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Peroxisomal Atg37 binds Atg30 or palmitoyl-CoA to regulate phagophore formation during pexophagy.

Nazarko TY, Ozeki K, Till A, Ramakrishnan G, Lotfi P, Yan M, Subramani S - J. Cell Biol. (2014)

Bottom Line: Palmitoyl-CoA competes with Atg30 for Atg37 binding.The human orthologue of Atg37, acyl-CoA-binding domain containing protein 5 (ACBD5), is also peroxisomal and is required specifically for pexophagy.We suggest that Atg37/ACBD5 is a new component and positive regulator of the pexophagic RPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Biology, Division of Biological Sciences, and 2 San Diego Center for Systems Biology, University of California, San Diego, La Jolla, CA 92093.

ABSTRACT
Autophagy is a membrane trafficking pathway that sequesters proteins and organelles into autophagosomes. The selectivity of this pathway is determined by autophagy receptors, such as the Pichia pastoris autophagy-related protein 30 (Atg30), which controls the selective autophagy of peroxisomes (pexophagy) through the assembly of a receptor protein complex (RPC). However, how the pexophagic RPC is regulated for efficient formation of the phagophore, an isolation membrane that sequesters the peroxisome from the cytosol, is unknown. Here we describe a new, conserved acyl-CoA-binding protein, Atg37, that is an integral peroxisomal membrane protein required specifically for pexophagy at the stage of phagophore formation. Atg30 recruits Atg37 to the pexophagic RPC, where Atg37 regulates the recruitment of the scaffold protein, Atg11. Palmitoyl-CoA competes with Atg30 for Atg37 binding. The human orthologue of Atg37, acyl-CoA-binding domain containing protein 5 (ACBD5), is also peroxisomal and is required specifically for pexophagy. We suggest that Atg37/ACBD5 is a new component and positive regulator of the pexophagic RPC.

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Atg37 is an integral PMP that binds Pex3. (A) Pex3 coimmunoprecipitates with Atg37. IP, immunoprecipitate. Cells were induced in methanol medium for 4 h. (B) Atg37ΔC binds Pex3ΔN in vitro. I, input; U, unbound; P, pull-down. (C) Atg37 is an integral membrane protein. PNS, postnuclear supernatant; 27S, 27,000 g supernatant; 27P, 27,000 g pellet; S, 200,000 g supernatant; P, 200,000 g pellet. (D) Atg37 localizes to the peroxisomal membranes (in WT) or ER-associated peroxisome remnants labeled with Pex3-mRFP (in Δpex19). DIC, differential interference contrast. Bar, 5 µm. (E) The TMD is essential for peroxisomal localization of Atg37. SKL, Ser-Lys-Leu, a peroxisomal targeting signal. Bar, 5 µm.
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fig1: Atg37 is an integral PMP that binds Pex3. (A) Pex3 coimmunoprecipitates with Atg37. IP, immunoprecipitate. Cells were induced in methanol medium for 4 h. (B) Atg37ΔC binds Pex3ΔN in vitro. I, input; U, unbound; P, pull-down. (C) Atg37 is an integral membrane protein. PNS, postnuclear supernatant; 27S, 27,000 g supernatant; 27P, 27,000 g pellet; S, 200,000 g supernatant; P, 200,000 g pellet. (D) Atg37 localizes to the peroxisomal membranes (in WT) or ER-associated peroxisome remnants labeled with Pex3-mRFP (in Δpex19). DIC, differential interference contrast. Bar, 5 µm. (E) The TMD is essential for peroxisomal localization of Atg37. SKL, Ser-Lys-Leu, a peroxisomal targeting signal. Bar, 5 µm.

Mentions: Coimmunoprecipitation experiments confirmed the association of Atg37 with Pex3, but not significantly with other peroxins tested (Fig. 1 A). The higher molecular weight species of Atg37-HA detected by immunoblotting represent phosphorylated forms of the protein, as indicated by their sensitivity to phosphatase treatment, similar to phosphorylated forms of Pex14 (Johnson et al., 2001; Fig. S1 A). In the yeast two-hybrid (Y2H) system, Pex3 interacted with Atg30 (Farré et al., 2008), but not with Atg37. We tried several truncations of Atg37, such as Atg37ΔC (aa 1–278), Atg37-131C (aa 279–409), and Atg37-80C (aa 330–409), but none of them interacted with Pex3 in the Y2H system (Fig. S1 B). However, the in vitro protein binding assays with Glutathione Sepharose beads demonstrated that purified His-Atg37ΔC was pulled down with GST-Pex3ΔN (aa 41–455, corresponding to the cytosolic domain), but not with GST (Fig. 1 B). Therefore, Atg37 directly binds Pex3.


