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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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Vps30BARA is required for efficient PAS targeting of Vps30 and Atg14.A, the vps30Δ mRFP-APE1 yeast cells (TKY1647) expressing Vps30 mutants fused to GFP were subjected to microscopic observation. Vps30-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. B, the vps30Δ mRFP-APE1 ATG14-GFP yeast cells (TKY1675) expressing Vps30 mutants were subjected to microscopic observation. Atg14-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. Arrows indicate the PAS. A bar indicates 2 μm.
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Figure 5: Vps30BARA is required for efficient PAS targeting of Vps30 and Atg14.A, the vps30Δ mRFP-APE1 yeast cells (TKY1647) expressing Vps30 mutants fused to GFP were subjected to microscopic observation. Vps30-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. B, the vps30Δ mRFP-APE1 ATG14-GFP yeast cells (TKY1675) expressing Vps30 mutants were subjected to microscopic observation. Atg14-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. Arrows indicate the PAS. A bar indicates 2 μm.

Mentions: Vps30BARA is crucial for autophagy; nevertheless, Vps30BARA was shown to be dispensable for the construction of PI 3-kinase complex I. What then is the function of Vps30BARA in autophagy? To examine the role of Vps30BARA in PAS targeting of PI 3-kinase complex I, we first monitored the localization of GFP-tagged Vps30 truncates in cells expressing mRFP-tagged Ape1 as a PAS marker (Fig. 5A).


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)

Vps30BARA is required for efficient PAS targeting of Vps30 and Atg14.A, the vps30Δ mRFP-APE1 yeast cells (TKY1647) expressing Vps30 mutants fused to GFP were subjected to microscopic observation. Vps30-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. B, the vps30Δ mRFP-APE1 ATG14-GFP yeast cells (TKY1675) expressing Vps30 mutants were subjected to microscopic observation. Atg14-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. Arrows indicate the PAS. A bar indicates 2 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Vps30BARA is required for efficient PAS targeting of Vps30 and Atg14.A, the vps30Δ mRFP-APE1 yeast cells (TKY1647) expressing Vps30 mutants fused to GFP were subjected to microscopic observation. Vps30-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. B, the vps30Δ mRFP-APE1 ATG14-GFP yeast cells (TKY1675) expressing Vps30 mutants were subjected to microscopic observation. Atg14-GFP and mRFP-Ape1 in cells with rapamycin treatment for 1 h were observed using fluorescent microscopy. Arrows indicate the PAS. A bar indicates 2 μm.
Mentions: Vps30BARA is crucial for autophagy; nevertheless, Vps30BARA was shown to be dispensable for the construction of PI 3-kinase complex I. What then is the function of Vps30BARA in autophagy? To examine the role of Vps30BARA in PAS targeting of PI 3-kinase complex I, we first monitored the localization of GFP-tagged Vps30 truncates in cells expressing mRFP-tagged Ape1 as a PAS marker (Fig. 5A).

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