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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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Assessment of autophagy and CPY-sorting activities.A, autophagic activity of pho8Δ60 vps30Δ yeast cells (TKY1308) expressing Vps30 mutants. Autophagic activity was measured using Pho8Δ60 alkaline phosphatase (ALP) assay as described under “Experimental Procedures.” Error bars indicate the standard deviation of three independent experiments. B, Ape1 maturation in vps30Δ cells expressing Vps30 mutants. Lysates of the vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to Western blotting and Ape1 and Vps30 bands were detected using anti-Ape1 and anti-Myc antibodies, respectively. As a loading control, Pgk1 was detected using anti-Pgk1 antibody. C, CPY sorting in vps30Δ cells expressing Vps30 mutants. The vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to CPY sorting assay as described under “Experimental Procedures.”
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Figure 3: Assessment of autophagy and CPY-sorting activities.A, autophagic activity of pho8Δ60 vps30Δ yeast cells (TKY1308) expressing Vps30 mutants. Autophagic activity was measured using Pho8Δ60 alkaline phosphatase (ALP) assay as described under “Experimental Procedures.” Error bars indicate the standard deviation of three independent experiments. B, Ape1 maturation in vps30Δ cells expressing Vps30 mutants. Lysates of the vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to Western blotting and Ape1 and Vps30 bands were detected using anti-Ape1 and anti-Myc antibodies, respectively. As a loading control, Pgk1 was detected using anti-Pgk1 antibody. C, CPY sorting in vps30Δ cells expressing Vps30 mutants. The vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to CPY sorting assay as described under “Experimental Procedures.”

Mentions: Because Vps30 BARA (Vps30BARA) is conserved among Vps30/Beclin 1 homologues, it is speculated that Vps30BARA plays important roles in the functioning of Vps30. To investigate the contribution of Vps30BARA on autophagy, cells expressing truncated forms of Vps30 were subjected to the Pho8Δ60 assay, a method commonly used for the assessment of autophagic activity (42, 43). This method utilizes a genetically engineered cytosolic form of an alkaline phosphatase, Pho8 (Pho8Δ60), which is delivered into the vacuole exclusively by autophagy, and activated. Thus, the autophagic activity correlates well with the phosphatase activity. As shown in Fig. 3A, wild-type cells as well as vps30Δ cells expressing wild-type Vps30 (Vps30WT cells) showed increased phosphatase activity in response to starvation conditions for 4 h, whereas vps30Δ cells showed no increase in the activity. Compared with these cells, Vps30ΔBARA or Vps30BARA cells showed only a slight increase in activity.


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)

Assessment of autophagy and CPY-sorting activities.A, autophagic activity of pho8Δ60 vps30Δ yeast cells (TKY1308) expressing Vps30 mutants. Autophagic activity was measured using Pho8Δ60 alkaline phosphatase (ALP) assay as described under “Experimental Procedures.” Error bars indicate the standard deviation of three independent experiments. B, Ape1 maturation in vps30Δ cells expressing Vps30 mutants. Lysates of the vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to Western blotting and Ape1 and Vps30 bands were detected using anti-Ape1 and anti-Myc antibodies, respectively. As a loading control, Pgk1 was detected using anti-Pgk1 antibody. C, CPY sorting in vps30Δ cells expressing Vps30 mutants. The vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to CPY sorting assay as described under “Experimental Procedures.”
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Assessment of autophagy and CPY-sorting activities.A, autophagic activity of pho8Δ60 vps30Δ yeast cells (TKY1308) expressing Vps30 mutants. Autophagic activity was measured using Pho8Δ60 alkaline phosphatase (ALP) assay as described under “Experimental Procedures.” Error bars indicate the standard deviation of three independent experiments. B, Ape1 maturation in vps30Δ cells expressing Vps30 mutants. Lysates of the vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to Western blotting and Ape1 and Vps30 bands were detected using anti-Ape1 and anti-Myc antibodies, respectively. As a loading control, Pgk1 was detected using anti-Pgk1 antibody. C, CPY sorting in vps30Δ cells expressing Vps30 mutants. The vps30Δ cells (KVY135) carrying pRS314-based VPS30 mutants were subjected to CPY sorting assay as described under “Experimental Procedures.”
Mentions: Because Vps30 BARA (Vps30BARA) is conserved among Vps30/Beclin 1 homologues, it is speculated that Vps30BARA plays important roles in the functioning of Vps30. To investigate the contribution of Vps30BARA on autophagy, cells expressing truncated forms of Vps30 were subjected to the Pho8Δ60 assay, a method commonly used for the assessment of autophagic activity (42, 43). This method utilizes a genetically engineered cytosolic form of an alkaline phosphatase, Pho8 (Pho8Δ60), which is delivered into the vacuole exclusively by autophagy, and activated. Thus, the autophagic activity correlates well with the phosphatase activity. As shown in Fig. 3A, wild-type cells as well as vps30Δ cells expressing wild-type Vps30 (Vps30WT cells) showed increased phosphatase activity in response to starvation conditions for 4 h, whereas vps30Δ cells showed no increase in the activity. Compared with these cells, Vps30ΔBARA or Vps30BARA cells showed only a slight increase in activity.

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