Peroxisomal Atg37 binds Atg30 or palmitoyl-CoA to regulate phagophore formation during pexophagy.

Nazarko TY, Ozeki K, Till A, Ramakrishnan G, Lotfi P, Yan M, Subramani S - J. Cell Biol. (2014)

Atg37 is an integral PMP that binds Pex3. (A) Pex3 coimmunoprecipitates with Atg37. IP, immunoprecipitate. Cells were induced in methanol medium for 4 h. (B) Atg37ΔC binds Pex3ΔN in vitro. I, input; U, unbound; P, pull-down. (C) Atg37 is an integral membrane protein. PNS, postnuclear supernatant; 27S, 27,000 g supernatant; 27P, 27,000 g pellet; S, 200,000 g supernatant; P, 200,000 g pellet. (D) Atg37 localizes to the peroxisomal membranes (in WT) or ER-associated peroxisome remnants labeled with Pex3-mRFP (in Δpex19). DIC, differential interference contrast. Bar, 5 µm. (E) The TMD is essential for peroxisomal localization of Atg37. SKL, Ser-Lys-Leu, a peroxisomal targeting signal. Bar, 5 µm.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3926955&req=5

fig1: Atg37 is an integral PMP that binds Pex3. (A) Pex3 coimmunoprecipitates with Atg37. IP, immunoprecipitate. Cells were induced in methanol medium for 4 h. (B) Atg37ΔC binds Pex3ΔN in vitro. I, input; U, unbound; P, pull-down. (C) Atg37 is an integral membrane protein. PNS, postnuclear supernatant; 27S, 27,000 g supernatant; 27P, 27,000 g pellet; S, 200,000 g supernatant; P, 200,000 g pellet. (D) Atg37 localizes to the peroxisomal membranes (in WT) or ER-associated peroxisome remnants labeled with Pex3-mRFP (in Δpex19). DIC, differential interference contrast. Bar, 5 µm. (E) The TMD is essential for peroxisomal localization of Atg37. SKL, Ser-Lys-Leu, a peroxisomal targeting signal. Bar, 5 µm.
Mentions: Coimmunoprecipitation experiments confirmed the association of Atg37 with Pex3, but not significantly with other peroxins tested (Fig. 1 A). The higher molecular weight species of Atg37-HA detected by immunoblotting represent phosphorylated forms of the protein, as indicated by their sensitivity to phosphatase treatment, similar to phosphorylated forms of Pex14 (Johnson et al., 2001; Fig. S1 A). In the yeast two-hybrid (Y2H) system, Pex3 interacted with Atg30 (Farré et al., 2008), but not with Atg37. We tried several truncations of Atg37, such as Atg37ΔC (aa 1–278), Atg37-131C (aa 279–409), and Atg37-80C (aa 330–409), but none of them interacted with Pex3 in the Y2H system (Fig. S1 B). However, the in vitro protein binding assays with Glutathione Sepharose beads demonstrated that purified His-Atg37ΔC was pulled down with GST-Pex3ΔN (aa 41–455, corresponding to the cytosolic domain), but not with GST (Fig. 1 B). Therefore, Atg37 directly binds Pex3.

Bottom Line: Palmitoyl-CoA competes with Atg30 for Atg37 binding.The human orthologue of Atg37, acyl-CoA-binding domain containing protein 5 (ACBD5), is also peroxisomal and is required specifically for pexophagy.We suggest that Atg37/ACBD5 is a new component and positive regulator of the pexophagic RPC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Molecular Biology, Division of Biological Sciences, and 2 San Diego Center for Systems Biology, University of California, San Diego, La Jolla, CA 92093.

ABSTRACT
Autophagy is a membrane trafficking pathway that sequesters proteins and organelles into autophagosomes. The selectivity of this pathway is determined by autophagy receptors, such as the Pichia pastoris autophagy-related protein 30 (Atg30), which controls the selective autophagy of peroxisomes (pexophagy) through the assembly of a receptor protein complex (RPC). However, how the pexophagic RPC is regulated for efficient formation of the phagophore, an isolation membrane that sequesters the peroxisome from the cytosol, is unknown. Here we describe a new, conserved acyl-CoA-binding protein, Atg37, that is an integral peroxisomal membrane protein required specifically for pexophagy at the stage of phagophore formation. Atg30 recruits Atg37 to the pexophagic RPC, where Atg37 regulates the recruitment of the scaffold protein, Atg11. Palmitoyl-CoA competes with Atg30 for Atg37 binding. The human orthologue of Atg37, acyl-CoA-binding domain containing protein 5 (ACBD5), is also peroxisomal and is required specifically for pexophagy. We suggest that Atg37/ACBD5 is a new component and positive regulator of the pexophagic RPC.

Show MeSH
Related in: MedlinePlus