down with anti-Myc antibody and protein G-Sepharose beads. As shown in Fig. 4, Vps30WT, Vps30ΔBARA, and Vps30CCD+BARA but not Vps30BARA interacted with Atg14. These results suggest that Vps30BARA is dispensable for the interaction with Atg14. Similarly, immunoprecipitation experiments using vps30Δ cells co-expressing truncated mutants of Vps30 and Vps38-HA-GFP showed that Vps30BARA is dispensable for the interaction with Vps38. Vps34 was coimmunoprecipitated with Vps30WT and truncated forms of Vps30 except for Vps30BARA, which is consistent with the previous report that Vps30 interacts with Vps34 through either Atg14 or Vps38 (5).

Bottom Line: Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain.On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting.These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbial Chemistry, Tokyo, Tokyo 141-0021, Japan. nn@bikaken.or.jp

ABSTRACT
Vacuolar protein sorting 30 (Vps30)/autophagy-related protein 6 (Atg6) is a common component of two distinct phosphatidylinositol 3-kinase complexes. In complex I, Atg14 links Vps30 to Vps34 lipid kinase and exerts its specific role in autophagy, whereas in complex II, Vps38 links Vps30 to Vps34 and plays a crucial role in vacuolar protein sorting. However, the molecular role of Vps30 in each pathway remains unclear. Here, we report the crystal structure of the carboxyl-terminal domain of Vps30. The structure is a novel globular fold comprised of three β-sheet-α-helix repeats. Truncation analyses showed that the domain is dispensable for the construction of both complexes, but is specifically required for autophagy through the targeting of complex I to the pre-autophagosomal structure. Thus, the domain is named the β-α repeated, autophagy-specific (BARA) domain. On the other hand, the N-terminal region of Vps30 was shown to be specifically required for vacuolar protein sorting. These structural and functional investigations of Vps30 domains, which are also conserved in the mammalian ortholog, Beclin 1, will form the basis for studying the molecular functions of this protein family in various biological processes.

Show MeSH