Search

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through UFMylation, a process similar to ubiquitylation. Growing lines of evidence regarding not only the structural basis of the components essential for UFMylation but also their biological properties shed light on crucial roles of the UFM1 system in the endoplasmic reticulum (ER), such as ER-phagy and ribosome-associated quality control at the ER, although there are some functions unrelated to the ER. Mouse genetics studies also revealed the indispensable roles of this system in hematopoiesis, liver development, neurogenesis, and chondrogenesis. Of critical importance, mutations of genes encoding core components of the UFM1 system in humans cause hereditary developmental epileptic encephalopathy and Schohat-type osteochondrodysplasia of the epiphysis. Here, we provide a multidisciplinary review of our current understanding of the mechanisms and cellular functions of the UFM1 system as well as its pathophysiological roles, and discuss issues that require resolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Yeast mitofissin Atg44 is a mitochondrial intermembrane space protein that causes membrane fission required for mitophagy. Here, we present a protocol for observing Atg44-mediated membrane fission. We describe steps for recombinant Atg44 purification, lipid nanotube preparation as model membranes, and Atg44-mediated membrane fission real-time observation. We then detail procedures for tube radius estimation using confocal microscopy. This protocol can also be adapted to the study of membrane fission by other proteins. For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

High-speed atomic force microscopy is a technique that allows real-time observation of biomolecules and biological phenomena reconstituted on a substrate. Here, we present a protocol for immobilizing lipid nanorods onto two-dimensional crystals of biotin-binding protein tamavidin 2. We describe steps for the preparation of tamavidin 2 protein, lipid nanorods, and two-dimensional crystals of tamavidin 2 formed on mica. Immobilized lipid nanorods are one of the useful tools for observation of specific proteins in action. For complete details on the use and execution of this protocol, please refer to Fukuda et al. (2023). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

In addition to membranous organelles, autophagy selectively degrades biomolecular condensates, in particular p62/SQSTM1 bodies, to prevent diseases including cancer. Evidence is growing regarding the mechanisms by which autophagy degrades p62 bodies, but little is known about their constituents. Here, we established a fluorescence-activated-particle-sorting-based purification method for p62 bodies using human cell lines and determined their constituents by mass spectrometry. Combined with mass spectrometry of selective-autophagy-defective mouse tissues, we identified vault, a large supramolecular complex, as a cargo within p62 bodies. Mechanistically, major vault protein directly interacts with NBR1, a p62-interacting protein, to recruit vault into p62 bodies for efficient degradation. This process, named vault-phagy, regulates homeostatic vault levels in vivo, and its impairment may be associated with non-alcoholic-steatohepatitis-derived hepatocellular carcinoma. Our study provides an approach to identifying phase-separation-mediated selective autophagy cargoes, expanding our understanding of the role of phase separation in proteostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Clearance of biomolecular condensates by selective autophagy is thought to play a crucial role in cellular homeostasis. However, the mechanism underlying selective autophagy of condensates and whether liquidity determines a condensate's susceptibility to degradation by autophagy remain unknown. Here, we show that the selective autophagic cargo aminopeptidase I (Ape1) undergoes phase separation to form semi-liquid droplets. The Ape1-specific receptor protein Atg19 localizes to the surface of Ape1 droplets both in vitro and in vivo, with the "floatability" of Atg19 preventing its penetration into droplets. In vitro reconstitution experiments reveal that Atg19 and lipidated Atg8 are necessary and sufficient for selective sequestration of Ape1 droplets by membranes. This sequestration is impaired by mutational solidification of Ape1 droplets or diminished ability of Atg19 to float. Taken together, we propose that cargo liquidity and the presence of sufficient amounts of autophagic receptor on cargo are crucial for selective autophagy of biomolecular condensates., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Selective autophagy mediates the degradation of various cargoes, including protein aggregates and organelles, thereby contributing to cellular homeostasis. Cargo receptors ensure selectivity by tethering specific cargo to lipidated Atg8 at the isolation membrane. However, little is known about the structural requirements underlying receptor-mediated cargo recognition. Here, we report structural, biochemical, and cell biological analysis of the major selective cargo protein in budding yeast, aminopeptidase I (Ape1), and its complex with the receptor Atg19. The Ape1 propeptide has a trimeric coiled-coil structure, which tethers dodecameric Ape1 bodies together to form large aggregates. Atg19 disassembles the propeptide trimer and forms a 2:1 heterotrimer, which not only blankets the Ape1 aggregates but also regulates their size. These receptor activities may promote elongation of the isolation membrane along the aggregate surface, enabling sequestration of the cargo with high specificity., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Multicellular organisms have multiple homologs of the yeast ATG8 gene, but the differential roles of these homologs in autophagy during development remain largely unknown. Here we investigated structure/function relationships in the two C. elegans Atg8 homologs, LGG-1 and LGG-2. lgg-1 is essential for degradation of protein aggregates, while lgg-2 has cargo-specific and developmental-stage-specific roles in aggregate degradation. Crystallography revealed that the N-terminal tails of LGG-1 and LGG-2 adopt the closed and open form, respectively. LGG-1 and LGG-2 interact differentially with autophagy substrates and Atg proteins, many of which carry a LIR motif. LGG-1 and LGG-2 have structurally distinct substrate binding pockets that prefer different residues in the interacting LIR motif, thus influencing binding specificity. Lipidated LGG-1 and LGG-2 possess distinct membrane tethering and fusion activities, which may result from the N-terminal differences. Our study reveals the differential function of two ATG8 homologs in autophagy during C. elegans development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The Helicobacter pylori type IV secretion effector CagA is a major bacterial virulence determinant and critical for gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA localizes to the inner face of the plasma membrane, where it acts as a pathogenic scaffold/hub that promiscuously recruits host proteins to potentiate oncogenic signaling. We find that CagA comprises a structured N-terminal region and an intrinsically disordered C-terminal region that directs versatile protein interactions. X-ray crystallographic analysis of the N-terminal CagA fragment (residues 1-876) revealed that the region has a structure comprised of three discrete domains. Domain I constitutes a mobile CagA N terminus, while Domain II tethers CagA to the plasma membrane by interacting with membrane phosphatidylserine. Domain III interacts intramolecularly with the intrinsically disordered C-terminal region, and this interaction potentiates the pathogenic scaffold/hub function of CagA. The present work provides a tertiary-structural basis for the pathophysiological/oncogenic action of H. pylori CagA., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The Atg12-Atg5 conjugate, which is formed by an ubiquitin-like conjugation system, is essential to autophagosome formation, a central event in autophagy. Despite its importance, the molecular mechanism of the Atg12-Atg5 conjugate formation has not been elucidated. Here, we report the solution and crystal structures of Atg10 and Atg5 homologs from Kluyveromyces marxianus (Km), a thermotolerant yeast. KmAtg10 comprises an E2-core fold with characteristic accessories, including two β strands, whereas KmAtg5 has two ubiquitin-like domains and a helical domain. The nuclear magnetic resonance experiments, mutational analyses, and crosslinking experiments showed that KmAtg10 directly recognizes KmAtg5, especially its C-terminal ubiquitin-like domain, by its characteristic two β strands. Kinetic analysis suggests that Tyr56 and Asn114 of KmAtg10 may place the side chain of KmAtg5 Lys145 into the optimal orientation for its conjugation reaction with Atg12. These structural features enable Atg10 to mediate the formation of the Atg12-Atg5 conjugate without a specific E3 enzyme., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer., (Copyright © 2011 Elsevier Inc. All rights reserved.)