You have to keep in mind that even when you suppress the expression level of Atg5 and/or Atg7, Atg-independent autophagy (also referred to as “alternative autophagy”) is likely to occur.

The treatment with etoposide, a topoisomerase inhibitor that induces genotoxic stress, has been shown to exhibit the formation of autophagic structures even in Atg5-deficient MEFs. The numbers and sizes of these autophagic vacuoles are equivalent in wild-type and Atg5-deficient MEFs, and the morphology of these etoposide-induced autophagic structures is indistinguishable from the autophagic vacuoles observed during rapamycin-induced conventional autophagy. Moreover, all stages of autophagosome biogenesis were observed in Atg5-deficient MEFs, including formation of isolation membranes (membrane cisternae curving around a part of the cytoplasm), autophagosomes (double-membrane vacuoles generated by sealing of the edges of the isolation membrane), and autolysosomes (single-membrane vacuoles generated by fusion of an autophagosome and lysosome) according to the previous report by my colleagues. To sum up, mammalian cells conduct Atg5-independent macroautophagy largely regulated by Ulk1.

Ref.

Nature. 2009 Oct 1;461(7264):654-8. doi: 10.1038/nature08455. Discovery of Atg5/Atg7-independent alternative macroautophagy.

My lab has published several papers which put emphasis on the physiological roles of the alternative autophagy.

It has been reported that fetal definitive reticulocytes from Ulk1-deficient and Ulk1/Atg5 double-deficient mice retain their mitochondria, whereas the mitochondria are engulfed and digested within autophagic structures in wild-type and Atg5-deficient mice. Mitochondrial retention by Ulk1-deficient reticulocytes is far less marked in primitive and adult definitive reticulocytes.

(Nature Commun. 2014 Jun 4;5:4004. doi: 10.1038/ncomms5004.)

It has been also shown that the budding yeast Saccharomyces cerevisiae, which lacks Atg5, undergoes bulk protein degradation using Golgi‐mediated structures to compensate for autophagy when treated with amphotericin B1, a polyene antifungal drug. This phenomenon was named as Golgi membrane‐associated degradation (GOMED) pathway. This process is driven by the disruption of PI(4)P‐dependent anterograde trafficking from the Golgi, and it also exists in Atg5‐deficient mammalian cells. Biologically, when an Atg5‐deficient beta‐cell line and Atg7‐deficient beta cells were cultured in glucose‐deprived medium, a disruption in the secretion of insulin granules from the Golgi occurred, and GOMED was induced to digest these (pro)insulin granules.

(EMBO J. 2016 Sep 15;35(18):1991-2007. doi: 10.15252/embj.201593191.)

In addition, it has been recently reported that Atg5-independent autophagic process mediates mitochondrial clearance and the blockage of such autophagy, but not canonical Atg-dependent autophagy, inhibits mitochondrial clearance, in turn, preventing induced pluripotent stem cells induction. Of note, AMPK seems to be the upstream molecule of this Atg5-independent autophagic pathway and can be targeted by small molecules to modulate mitochondrial clearance during metabolic reprogramming.

(Nat Cell Biol. 2015 Nov;17(11):1379-87. doi: 10.1038/ncb3256.)

Dear Dr. Yoshida, According to what you said, what should be done to completelly block autophagy? Using siRNA against ATG5 + other drugs?

Thank you

In fact, it is very difficult to specifically block autophagy without influencing on endosome signal pathway. To be sure, the treatment with bafilomycin A1 and/or chloroquine contributes to the blockage of the "autophagy flux," but those agents are cytotoxic mainly due to the disruption of endosome.

My colleagues and I now try to identify the novel marker to better understand Atg-independent alternative autophagy.