Direct uptake and degradation of DNA by lysosomes - PubMed (original) (raw)

Direct uptake and degradation of DNA by lysosomes

Yuuki Fujiwara et al. Autophagy. 2013 Aug.

Abstract

Lysosomes contain various hydrolases that can degrade proteins, lipids, nucleic acids and carbohydrates. We recently discovered "RNautophagy," an autophagic pathway in which RNA is directly taken up by lysosomes and degraded. A lysosomal membrane protein, LAMP2C, a splice variant of LAMP2, binds to RNA and acts as a receptor for this pathway. In the present study, we show that DNA is also directly taken up by lysosomes and degraded. Like RNautophagy, this autophagic pathway, which we term "DNautophagy," is dependent on ATP. The cytosolic sequence of LAMP2C also directly interacts with DNA, and LAMP2C functions as a receptor for DNautophagy, in addition to RNautophagy. Similarly to RNA, DNA binds to the cytosolic sequences of fly and nematode LAMP orthologs. Together with the findings of our previous study, our present findings suggest that RNautophagy and DNautophagy are evolutionarily conserved systems in Metazoa.

Keywords: DNA; DNautophagy; LAMP-2; LAMP-2C; LAMP2; LAMP2C; RNA; RNautophagy; autophagy.

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Figures

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Figure 1. Interactions of DNA with cytosolic sequences of LAMPs. Interactions of purified plasmid DNA (pCI-neo) with cytosolic sequences of human LAMP2s, nematode (Caenorhabditis elegans) LMP-1, and fly (Drosophila melanogaster) Lamp1. Pull-down assays were performed using 5 μg of DNA and 8 nmol of peptide constructs of cytosolic sequences corresponding to each LAMP, fused to biotin. DNA was detected with EtBr.

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Figure 2. Uptake and degradation of DNA by isolated lysosomes. (A) Isolated lysosomes were incubated with 0.5 μg of purified DNA for 5 min in the presence or absence of ATP (energy regenerating system), and pelleted by centrifugation. The levels of DNA remaining in solution outside of lysosomes, and the levels of DNA in the precipitated lysosomes were analyzed. (B) Isolated lysosomes were incubated with or without DNA for 5 min in the presence of ATP (energy regenerating system), and then immunogold labeling was performed using an anti-DNA antibody followed by anti-mouse IgG coupled with 10 nm of gold particles (left and middle, respectively). As a control assay, immunogold labeling was performed without primary antibody (right). Gold particles are observed in the lysosomes in the left panel, while no gold particles are observed in the lysosomes in the middle and the right panels. (C) Isolated lysosomes were incubated with 1 μg of purified DNA for 5 min in the presence or absence of ATP (energy regenerating system), and total levels of DNA in the incubated samples were analyzed.

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Figure 3. Effects of overexpression of LAMP2C and knockout of Lamp2 on DNautophagy. (A and B) Uptake of DNA by lysosomes isolated from HeLa cells transfected with LAMP2C expression vector or empty vector (n = 3) (A). Uptake of DNA by lysosomes isolated from the brains of wild-type and lamp2 knockout mice (n = 3) (B). Isolated lysosomes were incubated with purified DNA for 5 min in the presence of ATP (energy regenerating system). Levels of DNA uptake were measured by subtracting the levels of DNA remaining in solution outside of lysosomes from the levels of input DNA. *p < 0.05 and **p < 0.01, respectively (Student’s t-test).

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Figure 4. Uptake and degradation of mtDNA by isolated lysosomes. (A) mtDNA was isolated from HeLa cells. The mtDNA was confirmed by PCR (25 cycles) using primers specific for mtDNA. As a negative control, plasmid DNA (pCI-neo) was used as a template. (B and C) Isolated lysosomes were incubated with 0.5 μg of isolated mtDNA for 5 min in the presence or absence of ATP (energy regenerating system), and pelleted by centrifugation. The levels of DNA remaining in solution outside of lysosomes (B), and the levels of DNA in the precipitated lysosomes were analyzed (C). The mtDNA in the samples was confirmed by PCR using primers specific for mtDNA. (D) Isolated lysosomes were incubated with 0.5 μg of mtDNA for 5 min in the presence or absence of ATP (energy regenerating system), and total levels of DNA in the incubated samples were analyzed. The mtDNA in the samples was confirmed by PCR.

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