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

Macroautophagy (hereafter autophagy) is a ubiquitous process in eukaryotic cells that is integrally involved in various aspects of cellular and organismal physiology. The morphological hallmark of autophagy is the formation of double-membrane cytosolic vesicles, autophagosomes, which sequester cytoplasmic cargo and deliver it to the lysosome or vacuole. Thus, autophagy involves dynamic membrane mobilization, yet the source of the lipid that forms the autophagosomes and the mechanism of membrane delivery are poorly characterized. The TRAPP complexes are multimeric guanine nucleotide exchange factors (GEFs) that activate the Rab GTPase Ypt1, which is required for secretion. Here we describe another form of this complex (TRAPPIII) that acts as an autophagy-specific GEF for Ypt1. The Trs85 subunit of the TRAPPIII complex directs this Ypt1 GEF to the phagophore assembly site (PAS) that is involved in autophagosome formation. Consistent with the observation that a Ypt1 GEF is directed to the PAS, we find that Ypt1 is essential for autophagy. This is an example of a Rab GEF that is specifically targeted for canonical autophagosome formation.utophagy is a catabolic process in which damaged or superfluous cytoplasmic components are degraded in response to stress conditions; it is evolutionarily conserved in eukaryotes and is integrally involved in development and physiology (1, 2). The morphological hallmark of autophagy is the formation of doublemembrane cytosolic vesicles, autophagosomes, which sequester cytoplasm. The autophagosomes then fuse with the lysosome, resulting in the degradation of the cargo. The mechanism of autophagosome formation is distinct from that used for vesicle formation in the secretory or endocytic pathways and is said to be de novo in that it does not occur by direct budding from a preexisting organelle. Instead, a nucleating structure, the phagophore, appears to expand by the addition of membrane possibly through vesicular fusion. One consequence of this mechanism is that it allows the sequestration of essentially any sized cargo, including intact organelles or invasive microbes, and this capability is critical to autophagic function. When autophagy is induced there is a substantial demand for membrane, and a major question in the field concerns the membrane origin; nearly every organelle has been implicated in this role (3). The early secretory pathway is likely one such membrane source for autophagy (4, 5).Rab GTPases are key regulators of membrane traffic that mediate multiple events including vesicle tethering and membrane fusion. These molecular switches cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The yeast Rab Ypt1, which is essential for ER-Golgi and Golgi traffic (6), is activated by the multimeric guanine nucleotide exchange factor (GEF) called TRAPP (7,8). Two forms of the TRAPP complexes have been identified (9). These two complexes share several subunits, including four (Bet3, Bet5, Trs23, and Trs31) that are essential to activate Ypt1. How each of th...

Mitophagy is the process of selective mitochondrial degradation via autophagy, which has an important role in mitochondrial quality control. Very little is known, however, about the molecular mechanism of mitophagy. A genome-wide yeast mutant screen for mitophagy-defective strains identified 32 mutants with a block in mitophagy, in addition to the known autophagy-related (ATG) gene mutants. We further characterized one of these mutants, ylr356wDelta that corresponds to a gene whose function has not been identified. YLR356W is a mitophagy-specific gene that was not required for other types of selective autophagy or macroautophagy. The deletion of YLR356W partially inhibited mitophagy during starvation, whereas there was an almost complete inhibition at post-log phase. Accordingly, we have named this gene ATG33. The new mutants identified in this analysis will provide a useful foundation for researchers interested in the study of mitochondrial homeostasis and quality control.

The Rif1 protein, originally identified as a telomere-binding factor in yeast, has recently been implicated in DNA replication control from yeast to metazoans. Here, we show that budding yeast Rif1 protein inhibits activation of prereplication complexes (pre-RCs). This inhibitory function requires two N-terminal motifs, RVxF and SILK, associated with recruitment of PP1 phosphatase (Glc7). In G1 phase, we show both that Glc7 interacts with Rif1 in an RVxF/SILK-dependent manner and that two proteins implicated in pre-RC activation, Mcm4 and Sld3, display increased Dbf4-dependent kinase (DDK) phosphorylation in rif1 mutants. Rif1 also interacts with Dbf4 in yeast two-hybrid assays, further implicating this protein in direct modulation of pre-RC activation through the DDK. Finally, we demonstrate Rif1 RVxF/SILK motif-dependent recruitment of Glc7 to telomeres and earlier replication of these regions in cells where the motifs are mutated. Our data thus link Rif1 to negative regulation of replication origin firing through recruitment of the Glc7 phosphatase.

Autophagy mediates the bulk turnover of cytoplasmic constituents in lysosomes. During embryonic development in animals, a dramatic degradation of yolk proteins and synthesis of zygotic proteins takes place, leading to intracellular remodeling and cellular differentiation. Zebrafish represents a unique system to study autophagy due in part to its rapid embryonic development relative to other vertebrates. The technical advantages of this organism make it uniquely suited to various studies including high-throughput drug screens. To study autophagy in zebrafish, we identified two zebrafish Atg8 homologs, lc3 and gabarap, and generated two transgenic zebrafish lines expressing GFP-tagged versions of the corresponding proteins. Similar to yeast Atg8 and mammalian LC3, zebrafish Lc3 undergoes post-translational modification starting at the pharyngula stage during embryonic development. We observed a high level of autophagy activity in zebrafish embryos, which can be further upregulated by the TOR inhibitor rapamycin or the calpain inhibitor calpeptin. In addition, zebrafish Gabarap accumulates within lysosomes upon autophagy induction. Thus, we established a convenient zebrafish tool to assay autophagic activity during embryogenesis in vivo.