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

Autophagy, an intrinsically nonselective process, can also target selective cargo for degradation. The mechanism of selective peroxisome turnover by autophagy-related processes (pexophagy), termed micropexophagy and macropexophagy, is unknown. We show how a Pichia pastoris protein, PpAtg30, mediates peroxisome selection during pexophagy. It is necessary for pexophagy, but not for other selective and nonselective autophagy-related processes. It localizes at the peroxisome membrane via interaction with peroxins, and during pexophagy it colocalizes transiently at the preautophagosomal structure (PAS) and interacts with the autophagy machinery. PpAtg30 is required for formation of pexophagy intermediates, such as the micropexophagy apparatus (MIPA) and the pexophagosome (Ppg). During pexophagy, PpAtg30 undergoes multiple phosphorylations, at least one of which is required for pexophagy. PpAtg30 overexpression stimulates pexophagy even under peroxisome-induction conditions, impairing peroxisome biogenesis. Therefore, PpAtg30 is a key player in the selection of peroxisomes as cargo and in their delivery to the autophagy machinery for pexophagy.

Autophagy has burgeoned rapidly as a field of study because of its evolutionary conservation, the diversity of intracellular cargoes degraded and recycled by this machinery the mechanisms involved, as well as its physiological relevance to human health and disease. This self-eating process was initially viewed as a non-selective mechanism used by eukaryotic cells to degrade and recycle macromolecules in response to stress; we now know that various cellular constituents, as well as pathogens, can also undergo selective autophagy. In contrast to non-selective autophagy, selective autophagy pathways rely on a plethora of selective autophagy receptors (SARs) that recognize and direct intracellular protein aggregates, organelles and pathogens for specific degradation. Although SARs themselves are not highly conserved, their modes of action and the signalling cascades that activate and regulate them are. Recent yeast studies have provided novel mechanistic insights into selective autophagy pathways, revealing principles of how various cargoes can be marked and targeted for selective degradation.

The selective autophagy receptors Atg19 and Atg32 interact with two proteins of the core autophagic machinery: the scaffold protein Atg11 and the ubiquitin-like protein Atg8. We found that the Pichia pastoris pexophagy receptor, Atg30, also interacts with Atg8. Both Atg30 and Atg32 interactions are regulated by phosphorylation close to Atg8-interaction motifs. Extending this finding to Saccharomyces cerevisiae, we confirmed phosphoregulation for the mitophagy and pexophagy receptors, Atg32 and Atg36. Each Atg30 molecule must interact with both Atg8 and Atg11 for full functionality, and these interactions occur independently and not simultaneously, but rather in random order. We present a common model for the phosphoregulation of selective autophagy receptors.

Many organisms stringently regulate the number, volume and enzymatic content of peroxisomes (and other organelles). Understanding this regulation requires knowledge of how organelles are assembled and selectively destroyed in response to metabolic cues. In the past decade, considerable progress has been achieved in the elucidation of the roles of genes involved in peroxisome biogenesis, half of which are affected in human peroxisomal disorders. The recent discovery of intermediates and genes in peroxisome turnover by selective autophagy-related processes (pexophagy) opens the door to understanding peroxisome turnover and homeostasis. In this article, we summarize advances in the characterization of genes that are necessary for the transport and delivery of selective and nonselective cargos to the lysosome or vacuole by autophagy-related processes, with emphasis on peroxisome turnover by micropexophagy.During autophagy, cargos consisting of individual proteins, bulk cytosol and/or subcellular organelles, are degraded in the lysosome (or vacuole in yeast), with ensuing recycling of the amino acid and lipid precursors. Although autophagy was thought initially to enable cells to survive nutrient starvation by degradation and recycling of dispensable cellular components, it is now recognized to be involved in a plethora of cellular responses, including the regulation of organelle number [1][2][3] Autophagy is universal to all eukaryotic cells, including yeasts, worms, insects, plants and mammals [10]. In unicellular organisms, such as yeasts, it is observed primarily under nutrient starvation conditions or during the re-adaptation of cells switched from certain environments to others. Two morphologically distinct, but mechanistically related, forms of autophagy common to uni-and multicellular eukaryotes have been described. These are macroautophagy and microautophagy (Figure 1). A third form, called chaperone-mediated autophagy, has been found only in mammalian systems and is described elsewhere [9]. Autophagy is often a degradative process but it can be either selective or nonselective with respect to cargo that is turned over. The degradation of nonselective cargo is a mechanism for recycling any excess cellular components (e.g. cytosol and/or organelles) and might be a strategy for adaptation to different environments. By contrast, cells resort to selective autophagic turnover of nonfunctional or damaged organelles, or protein aggregates, that might be too large for other proteolysis machinery, such as the proteasome.Unlike the autophagic pathways, the related cytosol-tovacuole transport (Cvt) pathway, described to date only in Saccharomyces cerevisiae, delivers a limited set of specific, Morphological steps in the cytosol-to-vacuole transport (Cvt), macroautophagy and microautophagy pathways in yeast. In microautophagy, organelles and/or cytosolic proteins are engulfed by the vacuolar membrane in a Pac-Manelike fashion and degraded in the vacuolar lumen. Macroautophagy involves the formation of large (300-900 nm)...

