On autophagy

Autophagy (from the Greek “auto” and “phagy,” literally meaning “self-eating”) is an intracellular process in eukaryotes that involves the breakdown and recycling of organelles, proteins, and other molecules. Cellular components targeted for degradation are typically dysfunctional or inefficient (i.e. such degradation may represent a diversion of resources), and are enveloped by an autophagosome (a double-membraned vesicle) which, in mammalian cells, ultimately fuses with the lysosome.

Within the acidic environment of the lysosome, hydrolases break down such organelles or proteins into their constituent components (e.g. amino acids) which may then be used to fuel synthesis of new macromolecules and organelles. This process is critical to cellular function, increasing the availability of energy substrates during starvation, recycling aging and/or damaged organelles (thus mitigating stress-associated damage, e.g. excess ROS generation by dysfunctional mitochondria), and destroying pathogenic particles such as viruses and bacteria.

Much of the research elucidating the mechanisms by which autophagy takes place has centered on the yeast Saccharomyces cerevisiae, a widely-used model organism. In a trait similar to that of plant cells, a large central vacuole is typically the most conspicuous organelle in S. cerevisiae. In fungal cells, the vacuole is analogous to the animal lysosome, serving as the cell’s lytic site via autophagic pathways that were identified by Yoshinori Ohsumi and colleagues.1

Researchers in Ohsumi’s lab used nutrient deprivation to induce autophagy in S. cerevisiae, reasoning that mutants that lacked the ability to produce proteases present in the vacuole would not be able break down cellular components delivered there for destruction. In such mutants, they observed the intravacuolar accumulation of what were termed “autophagic bodies” (vesicles containing various cellular contents; now referred to as “autophagosomes”).

Their technique (preventing the breakdown of components by utilizing protease-deficient mutants) allowed them to gain insight into the contents of autophagic bodies. These autophagic bodies contain a wide variety of organelles and other molecules, including mitochondria, portions of the rough endoplasmic reticulum, ribosomes, and various “housekeeping enzymes” such as glucose-6-phosphate dehydrogenase. It was found that nitrogen-starvation-induced autophagy was non-selective, that is, cytosolic contents were being non-preferentially sequestered in autophagic bodies for degradation.

Using genetic crosses, Ohsumi’s team narrowed down the particular protease whose absence was responsible for the accumulation of autophagic bodies; the serine protease PrB, the product of the gene PRB1. This was further confirmed when it was found that the serine protease inhibitor PMSF induced accumulation of autophagic bodies in wild-type cells. They concluded that autophagy was a major driver of the degradation of proteins into their constituent amino acids, and that this protein turnover was dependent on PrB. Subsequent research by Ohsumi’s team would reveal 15 genes (including PRB1) involved in autophagy. Many molecular pathways underlying autophagy have since been described, and these processes have been directly linked to those in mammalian cells.

Farré and colleagues recently sought to elucidate the complex regulatory mechanisms underlying autophagy in S. cerevisiae and the methylotrophic yeast Pichia pastoris.2 While nutrient deprivation results in non-selective autophagy in yeast, selective autophagy is also available and is mediated by three receptors in S. cerevisiae; Atg19 (involved in cytoplasm-to-vacuole targeting; interacts with “cargo proteins” which are translocated into the autophagosome on a one-by-one basis), Atg32 (mitophagy, the breakdown of mitochondria), and Atg36 (responsible for mediating pexophagy, the degradation of the peroxisome). In P. pastoris, Atg30 (the only other autophagy receptor identified in yeast at the time of the research) is the analogous pexophagy receptor. A variety of other proteins (using the “Atg” prefix for “Autophagy-related protein”) interact with these receptors to facilitate autophagic processes in yeast, but at the time of Farré’s work, little was known about how these interactions were regulated.

Farré and his team used protein complex immunoprecipitation (co-immunoprecipitation or Co-IP; a technique by which an antibody is used to obtain a complex including a protein the antibody is already known to bind) to find that the ubiquitin-like Atg8 and Atg11 (previously identified in S. cerevisiae) selectively bind to Atg30. By inducing mutations to alter amino acid sequences in Atg30 (in P. pastoris) and Atg32 (in S. cerevisiae), they discovered that both receptors have an “AIM” (Atg8-family interacting motif) region, a phosphorylation-dependent site that binds Atg8 and Atg11. Further mutations showed that Atg8 and Atg11 must bind sequentially (rather than simultaneously) to Atg30, and that both must bind to the same Atg30 molecule for optimal pexophagy.

