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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/ACBD5 is dispensable for aggrephagy and nonselective autophagy. (A) ACBD5 is not required for the autophagy of Htt-Q72 aggregates in HeLa cells. Cells expressed EGFP-Htt-Q72. Arrows point to the EGFP-Htt-Q72 aggregates. Bar, 10 µm. (B) Quantitation of data in A. The percentage of cells with EGFP-Htt-Q72 aggregates is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. siATG7). (C) ACBD5 does not affect SQSTM1 degradation and LC3 lipidation. Short, short exposure; Long, long exposure; β-Actin, loading control. (D) ACBD5 is dispensable for LC3-positive vesicle formation in HeLa cells expressing fluorescently tagged LC3. Cells were treated with 20 µM chloroquine in EBSS medium for 3 h and permeabilized with 0.025% digitonin to enrich for the vesicle-bound LC3 (indicated by arrows). Bar, 10 µm. (E) Quantitation of data in D. The percentage of cells with the vesicle-bound LC3 is shown as the mean + SD (error bars; n ≥ 75; *, P < 0.05 vs. siATG16L1).
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fig10: ATG37/ACBD5 is dispensable for aggrephagy and nonselective autophagy. (A) ACBD5 is not required for the autophagy of Htt-Q72 aggregates in HeLa cells. Cells expressed EGFP-Htt-Q72. Arrows point to the EGFP-Htt-Q72 aggregates. Bar, 10 µm. (B) Quantitation of data in A. The percentage of cells with EGFP-Htt-Q72 aggregates is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. siATG7). (C) ACBD5 does not affect SQSTM1 degradation and LC3 lipidation. Short, short exposure; Long, long exposure; β-Actin, loading control. (D) ACBD5 is dispensable for LC3-positive vesicle formation in HeLa cells expressing fluorescently tagged LC3. Cells were treated with 20 µM chloroquine in EBSS medium for 3 h and permeabilized with 0.025% digitonin to enrich for the vesicle-bound LC3 (indicated by arrows). Bar, 10 µm. (E) Quantitation of data in D. The percentage of cells with the vesicle-bound LC3 is shown as the mean + SD (error bars; n ≥ 75; *, P < 0.05 vs. siATG16L1).

Mentions: To consider ACBD5 for future therapeutic applications, it is important to know how the knockdown of ACBD5 affects other autophagic pathways. We studied the role of ACBD5 in the selective autophagy of protein aggregates (aggrephagy) and nonselective autophagy. For the aggrephagy assay, we expressed the EGFP-tagged mutant huntingtin exon 1 with 72 CAG repeats, EGFP-Htt-Q72, reported to form hyperfluorescent inclusions (Furlong et al., 2000). These bright protein aggregates were easily detected in HeLa cells transfected with siATG7, but not with siACBD5 or control siRNA, 2 d after transfection with EGFP-Htt-Q72 (Fig. 10, A and B). Therefore, ACBD5 is not required for aggrephagy. Consistent with this conclusion, HeLa cells treated with ACBD5 siRNA had the same levels of SQSTM1/p62, the aggrephagy receptor that is degraded together with protein aggregates (Bjørkøy et al., 2005), as the cells treated with control siRNA (Fig. 10 C). In contrast, the knockdown of ATG7 prevented degradation of SQSTM1, as expected.


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/ACBD5 is dispensable for aggrephagy and nonselective autophagy. (A) ACBD5 is not required for the autophagy of Htt-Q72 aggregates in HeLa cells. Cells expressed EGFP-Htt-Q72. Arrows point to the EGFP-Htt-Q72 aggregates. Bar, 10 µm. (B) Quantitation of data in A. The percentage of cells with EGFP-Htt-Q72 aggregates is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. siATG7). (C) ACBD5 does not affect SQSTM1 degradation and LC3 lipidation. Short, short exposure; Long, long exposure; β-Actin, loading control. (D) ACBD5 is dispensable for LC3-positive vesicle formation in HeLa cells expressing fluorescently tagged LC3. Cells were treated with 20 µM chloroquine in EBSS medium for 3 h and permeabilized with 0.025% digitonin to enrich for the vesicle-bound LC3 (indicated by arrows). Bar, 10 µm. (E) Quantitation of data in D. The percentage of cells with the vesicle-bound LC3 is shown as the mean + SD (error bars; n ≥ 75; *, P < 0.05 vs. siATG16L1).
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fig10: ATG37/ACBD5 is dispensable for aggrephagy and nonselective autophagy. (A) ACBD5 is not required for the autophagy of Htt-Q72 aggregates in HeLa cells. Cells expressed EGFP-Htt-Q72. Arrows point to the EGFP-Htt-Q72 aggregates. Bar, 10 µm. (B) Quantitation of data in A. The percentage of cells with EGFP-Htt-Q72 aggregates is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. siATG7). (C) ACBD5 does not affect SQSTM1 degradation and LC3 lipidation. Short, short exposure; Long, long exposure; β-Actin, loading control. (D) ACBD5 is dispensable for LC3-positive vesicle formation in HeLa cells expressing fluorescently tagged LC3. Cells were treated with 20 µM chloroquine in EBSS medium for 3 h and permeabilized with 0.025% digitonin to enrich for the vesicle-bound LC3 (indicated by arrows). Bar, 10 µm. (E) Quantitation of data in D. The percentage of cells with the vesicle-bound LC3 is shown as the mean + SD (error bars; n ≥ 75; *, P < 0.05 vs. siATG16L1).
Mentions: To consider ACBD5 for future therapeutic applications, it is important to know how the knockdown of ACBD5 affects other autophagic pathways. We studied the role of ACBD5 in the selective autophagy of protein aggregates (aggrephagy) and nonselective autophagy. For the aggrephagy assay, we expressed the EGFP-tagged mutant huntingtin exon 1 with 72 CAG repeats, EGFP-Htt-Q72, reported to form hyperfluorescent inclusions (Furlong et al., 2000). These bright protein aggregates were easily detected in HeLa cells transfected with siATG7, but not with siACBD5 or control siRNA, 2 d after transfection with EGFP-Htt-Q72 (Fig. 10, A and B). Therefore, ACBD5 is not required for aggrephagy. Consistent with this conclusion, HeLa cells treated with ACBD5 siRNA had the same levels of SQSTM1/p62, the aggrephagy receptor that is degraded together with protein aggregates (Bjørkøy et al., 2005), as the cells treated with control siRNA (Fig. 10 C). In contrast, the knockdown of ATG7 prevented degradation of SQSTM1, as expected.

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