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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 essential for pexophagy in human cells. (A and C) ACBD5 is needed for the delivery of peroxisomes to the lysosomes in HeLa (A) and Ntera-2 (C) cells expressing peroxisomally targeted mRFP-EGFP-SKL. Arrows point to the autolysosomes with acid-resistant mRFP fluorescence. Bars, 10 µm. (B) Quantitation of data in A. The percentage of cells with mRFP-labeled autolysosomes is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. control siRNA). (D) Quantitation of data in C. The ratio of mRFP and EGFP intensities per cell reflects the occurrence of pexophagy and is presented as the mean + SD (error bars; n ≥ 10 images; *, P < 0.05 vs. control siRNA).
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fig9: ATG37/ACBD5 is essential for pexophagy in human cells. (A and C) ACBD5 is needed for the delivery of peroxisomes to the lysosomes in HeLa (A) and Ntera-2 (C) cells expressing peroxisomally targeted mRFP-EGFP-SKL. Arrows point to the autolysosomes with acid-resistant mRFP fluorescence. Bars, 10 µm. (B) Quantitation of data in A. The percentage of cells with mRFP-labeled autolysosomes is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. control siRNA). (D) Quantitation of data in C. The ratio of mRFP and EGFP intensities per cell reflects the occurrence of pexophagy and is presented as the mean + SD (error bars; n ≥ 10 images; *, P < 0.05 vs. control siRNA).

Mentions: Next, we addressed the functional role of human ACBD5. To measure pexophagy in human cells, we used the tandem fluorochrome pexophagy assay with mRFP-EGFP-SKL as a reporter. It is based on the same principle as the recently published RG-lysosome pexophagy assay with EGFP-mCherry-TMDPEX26 (Deosaran et al., 2013). In both assays, the “red-green” peroxisomal reporter becomes “red only” after the delivery of the peroxisome to the lysosome, whose pH is acidic and causes quenching of the EGFP fluorescence. In the control treatment of HeLa cells with a nontargeting pool of siRNAs, we could readily detect the cells containing autolysosomes with acid-resistant mRFP fluorescence (Fig. 9, A and B). However, the cells treated with either siATG7 or siACBD5 did not display such “red only” structures, which suggests that, like ATG7, ACBD5 is required for the autophagic delivery of peroxisomes to the lysosomes. We also expressed mRFP-EGFP-SKL in Ntera-2 cells and calculated the ratio of mRFP and EGFP intensities per cell after knockdown of ATG16L1 or ACBD5 (Fig. 9, C and D). This experiment confirmed the appearance of “red only” structures exclusively in the cells treated with control siRNA. The mRFP/EGFP ratio in the cells treated with siACBD5 remained as low as that in the siATG16L1-treated cells, proving that ACBD5 plays an essential role in pexophagy.


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 essential for pexophagy in human cells. (A and C) ACBD5 is needed for the delivery of peroxisomes to the lysosomes in HeLa (A) and Ntera-2 (C) cells expressing peroxisomally targeted mRFP-EGFP-SKL. Arrows point to the autolysosomes with acid-resistant mRFP fluorescence. Bars, 10 µm. (B) Quantitation of data in A. The percentage of cells with mRFP-labeled autolysosomes is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. control siRNA). (D) Quantitation of data in C. The ratio of mRFP and EGFP intensities per cell reflects the occurrence of pexophagy and is presented as the mean + SD (error bars; n ≥ 10 images; *, P < 0.05 vs. control siRNA).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig9: ATG37/ACBD5 is essential for pexophagy in human cells. (A and C) ACBD5 is needed for the delivery of peroxisomes to the lysosomes in HeLa (A) and Ntera-2 (C) cells expressing peroxisomally targeted mRFP-EGFP-SKL. Arrows point to the autolysosomes with acid-resistant mRFP fluorescence. Bars, 10 µm. (B) Quantitation of data in A. The percentage of cells with mRFP-labeled autolysosomes is shown as the mean + SD (error bars; n ≥ 50; *, P < 0.05 vs. control siRNA). (D) Quantitation of data in C. The ratio of mRFP and EGFP intensities per cell reflects the occurrence of pexophagy and is presented as the mean + SD (error bars; n ≥ 10 images; *, P < 0.05 vs. control siRNA).
Mentions: Next, we addressed the functional role of human ACBD5. To measure pexophagy in human cells, we used the tandem fluorochrome pexophagy assay with mRFP-EGFP-SKL as a reporter. It is based on the same principle as the recently published RG-lysosome pexophagy assay with EGFP-mCherry-TMDPEX26 (Deosaran et al., 2013). In both assays, the “red-green” peroxisomal reporter becomes “red only” after the delivery of the peroxisome to the lysosome, whose pH is acidic and causes quenching of the EGFP fluorescence. In the control treatment of HeLa cells with a nontargeting pool of siRNAs, we could readily detect the cells containing autolysosomes with acid-resistant mRFP fluorescence (Fig. 9, A and B). However, the cells treated with either siATG7 or siACBD5 did not display such “red only” structures, which suggests that, like ATG7, ACBD5 is required for the autophagic delivery of peroxisomes to the lysosomes. We also expressed mRFP-EGFP-SKL in Ntera-2 cells and calculated the ratio of mRFP and EGFP intensities per cell after knockdown of ATG16L1 or ACBD5 (Fig. 9, C and D). This experiment confirmed the appearance of “red only” structures exclusively in the cells treated with control siRNA. The mRFP/EGFP ratio in the cells treated with siACBD5 remained as low as that in the siATG16L1-treated cells, proving that ACBD5 plays an essential role in pexophagy.

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