a b s t r a c tPexophagy is a selective autophagy process wherein damaged and/or superfluous peroxisomes undergo vacuolar degradation. In methylotropic yeasts, where pexophagy has been studied most extensively, this process occurs by either micro-or macropexophagy: processes analogous to microand macroautophagy. Recent studies have identified specific factors and illustrated mechanisms involved in pexophagy. Although mechanistically pexophagy relies heavily on the core autophagic machinery, the latest findings about the role of auxiliary pexophagy factors have highlighted specialized membrane structures required for micropexophagy, and shown how cargo selectivity is achieved and how cargo size dictates the requirement for these factors during pexophagy. These insights and additional observations in the literature provide a framework for an understanding of the physiological role(s) of pexophagy.

Peroxisomes are conserved organelles of eukaryotic cells with important roles in cellular metabolism, human health, redox homeostasis, as well as intracellular metabolite transfer and signaling. We review here the current status of the different co‐existing modes of biogenesis of peroxisomal membrane proteins demonstrating the fascinating adaptability in their targeting and sorting pathways. While earlier studies focused on peroxisomes as autonomous organelles, the necessity of the ER and potentially even mitochondria as sources of peroxisomal membrane proteins and lipids has come to light in recent years. Additionally, the intimate physical juxtaposition of peroxisomes with other organelles has transitioned from being viewed as random encounters to a growing appreciation of the expanding roles of such inter‐organellar membrane contact sites in metabolic and regulatory functions. Peroxisomal quality control mechanisms have also come of age with a variety of mechanisms operating both during biogenesis and in the cellular response to environmental cues.

RNA editing in higher plant mitochondria modifies mRNA sequences by means of C-to-U conversions at highly specific sites. To determine the cis elements involved in recognition of an editing site in plant mitochondria, deletion and site-directed mutation constructs containing the cognate cox II mitochondrial gene were introduced into purified mitochondria by electroporation. The RNA editing status was analyzed for precursor and spliced transcripts from the test construct. We found that only a restricted number of nucleotides in the vicinity of the target C residue were necessary for recognition by the editing machinery and that the nearest neighbor 3 residues were crucial for the editing process. We provide evidence that two functionally distinguishable sequences can be defined: the 16-nucleotide 5 region, which can be replaced with the same region from another editing site, and a 6-nucleotide 3 region specific to the editing site. The latter region may play a role in positioning the actual editing residue.RNA editing refers to a process whereby the genetic message is changed at single nucleotides in a very specific manner. This process involves a variety of genetic systems and occurs by different mechanisms (reference 6 and references therein). In trypanosome kinetoplasts, RNA editing proceeds by insertion and deletion of uridine nucleotides in mRNAs (2); the insertion of C residues has been described for Physarum polycephalum mitochondria, and the insertion of some G residues occurs in paramyxovirus (25,36). Another type of RNA editing is base conversion, occurring in mammalian nuclei (31) and plant organelles. C-to-U conversions have been described for higher plant mitochondria (9, 13, 16) and to a lesser extent for chloroplasts (18,21).RNA editing is a posttranscriptional event in plant organelles. It is essential in plant mitochondrion gene expression processes such as the maturation step of organellar transcripts (26,27) or the synthesis of functional proteins, since the nucleotide conversions usually alter the coding properties of the mRNA (1). The editing systems in higher plant organelles, mitochondria, and chloroplasts share many similar features, but promiscuous chloroplast sequences are not edited in mitochondria (39); conversely, a mitochondrial sequence carrying an editing site does not sustain editing when transcribed into chloroplasts (35). These results indicate that editing recognition signals are specific to each organelle. The sequences flanking target C residues lack any apparent conserved consensus elements at the primary or secondary structure level. An essential problem is to define the signals that determine the specific recognition of every editing site.A number of in vivo studies of transgenic chloroplasts have demonstrated that mRNA sequences flanking the editing site are involved in RNA editing (4, 5). RNA editing has been determined to proceed by deamination of the C residue in wheat (3) and pea (38). However, the molecular determinants for editing-site recognition have not yet been ...