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In autophagy, a membrane cisterna called the isolation membrane expands, bends, becomes spherical, and closes to sequester cytoplasmic constituents into the resulting double-membrane vesicle autophagosome for lysosomal/vacuolar degradation. Here, we discover a mechanism that allows the isolation membrane to expand with a large opening to ensure non-selective cytoplasm sequestration within the autophagosome. A sorting nexin complex that localizes to the opening edge of the isolation membrane plays a critical role in this process. Without the complex, the isolation membrane expands with a small opening that prevents the entry of particles larger than about 25 nm, including ribosomes and proteasomes, although autophagosomes of nearly normal size eventually form. This study sheds light on membrane morphogenesis during autophagosome formation and selectivity in autophagic degradation., (© 2023. Springer Nature Limited.)

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.

We generated a new transgenic mouse model that expresses a pHluorin-mKate2 fluorescent protein fused with human LC3B (PK-LC3 mice) for monitoring autophagy activity in neurons of the central nervous system. Histological analysis revealed fluorescent puncta in neurons of the cerebral cortex, hippocampus, cerebellar Purkinje cells, and anterior spinal regions. Using CLEM analysis, we confirmed that PK-LC3-positive puncta in the perikarya of Purkinje cells correspond to autophagic structures. To validate the usability of PK-LC3 mice, we quantified PK-LC3 puncta in Purkinje cells of mice kept in normal feeding conditions and of mice starved for 24 hours. Our results showed a significant increase in autophagosome number and in individual puncta areal size following starvation. To confirm these results, we used morphometry at the electron microscopic level to analyze the volume densities of autophagosomes and lysosomes/autolysosomes in Purkinje cells of PK-LC3 mice. The results revealed that the volume densities of autophagic structures increase significantly after starvation. Together, our data show that PK-LC3 mice are suitable for monitoring autophagy flux in Purkinje cells of the cerebellum, and potentially other areas in the central nervous system.

In innate immunity, multiple autophagic processes eliminate intracellular pathogens, but it remains unclear whether noncanonical autophagy and xenophagy are coordinated, and whether they occur concomitantly or sequentially. Here, we show that Streptococcus pneumoniae, a causative of invasive pneumococcal disease, can trigger FIP200-, PI3P-, and ROS-independent pneumococcus-containing LC3-associated phagosome (LAPosome)-like vacuoles (PcLVs) in an early stage of infection, and that PcLVs are indispensable for subsequent formation of bactericidal pneumococcus-containing autophagic vacuoles (PcAVs). Specifically, we identified LC3- and NDP52-delocalized PcLV, which are intermediates between PcLV and PcAV. Atg14L, Beclin1, and FIP200 were responsible for delocalizing LC3 and NDP52 from PcLVs. Thus, multiple noncanonical and canonical autophagic processes are deployed sequentially against intracellular S. pneumoniae. The Atg16L1 WD domain, p62, NDP52, and poly-Ub contributed to PcLV formation. These findings reveal a previously unidentified hierarchical autophagy mechanism during bactericidal xenophagy against intracellular bacterial pathogens, and should improve our ability to control life-threating pneumococcal diseases.

Renal proximal tubular epithelial cells are significantly damaged during acute kidney injury. Renal proximal tubular cell-specific autophagy-deficient mice show increased sensitivity against renal injury, while showing few pathological defects under normal fed conditions. Considering that autophagy protects the proximal tubular cells from acute renal injury, it is reasonable to assume that autophagy contributes to the maintenance of renal tubular cells under normal fed conditions. To clarify this possibility, we generated a knock out mouse model which lacks Atg7, a key autophagosome forming enzyme, in renal proximal tubular cells ( Atg7 flox/flox ;KAP-Cre + ). Analysis of renal tissue from two months old Atg7 flox/flox ;KAP-Cre + mouse revealed an accumulation of LC3, binding protein p62/sequestosome 1 (a selective substrate for autophagy), and more interestingly, Kim-1, a biomarker for early kidney injury, in the renal proximal tubular cells under normal fed conditions. TUNEL (TdT-mediated dUTP Nick End Labeling)-positive cells were also detected in the autophagy-deficient renal tubular cells. Analysis of renal tissue from Atg7 flox/flox ;KAP-Cre + mice at different age points showed that tubular cells positive for p62 and Kim-1 continually increase in number in an age-dependent manner. Ultrastructural analysis of tubular cells from Atg7 flox/flox ;KAP-Cre + revealed the presence of intracellular inclusions and abnormal structures. These results indicated that autophagy-deficiency in the renal proximal epithelial tubular cells leads to an increase in injured cells in the kidney even under normal fed conditions.

Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in the autophagy field but also in other fields., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Phosphatidylinositol 3-kinase is indispensable for autophagy but it is not well understood how its product, phosphatidylinositol 3-phosphate (PtdIns(3)P), participates in the biogenesis of autophagic membranes. Here, by using quick-freezing and freeze-fracture replica labelling, which enables determination of the nanoscale distributions of membrane lipids, we show that PtdIns(3)P in yeast autophagosomes is more abundant in the luminal leaflet (the leaflet facing the closed space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. This distribution is drastically different from that of the mammalian autophagosome in which PtdIns(3)P is confined to the cytoplasmic leaflet. In mutant yeast lacking two cytoplasmic phosphatases, ymr1Δ and sjl3Δ, PtdIns(3)P in the autophagosome is equally abundant in the two membrane leaflets, suggesting that the PtdIns(3)P asymmetry in wild-type yeast is generated by unilateral hydrolysis. The observed differences in PtdIns(3)P distribution suggest that autophagy in yeast and mammals may involve substantially different processes.

Atg9 is a transmembrane protein that is essential for autophagy. In the budding yeast Saccharomyces cerevisiae, it has recently been revealed that Atg9 exists on cytoplasmic small vesicles termed Atg9 vesicles. To identify the components of Atg9 vesicles, we purified the Atg9 vesicles and subjected them to mass spectrometry. We found that their protein composition was distinct from other organellar membranes and that Atg9 and Atg27 in particular are major components of Atg9 vesicles. In addition to these two components, Trs85, a specific subunit of the transport protein particle III (TRAPPIII) complex, and the Rab GTPase Ypt1 were also identified. Trs85 directly interacts with Atg9, and the Trs85-containing TRAPPIII complex facilitates the association of Ypt1 onto Atg9 vesicles. We also showed that Trs85 and Ypt1 are localized to the preautophagosomal structure in an Atg9-dependent manner. Our data suggest that Atg9 vesicles recruit the TRAPPIII complex and Ypt1 to the preautophagosomal structure. The vesicle-tethering machinery consequently acts in the process of autophagosome formation.

In autophagy, a cup-shaped membrane called the isolation membrane is formed, expanded, and sealed to complete a double membrane-bound vesicle called the autophagosome that encapsulates cellular constituents to be transported to and degraded in the lysosome/vacuole. The formation of the autophagosome requires autophagy-related (Atg) proteins. Atg8 is a ubiquitin-like protein that localizes to the isolation membrane; a subpopulation of this protein remains inside the autophagosome and is transported to the lysosome/vacuole. In the budding yeast Saccharomyces cerevisiae, Atg1 is a serine/threonine kinase that functions in the initial step of autophagosome formation and is also efficiently transported to the vacuole via autophagy. Here, we explore the mechanism and significance of this autophagic transport of Atg1. In selective types of autophagy, receptor proteins recognize degradation targets and also interact with Atg8, via the Atg8 family interacting motif (AIM), to link the targets to the isolation membrane. We find that Atg1 contains an AIM and directly interacts with Atg8. Mutations in the AIM disrupt this interaction and abolish vacuolar transport of Atg1. These results suggest that Atg1 associates with the isolation membrane by binding to Atg8, resulting in its incorporation into the autophagosome. We also show that mutations in the Atg1 AIM cause a significant defect in autophagy, without affecting the functions of Atg1 implicated in triggering autophagosome formation. We propose that in addition to its essential function in the initial stage, Atg1 also associates with the isolation membrane to promote its maturation into the autophagosome.

Selective autophagy of bacterial pathogens represents a host innate immune mechanism. Selective autophagy has been characterized on the basis of distinct cargo receptors but the mechanisms by which different cargo receptors are targeted for autophagic degradation remain unclear. In this study we identified a highly conserved Tectonin domain-containing protein, Tecpr1, as an Atg5 binding partner that colocalized with Atg5 at Shigella-containing phagophores. Tecpr1 activity is necessary for efficient autophagic targeting of bacteria, but has no effect on rapamycin- or starvation-induced canonical autophagy. Tecpr1 interacts with WIPI-2, a yeast Atg18 homolog and PI(3)P-interacting protein required for phagophore formation, and they colocalize to phagophores. Although Tecpr1-deficient mice appear normal, Tecpr1-deficient MEFs were defective for selective autophagy and supported increased intracellular multiplication of Shigella. Further, depolarized mitochondria and misfolded protein aggregates accumulated in the Tecpr1-knockout MEFs. Thus, we identify a Tecpr1-dependent pathway as important in targeting bacterial pathogens for selective autophagy., (Copyright © 2011 Elsevier Inc. All rights reserved.)