Meiyan Jin

Regulation of autophagy: modulation of the size and number of autophagosomes.

Autophagy as a conserved degradation and recycling process in eukaryotic cells, occurs constitutively, but is induced by stress. A fine regulation of autophagy in space, time, and intensity is critical for maintaining normal energy homeostasis and metabolism, and to allow for its therapeutic modulation in various autophagy‐related human diseases. Autophagy activity is regulated in both transcriptional and post‐translational manners. In this review, we summarize the cytosolic regulation of autophagy via its molecular machinery, and nuclear regulation by transcription factors. Specifically, we consider Ume6‐ATG8 and Pho23‐ATG9 transcriptional regulation in detail, as examples of how nuclear transcription factors and cytosolic machinery cooperate to determine autophagosome size and number, which are the two main mechanistic factors through which autophagy activity is regulated.

Ume6 transcription factor is part of a signaling cascade that regulates autophagy

Autophagy has been implicated in a number of physiological processes important for human heath and disease. Autophagy involves the formation of a double-membrane cytosolic vesicle, an autophagosome. Central to the formation of the autophagosome is the ubiquitin-like protein autophagy-related (Atg)8 (microtubule-associated protein 1 light chain 3/LC3 in mammalian cells). Following autophagy induction, Atg8 shows the greatest change in expression of any of the proteins required for autophagy. The magnitude of autophagy is, in part, controlled by the amount of Atg8; thus, controlling Atg8 protein levels is one potential mechanism for modulating autophagy activity. We have identified a negative regulator of ATG8 transcription, Ume6, which acts along with a histone deacetylase complex including Sin3 and Rpd3 to regulate Atg8 levels; deletion of any of these components leads to an increase in Atg8 and a concomitant increase in autophagic activity. A similar regulatory mechanism is present in mammalian cells, indicating that this process is highly conserved.

Proteolytic processing of Atg32 by the mitochondrial i-AAA protease Yme1 regulates mitophagy.

Mitophagy, the autophagic removal of mitochondria, occurs through a highly selective mechanism. In the yeast Saccharomyces cerevisiae, the mitochondrial outer membrane protein Atg32 confers selectivity for mitochondria sequestration as a cargo by the autophagic machinery through its interaction with Atg11, a scaffold protein for selective types of autophagy. The activity of mitophagy in vivo must be tightly regulated considering that mitochondria are essential organelles that produce most of the cellular energy, but also generate reactive oxygen species that can be harmful to cell physiology. We found that Atg32 was proteolytically processed at its C terminus upon mitophagy induction. Adding an epitope tag to the C terminus of Atg32 interfered with its processing and caused a mitophagy defect, suggesting the processing is required for efficient mitophagy. Furthermore, we determined that the mitochondrial i-AAA protease Yme1 mediated Atg32 processing and was required for mitophagy. Finally, we found that the interaction between Atg32 and Atg11 was significantly weakened in yme1∆ cells. We propose that the processing of Atg32 by Yme1 acts as an important regulatory mechanism of cellular mitophagy activity.

SnapShot: Selective Autophagy

There are various types of autophagy, which can be categorized as nonselective or selective. Macroautophagy is an evolutionarily conserved process through which cells degrade and recycle cytoplasm. Nonselective macroautophagy randomly engulfs a portion of the cytoplasm into autophagosomes and then delivers them to the vacuole (in fungi or plants) or the lysosome (in other higher eukaryotes) for degradation. Selective macroautophagy, however, specifically recognizes and degrades a particular cargo, either a protein complex, an organelle, or an invading microbe. The morphological hallmark of macroautophagy is the formation of an initial sequestering compartment, the phagophore, which expands into the double-membrane autophagosome; the initial sequestration occurs in a compartment that is separate from the degradative organelle. Selective microautophagy utilizes the same cellular machinery, but in this case, the sequestration event takes place directly at the limiting membrane of the lysosome/vacuole. In higher eukaryotes, selective types of autophagy also include chaperone-mediated autophagy (CMA), and two similar processes, endosomal microautophagy (e-MI) and chaperone-assisted selective autophagy (CASA), each of which involves uptake at the limiting membrane of either the lysosome or endosome. In all cases, how a substrate is targeted for sequestration and segregated from other parts of the cell is one of the major questions in this research field. Nonselective autophagy is primarily a starvation response, whereas cells use selective autophagy for a variety of purposes, including remodeling to adapt to changing environmental/nutritional conditions and to eliminate damaged organelles. Accordingly, defects in selective autophagy are associated with a range of pathophysiologies in humans, including certain types of neurodegenerative diseases.

