Macromusophagy: A solo piano musical representation of macroautophagy

Macroautophagy is a complex process involving dynamic membrane rearrangements in which parts of the cytoplasm are sequestered within double-membrane phagophores. Upon completion, these structures mature into autophagosomes that fuse with the yeast vacuole or mammalian lysosome, leading to degradation of the cargo and release of the resulting macromolecules back into the cytosol. How can the complexities of macroautophagy best be conveyed to an audience that is composed primarily of people who are not experts in this topic, and possibly not even scientists? The literature on learning is vast, and difficult to summarize, but there are certain themes that frequently appear. First, people learn in different ways. Thus, for example, while lectures are effective for conveying information to part of the audience, some will benefit tremendously from alternative methods of presentation. The latter can be visual (taking the form of illustrations, videos, or even physical movement), tactile or audible. Second, a line of research suggests that the engagement of more than one part of the brain (dual channels) improves learning. We decided to explore these concepts focusing on an audible format through a collaborative approach by combining a scientific explanation of macroautophagy with a musical score that was composed specifically to represent this process.     

The proteasome subunit RPN10 functions as a specific receptor for degradation of the 26S proteasome by macroautophagy in Arabidopsis

The ubiquitin-proteasome system (UPS) and macroautophagy/autophagy are 2 main degradative routes, which are important for cellular homeostasis. In a study conducted by Marshall et al., the authors demonstrated that the UPS and autophagy converge in Arabidopsis (see the punctum in issue #11–10). In particular, they found that the 26S proteasome is degraded by autophagy, either nonselectively (induced by nitrogen starvation) or selectively (induced by proteasome inhibition). The selective phenotype is mediated through the proteasome subunit RPN10, which can bind both ubiquitin and ATG8. This newly identified autophagic degradation of the proteasome is termed “proteaphagy,” and the process reveals an interesting relationship between these degradative systems.     

Guidelines for the use and interpretation of assays for monitoring autophagy

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.     

Guidelines for the use and interpretation of assays for monitoring autophagy

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 in plants

Autophagy is a highly conserved processing mechanism in eukaryotes whereby cytoplasmic components are engulfed in double-membrane vesicles called autophagosomes and are delivered into organelles such as lysosomes (mammal) or vacuoles (yeast/plant) for degradation and recycling of the resulting molecules. Isolation of yeastAUTOPHAGY (ATG) genes has facilitated the identification of correspondingArabidopsis ATG genes based on sequence similarity. Genetic and molecular analyses using knockout and/or knockdown mutants of those genes have unraveled the biological functions of autophagy during plant development, nutrient recycling, and environmental stress responses. Additional roles for autophagy have been suggested in the degradation of oxidized proteins during oxidative stress and the regulation of hypersensitive response (HR)-programmed cell death (PCD) during innate immunity. Our review summarizes knowledge about the structure and function of autophagic pathways andATG components, and the biological roles of autophagy in plants.     

The late stage of autophagy: cellular events and molecular regulation

Autophagy is an intracellular degradation system that delivers cytoplasmic contents to the lysosome for degradation. It is a “self-eating” process and plays a “house-cleaner” role in cells. The complex process consists of several sequential steps—induction, autophagosome formation, fusion of lysosome and autophagosome, degradation, efflux transportation of degradation products, and autophagic lysosome reformation. In this review, the cellular and molecular regulations of late stage of autophagy, including cellular events after fusion step, are summarized.     

Macromusophagy

The authors recently reported a novel role for autophagy in late-stage quality control of a secreted protein, apolipoprotein-B₁₀₀ (apoB). Hepatocytes assemble this protein with triglycerides, cholesterol, and other lipids into macromolecular complexes called lipoproteins. In what appears to be a normal response to diets rich in polyunsaturated fatty acids, which are readily peroxidized, apoB comes into contact with lipid peroxides in or after the Golgi apparatus. The protein becomes oxidatively damaged, aggregates, and is diverted out of the secretory pathway by autophagosomes, which deliver it to lysosomes for destruction. ApoB secretory control via autophagosomes is likely a key component of normal and pathological regulation of plasma lipoprotein levels, as well as a means for remarkably late-stage quality control of a secreted protein.

Addendum to: Pan M, Maitin V, Parathath S, et al. Pre-secretory oxidation, aggregation, and autophagosomal destruction of apolipoprotein-B: A pathway for late-stage quality control. Proc Natl Acad Sci USA 2008; 105:5862-7.     

The Atg17-Atg31-Atg29 complex and Atg11 regulate autophagosome-vacuole fusion

The macroautophagy (hereafter autophagy) process involves de novo formation of double-membrane autophagosomes; after sequestering cytoplasm these transient organelles fuse with the vacuole/lysosome. Genetic studies in yeasts have characterized more than 40 autophagy-related (Atg) proteins required for autophagy, and the majority of these proteins play roles in autophagosome formation. The fusion of autophagosomes with the vacuole is mediated by the Rab GTPase Ypt7, its guanine nucleotide exchange factor Mon1-Ccz1, and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, these factors are not autophagosome-vacuole fusion specific. We recently showed that 2 autophagy scaffold proteins, the Atg17-Atg31-Atg29 complex and Atg11, regulate autophagosome-vacuole fusion by recruiting the vacuolar SNARE Vam7 to the phagophore assembly site (PAS), where an autophagosome forms in yeast.     

Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole

Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.     

The vacuole vs. the lysosome

The morphometric examination of autophagic bodies provides useful information about the mechanism and magnitude of macroautophagy, and yeast researchers frequently utilize various measurements of these structures as part of their quantification of the process. The utility of this approach, however, has led to the common misconception that autophagic bodies can be found in the mammalian lysosome, which is generally not correct.     

Editorial: The vacuole vs. the lysosome: When size matters

The morphometric examination of autophagic bodies provides useful information about the mechanism and magnitude of macroautophagy, and yeast researchers frequently utilize various measurements of these structures as part of their quantification of the process. The utility of this approach, however, has led to the common misconception that autophagic bodies can be found in the mammalian lysosome, which is generally not correct.     

Downregulation of autophagy through CUL3-KLHL20-mediated turnover of the ULK1 and PIK3C3/VPS34 complexes

The molecular mechanism of macroautophagy/autophagy induction has been intensively studied, but little is known about downregulation of autophagy and how this process is restricted. In particular, how is autophagy maintained at an appropriate homeostatic level when cells are subjected to prolonged stress? In this study (see the related punctum in Autophagy 12–5), Liu et al. report a function of the CUL3-KLHL20 ubiquitin ligase in feedback regulation, leading to the downregulation of autophagy through the degradation of the ULK1 and PIK3C3/VPS34 complexes.