The authors extended their findings to S. cerevisiae, identifying an AIM sequence on Atg32 and 36 (the receptors responsible for mitophagy and pexophagy respectively in S. cerevisiae). They found that, similarly to P. pastoris, Atg8 and Atg11 bind sequentially (in a phosphorylation-dependent manner) to Atg32 and Atg36. The authors conclude by outlining their putative regulatory model for pexophagy in both organisms; the receptor’s Atg11-binding site is phosphorylated, interacting with either Atg11 or Atg8; whichever binds first then dissociates, and a second phosphorylation event spurs the binding of the other protein, effectively regulating selective autophagy in these organisms (i.e. mitophagy and pexophagy).

In 2015, shortly before being awarded the Nobel Prize for his work on autophagy decades before, Oshumi and his collaborators were still providing deep insights into the autophagic pathways involved in organelle degradation. In recently published work,3 researchers at Ohsumi’s lab were interested in identifying proteins involved in the regulation of selective autophagy of the rough endoplasmic reticulum (what they call “ER-phagy”) and the nucleus (nucleophagy) in S. cerevisiae. This research represents the first description of the proteins Atg39 and Atg40, two receptors Ohsumi’s team identified as vital to these selective degradation pathways.

Ohsumi and colleagues initially discovered two unknown proteins via mass spectrometry of Atg8 immunoprecipitates (that is, proteins determined to bind Atg8 by use of an Atg8-specific antibody); these proteins were dubbed Atg39 and Atg40. As Farré had done, Ohsumi and colleagues used co-immunoprecipitation to confirm that Atg39 and Atg40 both interacted with Atg8. AIM (Atg8-family interacting motif) regions were identified in both proteins, and mutation of these regions eliminated Atg8 binding. An Atg11-binding sequence was discovered in Atg39. However, the team found that cells lacking Atg39 and Atg40 were able to carry out non-selective autophagy under nutrient deprivation as well as selective autophagy of mitochondria, peroxisomes, and vacuolar enzymes as normal.

The researchers used a GFP-tagged ER membrane protein (Sec63) and observed that loss of Atg39 and Atg40 caused accumulation of GFP-fluorescent ER fragments in the vacuole. They then tagged Atg39 and Atg40 with GFP, finding that these proteins localized to the rough ER where they bound to Atg8 and Atg11, leading to the conclusion that these proteins are ER-phagy receptors.

Since the ER is continuous with the nuclear envelope, the team next investigated the possibility that these proteins might also mediate nucelophagy. In yeast, ER is divided into two distinct regions; the perinuclear ER (pnER, equivalent to the nuclear envelope) and the cortical ER (cER). They found that Atg39 localized to the pnER while a majority of Atg40 localized to the cER, and that the two proteins are responsible for autophagy of these distinct regions respectively. Finally, since Atg39 mediates degradation of fragments of the nuclear envelope, the authors determined that it is largely responsible for selective degradation of the nucleus.

These recent studies reveal the intricacy of receptor-mediated selective autophagy in yeast. The identification of analogous receptors in mammalian cells might pave the way not only for greater understanding of autophagic pathways in humans, but also the potential for novel therapies targeting such pathways in order to treat lysosomal storage diseases.

[1] Takeshige K, Baba M, Tsuboi S, Noda T, Ohsumi Y. Autophagy in yeast demonstrated with proteinase-deficient mutants and conditions for its induction. J Cell Biol. 1992;119(2):301-11. doi:10.1083/jcb.119.2.301. PubMed PMID: 1400575; PubMed Central PMCID: PMC2289660

[2] Farré JC, Burkenroad A, Burnett SF, Subramani S. Phosphorylation of mitophagy and pexophagy receptors coordinates their interaction with Atg8 and Atg11. EMBO Rep. 2013;14(5):441-9. doi:10.1038/embor.2013.40. PubMed PMID: 23559066; PubMed Central PMCID: PMC3642380

[3] Mochida K, Oikawa Y, Kimura Y, et al. Receptor-mediated selective autophagy degrades the endoplasmic reticulum and the nucleus. Nature. 2015;522(7556):359-62. doi:10.1038/nature14506. PubMed PMID: 26040717