Transcriptional Regulation by Pho23 Modulates the Frequency of Autophagosome Formation

BACKGROUND Autophagy as a conserved lysosomal/vacuolar degradation and recycling pathway is important in normal development and physiology, and defects in this process are linked to many kinds of disease. Because too much or too little autophagy can be detrimental, the process must be tightly regulated both temporally and in magnitude. Two parameters that affect this regulation are the size and the number of autophagosomes; however, although we know that the amount of Atg8 affects the size of autophagosomes, the mechanism for regulating their number has not been elucidated. The transcriptional induction and repression of the autophagy-related (ATG) genes is one crucial aspect of autophagy regulation, but the transcriptional regulators that modulate autophagy are not well characterized. RESULTS We detected increased expression levels of ATG genes, and elevated autophagy activity, in cells lacking the transcriptional regulator Pho23. Using transmission electron microscopy, we found that PHO23 null mutant cells contain significantly more autophagosomes than the wild-type. By RNA sequencing transcriptome profiling, we identified ATG9 as one of the key targets of Pho23, and our studies with strains expressing modulated levels of Atg9 show that the amount of this protein directly correlates with the frequency of autophagosome formation and the level of autophagy activity. CONCLUSIONS Our results identified Pho23 as a master transcriptional repressor for autophagy that regulates the frequency of autophagosome formation through its negative regulation of ATG9.

Transcriptional regulation of ATG9 by the Pho23-Rpd3 complex modulates the frequency of autophagosome formation

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.

A large-scale analysis of autophagy-related gene expression identifies new regulators of autophagy

Autophagy is a pathway mediating vacuolar degradation and recycling of proteins and organelles, which plays crucial roles in cellular physiology. To ensure its proper cytoprotective function, the induction and amplitude of autophagy are tightly regulated, and defects in its regulation are associated with various diseases. Transcriptional control of autophagy is a critical aspect of autophagy regulation, which remains largely unexplored. In particular, very few transcription factors involved in the activation or repression of autophagy-related gene expression have been characterized. To identify such regulators, we analyzed the expression of representative ATG genes in a large collection of DNA-binding mutant deletion strains in growing conditions as well as after nitrogen or glucose starvation. This analysis identified several proteins involved in the transcriptional control of ATG genes. Further analyses showed a correlation between variations in expression and autophagy magnitude, thus identifying new positive and negative regulators of the autophagy pathway. By providing a detailed analysis of the regulatory network of the ATG genes our study paves the way for future research on autophagy regulation and signaling.

The Core Molecular Machinery of Autophagosome Formation

Autophagy is a conserved cytoplasmic process from yeast to mammals, by which cells degrade and recycle their intracellular components. During macroautophagy, a unique compartment, named the autophagosome, is formed to engulf the cargos and send them to the vacuole or lysosome. Whether the cargos are nonspecifically sequestered, as occurs in most types of macroautophagy, or specifically selected, such as in the cytoplasm-to-vacuole targeting pathway or selective mitochondria degradation, a common set of molecular machinery is required for the formation of the autophagosome. In this chapter, we summarize our knowledge about the roles and regulation of these core machinery components in autophagosome formation, in both yeast and mammalian systems.