| Literature DB >> 27846365 |
Shu Aizawa1, Viorica Raluca Contu1,2, Yuuki Fujiwara1, Katsunori Hase1, Hisae Kikuchi1, Chihana Kabuta1, Keiji Wada1, Tomohiro Kabuta1.
Abstract
Lysosomes degrade macromolecules such as proteins and nucleic acids. We previously identified 2 novel types of autophagy, RNautophagy and DNautophagy, where lysosomes directly take up RNA and DNA, in an ATP-dependent manner, for degradation. We have also reported that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference defective-1), mediates RNA translocation during RNautophagy. In this addendum, we report that SIDT2 also mediates DNA translocation in the process of DNautophagy. These findings help elucidate the mechanisms underlying the direct uptake of nucleic acids by lysosomes and the physiological functions of DNautophagy.Entities:
Keywords: DNA; DNautophagy; RNA; RNautophagy; autophagy; lysosome; membrane protein
Mesh:
Substances:
Year: 2016 PMID: 27846365 PMCID: PMC5245770 DOI: 10.1080/15548627.2016.1248019
Source DB: PubMed Journal: Autophagy ISSN: 1554-8627 Impact factor: 16.016
Figure 1.Effects of SIDT2 overexpression on DNA uptake and degradation by lysosomes. (A) Lysosomes were isolated from SIDT2-overexpressing cells (SIDT2-overexpressing lysosomes) or control (empty vector transfected) cells (control lysosomes) and protein levels in lysosomal samples were analyzed by immunoblotting. (B) Intactness of SIDT2-overexpressing or control lysosomes was confirmed. Isolated lysosomes (∼25 µg protein) were incubated in the presence of an ATP regeneration system for 5 min at 37°C. Lysosomes were pelleted by centrifugation at 17,700 g for 1 min, and the supernatant fraction was incubated with 1 µg of plasmid DNA under the same conditions, then analyzed by agarose gel electrophoresis. DNA levels were quantified using ImageJ software. One hundred percent of input DNA was electrophoresed. Migration time was 5 min. Mean ± SEM (n = 3). n.s., not significant. (C) A DNA uptake assay was performed using SIDT2-overexpressing or control lysosomes as described in Materials and Methods. Mean ± SEM (n = 3). ***, P < 0.001. (D) SIDT2-overexpressing or control lysosomes were prepared and incubated with DNA as described in Materials and Methods. Post-embedding immunoelectron microscopy of the lysosomes was performed using an anti-DNA antibody followed by anti-mouse IgG coupled with 12-nm gold particles. Gold particles were observed in the lysosomes and the number of gold particles per lysosome counted. Mean ± SD (n = 20). ***, P < 0.001. Scale bars: 100 nm. (E) Lysosomes isolated from S564A mutant SIDT2-, wild-type SIDT2-overexpressing cells or control cells were subjected to a DNA uptake assay as described in Materials and Methods. Mean ± SEM (n = 3). *, P < 0.05. (F) A DNA degradation assay was performed as described in Materials and Methods using isolated lysosomes derived from Neuro2a cells transfected with SIDT2-expression or control empty vectors. Mean ± SEM (n = 3). **, P < 0.01. (G) SIDT2-overexpressing or control lysosomes were prepared and incubated without DNA as described in Materials and Methods.
Figure 2.Effects of SIDT2 knockdown on DNA uptake and degradation by lysosomes. (A and F) Lysosomes derived from SIDT2-knockdown HeLa cells were analyzed by immunoblotting as described in Fig. 1A. (B and G) Intactness of lysosomes isolated from HeLa cells was confirmed as described in Fig. 1B. (C and H) DNA uptake assays were performed using isolated lysosomes derived from SIDT2-knockdown HeLa cells (transfected with SIDT2 siRNA) or control HeLa cells (transfected with control siRNA) as described in Materials and Methods. Mean ± SEM (n = 3). ***, P < 0.001. (D and I) DNA degradation assays were performed using isolated lysosomes derived from SIDT2-knockdown HeLa cells or control HeLa cells as described in the Materials and Methods. Mean ± SEM (n = 3). *, P < 0.05. (E and J) Isolated lysosomes derived from SIDT2 knockdown HeLa cells or control HeLa cells were prepared and incubated without DNA as described in Materials and Methods.
Figure 3.Uptake efficacy of SIDT2 toward different types of nucleic acids. (A) A DNA uptake assay was performed using SIDT2-overexpressing and control lysosomes as described in Materials and Methods. One microgram of circular (plasmid) DNA or linear DNA was used as a substrate. Mean ± SEM (n = 3). ***, P < 0.001; **, P < 0.01; *, P < 0.05. (B) RNA and DNA uptake assays were performed. SIDT2-overexpressing or control lysosomes were incubated with 5 µg of mouse total RNA or linear DNA in the presence of an ATP regeneration system for 5 min at 37°C. Lysosomes were pelleted by centrifugation at 17,700 g for 1 min, and nucleic acids in the supernatant fraction were analyzed by agarose gel electrophoresis. Migration time was 15 min. Mean ± SEM (n = 3). *, P < 0.05.