In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Mitochondrial quality control is important to maintain proper cellular homeostasis. Although selective mitochondrial degradation by autophagy (mitophagy) is suggested to have an important role for quality control and there is evidence for a direct relation between mitophagy and neurodegenerative diseases, the molecular mechanism of mitophagy is poorly understood. Using a screen for mitophagy-deficient mutants, we found that YIL146C/ECM37 is essential for mitophagy. This gene is not required for other types of selective autophagy or for nonspecific macroautophagy. We designated this autophagy-related (ATG) gene as ATG32. The Atg32 protein localizes on mitochondria. Following the induction of mitophagy, Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and then Atg32 is recruited to and imported into the vacuole along with mitochondria. Therefore, Atg32 confers selectivity for mitochondrial sequestration as a cargo and is necessary for recruitment of this organelle by the autophagy machinery for mitophagy.

The regulation of mitochondrial degradation through autophagy is expected to be a tightly controlled process, considering the significant role of this organelle in many processes ranging from energy production to cell death. However, very little is known about the specific nature of the degradation process. We developed a new method to detect mitochondrial autophagy (mitophagy) by fusing the green fluorescent protein at the C terminus of two endogenous mitochondrial proteins and monitored vacuolar release of green fluorescent protein. Using this method, we screened several atg mutants and found that ATG11, a gene that is essential only for selective autophagy, is also essential for mitophagy. In addition, we found that mitophagy is blocked even under severe starvation conditions, if the carbon source makes mitochondria essential for metabolism. These findings suggest that the degradation of mitochondria is a tightly regulated process and that these organelles are largely protected from nonspecific autophagic degradation.The mitochondrion is an organelle that carries out a number of important metabolic processes such as fatty acid oxidation, the Krebs cycle, and oxidative phosphorylation. Mitochondria also have a key role in the regulation of apoptosis (1). Mitochondrial oxidative phosphorylation supplies a large amount of energy that contributes to a range of cellular activities. However, this organelle is also the major source of cellular reactive oxygen species that cause damage to mitochondrial lipid, DNA, and proteins, and the accumulation of this damage is related to aging, cancer, and neurodegenerative diseases (2). Thus, quality control of mitochondria is important to maintain cellular homeostasis. In fact, mitochondria have some of their own quality control systems including a protein degradation system (3), DNA repair enzymes (4, 5), and phospholipid hydroperoxide glutathione peroxidase (6). In addition, it has long been assumed that autophagy is the pathway for mitochondrial recycling, and various theories suggest that a specific targeting of damaged mitochondria to vacuoles or lysosomes occurs by autophagy (7), although there is little direct experimental evidence for selective recognition of mitochondria. Very recently, several studies suggest that selective mitochondrial degradation via autophagy (mitophagy) might play an important role for mitochondrial quality control (8 -12).Macroautophagy is the bulk degradation of cytoplasmic components that allows cells to respond to various types of stress and to adapt to changing nutrient conditions (13,14). After certain environmental cues such as nutrient deprivation or hormonal stimuli, cells dynamically sequester portions of the cytoplasm within double-membrane cytosolic vesicles, called autophagosomes, and the completed vesicles subsequently fuse with lysosomes/vacuoles (15, 16). There are a number of selective autophagy pathways that appear to target specific cellular components, and the cytoplasm to vacuole targeting (Cvt) 2 pathway and pexophagy are ...