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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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Vps30NTD is required for endosome-targeting of Vps38. The VPS38-GFP vps30Δ yeast cells (TKY1307) expressing Vps30 mutants in logarithmic phase were subjected to microscopic observation. Vps38-yEGFP was observed using fluorescent microscopy. A bar indicates 2 μm.
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Figure 7: Vps30NTD is required for endosome-targeting of Vps38. The VPS38-GFP vps30Δ yeast cells (TKY1307) expressing Vps30 mutants in logarithmic phase were subjected to microscopic observation. Vps38-yEGFP was observed using fluorescent microscopy. A bar indicates 2 μm.

Mentions: Vps30NTD was shown to be required for vacuolar protein sorting, but not autophagy (Fig. 3), which can be attributed to Vps30NTD for interaction with Vps38, but not with Atg14 (Fig. 4). To study the significance of the Vps30-Vps38 interaction through Vps30NTD on endosome targeting of Vps38, we monitored the intracellular localization of Vps38-GFP in cells expressing truncated forms of Vps30. As previously reported (18), Vps38 was localized to the punctate structures corresponding to endosomes in Vps30WT cells (Fig. 7). In Vps30ΔBARA cells, Vps38 showed a localization pattern similar to that in cells expressing Vps30WT, although the signal intensity was much lower (Fig. 7). In Vps30BARA or Vps30CCD+BARA cells, Vps38 was diffused in the cytosol and the signal intensity was decreased to an undetectable level as in the case of vps30Δ cells containing a control vector (Fig. 7). These results, together with those of the immunoprecipitation experiments, suggest that the Vps30-Vps38 interaction through Vps30NTD (and possibly Vps30CCD) is crucial for the interaction and stabilization of Vps38 and its targeting to endosomes.


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)

Vps30NTD is required for endosome-targeting of Vps38. The VPS38-GFP vps30Δ yeast cells (TKY1307) expressing Vps30 mutants in logarithmic phase were subjected to microscopic observation. Vps38-yEGFP was observed using fluorescent microscopy. A bar indicates 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Vps30NTD is required for endosome-targeting of Vps38. The VPS38-GFP vps30Δ yeast cells (TKY1307) expressing Vps30 mutants in logarithmic phase were subjected to microscopic observation. Vps38-yEGFP was observed using fluorescent microscopy. A bar indicates 2 μm.
Mentions: Vps30NTD was shown to be required for vacuolar protein sorting, but not autophagy (Fig. 3), which can be attributed to Vps30NTD for interaction with Vps38, but not with Atg14 (Fig. 4). To study the significance of the Vps30-Vps38 interaction through Vps30NTD on endosome targeting of Vps38, we monitored the intracellular localization of Vps38-GFP in cells expressing truncated forms of Vps30. As previously reported (18), Vps38 was localized to the punctate structures corresponding to endosomes in Vps30WT cells (Fig. 7). In Vps30ΔBARA cells, Vps38 showed a localization pattern similar to that in cells expressing Vps30WT, although the signal intensity was much lower (Fig. 7). In Vps30BARA or Vps30CCD+BARA cells, Vps38 was diffused in the cytosol and the signal intensity was decreased to an undetectable level as in the case of vps30Δ cells containing a control vector (Fig. 7). These results, together with those of the immunoprecipitation experiments, suggest that the Vps30-Vps38 interaction through Vps30NTD (and possibly Vps30CCD) is crucial for the interaction and stabilization of Vps38 and its targeting to endosomes.

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