Molecular mechanisms and physiological roles of Atg5/Atg7-independent alternative autophagy

Abstract

ATG5 and ATG7 are considered to be essential molecules for the induction of autophagy. However, we found that cells lacking ATG5 or ATG7 can still form autophagosomes/autolysosomes and perform autophagic protein degradation when subjected to certain types of stress. Although the lipidation of LC3 is accepted as a good indicator of autophagy, this did not occur during ATG5/ATG7-independent alternative autophagy. Unlike conventional autophagy, autophagosomes appeared to be generated in a Rab9-dependent manner by the fusion of the phagophores with vesicles derived from the trans-Golgi and late endosomes. Therefore, mammalian autophagy can occur via at least two different pathways; the ATG5/ATG7-dependent conventional pathway and an ATG5/ATG7-independent alternative pathway.

Keywords: Atg5-independent autophagy; alternative autophagy; reticulocytes.

Figure 1.

Hypothetical model of macroautophagy. There are at least two modes of macroautophagy, i.e., conventional and alternative autophagy. Conventional autophagy requires Atg5 and Atg7, is associated with LC3 modification, and is thought to originate from the ER membrane. In contrast, alternative autophagy occurs independently of Atg5 and Atg7, as well as LC3 modification. The generation of autophagic vacuoles in alternative autophagy is mediated by the fusion of isolation membranes with vesicles derived from the trans-Golgi as well as late endosomes, in a Rab9-dependent manner. Modified from Int. J. Mol. Sci. 2014, 15(2), 3154–3171.

Figure 2.

Induction of autophagy in Atg5-deficient MEFs by etoposide. (A) (Top row) Electron micrographs of wild-type MEFs and Atg5-deficient MEFs treated with etoposide (10 µM) for 18 h. Bar = 5 µm. (Bottom row) Magnified images of the areas enclosed in boxes in the top row. Typical autophagic structures were observed (arrows). (B) Wild-type MEFs and Atg5-deficient MEFs were incubated with etoposide and immunostained with anti-Lamp2 (red) or anti-LC3 (green) antibodies. Nuclei were counterstained (blue). Punctate GFP-LC3 fluorescence was observed in etoposide-treated wild-type MEFs but not Atg5-deficient MEFs.

Figure 3.

Involvement of alternative autophagy in mitochondrial clearance during erythrocyte maturation. (A) The final stage of red blood cell maturation. During erythrocyte maturation, erythroblasts lose their nuclei to become reticulocytes, and reticulocytes are transformed into erythrocytes by the elimination of their mitochondria. Autophagy is involved in the latter process. Modified from Dojin News, 2016, No. 160. (B) Electron micrographs of wild-type, Atg5-deficient, and Ulk1-deficient erythrocytes. Remaining mitochondria are observed in the Ulk1-deficient, but not wild-type and Atg5-deficient erythrocytes. Autophagosomes engulfing mitochondria (arrows), autolysosomes degrading mitochondria (arrowheads) and isolation membranes (asterisk) are shown. (C) Mechanism of mitochondrial elimination during erythrocyte maturation. Mitochondrial elimination mainly occurs via Ulk1-dependent alternative autophagy and only partially via Atg5-dependent conventional autophagy.