Abstract
Studies of the physiological and pathological roles of autophagy have revealed that too little or too much autophagy can be detrimental, and therefore autophagy activity needs to be tightly regulated. Altered transcription of autophagy-related (ATG) genes has been reported in many diseases, and ATG genes can be the most direct targets for the treatment of autophagy-associated diseases. Thus, it is important to understand how the amounts of different Atg proteins affect autophagy, and how the expression of their corresponding genes is regulated. Using budding yeast as the model, we showed that Pho23, a component of the Rpd3 large (Rpd3L) complex, represses the transcription of several ATG genes including ATG9, the expression of which regulates the frequency of autophagosome formation. More autophagosomes are formed in PHO23 null cells or in those overexpressing Atg9; conversely, there are fewer autophagosomes seen in cells with reduced Atg9 expression.
Keywords: autophagy, lysosome, phagophore, stress, vacuole, yeast
Autophagosomes are the structural hallmark of autophagy, and their size and number directly determine autophagy activity. The size of an autophagosome is regulated in part by the Atg8 protein level; smaller autophagosomes form when less Atg8 is expressed, although the number of autophagosomes remains unchanged. Ume6, a DNA-binding protein associated with the Rpd3L complex, represses the transcription of ATG8 by directly binding to its promoter. Accordingly, larger autophagosomes form in cells with a deletion of UME6. Besides ATG8, several ATG genes show an increase of transcription when autophagy is induced. These observations led us to ask the following: 1) How does the transcriptional regulation of the ATG genes regulate the size and/or number of autophagosomes? 2) What other transcription regulators target the ATG genes, thus modulating autophagy activity?
From a screen for transcriptional regulators of autophagy, we identified Pho23 as a repressor of autophagy. The mRNA (and corresponding protein) levels of ATG genes, such as ATG1, ATG7, ATG8, ATG9, ATG14, and ATG29 are elevated in pho23∆ cells in nutrient-rich conditions. In contrast, after nitrogen starvation, when the transcription of these ATG genes is normally upregulated, the difference of ATG mRNA level between the mutant and wild type is diminished, indicating that Pho23 represses ATG gene transcription primarily during vegetative growth, and this repression is impaired by nitrogen starvation. Increased autophagy activity is also detected in pho23∆ cells.
Interestingly, with the absence of Pho23, which is also a component of the Rpd3L complex similar to Ume6, we observed more, but not larger, autophagosomes. This phenotype is different from what is observed in ume6∆ cells; namely, larger autophagosomes. This easily ignored but important difference indicates that Pho23 may regulate autophagy differently from Ume6, although they are both associated with the Rpd3L complex. Indeed, the mRNA level of some of the ATG genes encoding components of the core autophagy machinery is increased in rpd3∆ as well as in pho23∆ cells. Furthermore, an additive increase in the amount of the ATG mRNAs is not detected in pho23∆ rpd3∆ double-deletion cells, indicating that Pho23 regulates ATG transcription in an Rpd3-dependent manner. In addition, the mRNA level of some of these ATG target genes, including ATG7, ATG9, and ATG14, is increased significantly in pho23∆ or rpd3∆ but not in ume6∆ cells, indicating that Rpd3 associates with Pho23 and Ume6 to form functionally distinguishable complexes that target different subgroups of ATG genes to regulate the size and number of autophagosomes relatively independently.
To test which ATG target(s) of Pho23 regulates the number of autophagosomes, we need to modulate the expression of individual ATG genes. We chose ATG9 as our first candidate because its expression shows the greatest magnitude and extended duration of increase in the absence of Pho23. We generated strains with different levels of Atg9 expression by changing the promoter of ATG9. We found that with lower Atg9 expression, lower autophagy activity was detected, and we observed fewer autophagosomes. In contrast, with higher Atg9 expression we measured higher autophagy activity and more autophagosomes, indicating that Atg9 regulates the number of autophagosomes.
As the Atg proteins function in different steps of autophagosome formation, autophagy may be regulated differently through modulating the size and/or number of autophagosomes by transcriptional regulation of different ATG genes. Besides ATG8 and ATG9, the biological significance of the increased transcription of other ATG genes, such as ATG1, ATG7, ATG14, and ATG29 upon autophagy induction has not been fully studied. The fine dissection of the roles of these Atg proteins may help to fine-tune autophagy activity temporally, spatially, and in magnitude.
As Pho23 cannot bind with DNA directly, it is still not clear how this protein targets the ATG genes. This binding may be through an interaction of Pho23 with methylated histone, or through recruitment by a DNA-binding protein to the target DNA. It is also not clear how the repression of ATG transcription by Pho23 is released by nitrogen starvation. To date, Rpd3 has been reported to regulate autophagy by at least 3 different, but not exclusive, methods: regulating the size of autophagosomes through Ume6, regulating the frequency of autophagosome formation through Pho23, and controlling activity through direct deacetylation of Atg proteins. Do these roles compete or cooperate with each other? Does Rpd3 play different roles to regulate autophagy in different conditions? Do the mammalian homologs of Pho23 and Rpd3 play conserved roles to regulate autophagy?
Our study reveals that the Atg9 protein amount regulates the frequency of autophagosome formation; however, the detailed mechanism is not yet clear. Atg9, a transmembrane protein, is considered as a potential membrane carrier, which transports membrane from other organelles to the phagophore to facilitate nucleation and/or expansion. Thus, one possible explanation is that the Atg9 protein level regulates the membrane flux to the phagophore, and therefore regulates its rate of expansion. Another possibility is that the Atg9 protein amount regulates the frequency of autophagosome initiation, because Atg9 also participates in an early stage of autophagosome formation. Finally, Atg9 may function in autophagosome completion, so with less Atg9, delay of completion results in a reduced number of autophagosomes being generated.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
This work was supported by NIH grant GM053396 to DJK, and was funded in part through the Protein Folding Diseases FastForward Initiative, University of Michigan.