The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy.

In mammals, autophagosome formation, a central event in autophagy, is initiated by the ULK complex comprising ULK1/2, FIP200, ATG13, and ATG101. However, the structural basis and mechanism underlying the ULK complex assembly have yet to be fully clarified. Here, we predicted the core interactions organizing the ULK complex using AlphaFold, which proposed that the intrinsically disordered region of ATG13 engages the bases of the two UBL domains in the FIP200 dimer via two phenylalanines and also binds the tandem microtubule-interacting and transport domain of ULK1, thereby yielding the 1:1:2 stoichiometry of the ULK1-ATG13-FIP200 complex.

Structural Insights into Autophagy in the AlphaFold Era.

Autophagy, a lysosomal intracellular degradation system, is characterized by the de novo biogenesis of autophagosomes. This biogenesis is mediated by approximately 20 core Atg proteins, exhibiting a high degree of conservation from yeast to humans. Over the preceding two decades, the structural biology of autophagy has been investigated predominantly through X-ray crystallography, NMR spectroscopy, and, more recently, cryo-electron microscopy, collectively contributing to the elucidation of the structural basis of core Atg proteins.

The autophagy protein ATG-9 regulates lysosome function and integrity.

The transmembrane autophagy protein ATG9 has multiple functions essential for autophagosome formation. Here, we uncovered a novel function of ATG-9 in regulating lysosome biogenesis and integrity in Caenorhabditis elegans. Through a genetic screen, we identified that mutations attenuating the lipid scrambling activity of ATG-9 suppress the autophagy defect in epg-5 mutants, in which non-degradative autolysosomes accumulate.

Mechanisms of autophagosome formation.

The formation of autophagosomes is a pivotal step in autophagy, a lysosomal degradation system that plays a crucial role in maintaining cellular homeostasis. After autophagy induction, phase separation of the autophagy-related (Atg) 1 complex occurs, facilitating the gathering of Atg proteins and organizes the autophagosome formation site, where the initial isolation membrane (IM)/phagophore is generated. The IM then expands after receiving phospholipids from endomembranes such as the endoplasmic reticulum.

A role for condensin-mediator interaction in mitotic chromosomal organization.

Eukaryotic genomes are organized by condensin into 3D chromosomal architectures suitable for chromosomal segregation during mitosis. However, molecular mechanisms underlying the condensin-mediated chromosomal organization remain largely unclear. Here, we investigate the role of newly identified interaction between the Cnd1 condensin and Pmc4 mediator subunits in fission yeast, Schizosaccharomyces pombe.

Atg8 family proteins, LIR/AIM motifs and other interaction modes.

The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, and to humans.

The UFM1 system: Working principles, cellular functions, and pathophysiology.

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.

Complete set of the Atg8-E1-E2-E3 conjugation machinery forms an interaction web that mediates membrane shaping.

Atg8, a ubiquitin-like protein, is conjugated with phosphatidylethanolamine (PE) via Atg7 (E1), Atg3 (E2) and Atg12-Atg5-Atg16 (E3) enzymatic cascade and mediates autophagy. However, its molecular roles in autophagosome formation are still unclear. Here we show that Saccharomyces cerevisiae Atg8-PE and E1-E2-E3 enzymes together construct a stable, mobile membrane scaffold.

Immobilization of lipid nanorods onto two-dimensional crystals of protein tamavidin 2 for high-speed atomic force microscopy.

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.

Mechanisms of mitochondrial reorganization.

The cytoplasm of eukaryotes is dynamically zoned by membrane-bound and membraneless organelles. Cytoplasmic zoning allows various biochemical reactions to take place at the right time and place. Mitochondrion is a membrane-bound organelle that provides a zone for intracellular energy production and metabolism of lipids and iron.

Structural biology of the Atg8 and Atg12 conjugation systems.

Atg8 and Atg12 are ubiquitin-like proteins, conjugated to phosphatidylethanolamine (PE) and Atg5, respectively, through enzymatic reactions similar to ubiquitylation. The resultant Atg8-PE and Atg12-Atg5 conjugates play crucial roles in autophagy. Structural studies have been extensively performed on all Atg proteins (Atg3, Atg4, Atg5, Atg7, Atg8, Atg10, Atg12, Atg16) involved in these conjugation systems.

Structural view on autophagosome formation.

Autophagy is a conserved intracellular degradation system in eukaryotes, involving the sequestration of degradation targets into autophagosomes, which are subsequently delivered to lysosomes (or vacuoles in yeasts and plants) for degradation. In budding yeast, starvation-induced autophagosome formation relies on approximately 20 core Atg proteins, grouped into six functional categories: the Atg1/ULK complex, the phosphatidylinositol-3 kinase complex, the Atg9 transmembrane protein, the Atg2-Atg18/WIPI complex, the Atg8 lipidation system, and the Atg12-Atg5 conjugation system. Additionally, selective autophagy requires cargo receptors and other factors, including a fission factor, for specific sequestration.

Protocol for real-time imaging of membrane fission by mitofissin.

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.

Mechanistic insights into the roles of the UFM1 E3 ligase complex in ufmylation and ribosome-associated protein quality control.

Ubiquitin-fold modifier 1 (UFM1) is a ubiquitin-like protein covalently conjugated with intracellular proteins through ufmylation, similar to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control (RQC) at the ER (ER-RQC), and ER-phagy. However, it remains unclear how ufmylation regulates such distinct ER-related functions.

Mitofissin: a novel mitochondrial fission protein that facilitates mitophagy.

Atg: autophagy related; IMM: inner mitochondrial membrane; IMS: intermembrane space; PAS: phagophore assembly site; SAR: selective autophagy receptor.

The Atg1 complex, Atg9, and Vac8 recruit PI3K complex I to the pre-autophagosomal structure.

In macroautophagy, cellular components are sequestered within autophagosomes and transported to lysosomes/vacuoles for degradation. Although phosphatidylinositol 3-kinase complex I (PI3KCI) plays a pivotal role in the regulation of autophagosome biogenesis, little is known about how this complex localizes to the pre-autophagosomal structure (PAS). In Saccharomyces cerevisiae, PI3KCI is composed of PI3K Vps34 and conserved subunits Vps15, Vps30, Atg14, and Atg38.

Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.

NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear.

Autophagy and cancer: Basic mechanisms and inhibitor development.

Autophagy is a lysosomal degradation system of cytoplasmic components that contributes to cellular homeostasis through the turnover of various biomolecules and organelles, often in a selective manner. Autophagy is closely related to cancer, but its roles in cancer are complicated. It works as either a promoter or suppressor, depending on the stage and type of cancer.

The UFM1 system regulates ER-phagy through the ufmylation of CYB5R3.

Protein modification by ubiquitin-like proteins (UBLs) amplifies limited genome information and regulates diverse cellular processes, including translation, autophagy and antiviral pathways. Ubiquitin-fold modifier 1 (UFM1) is a UBL covalently conjugated with intracellular proteins through ufmylation, a reaction analogous to ubiquitylation. Ufmylation is involved in processes such as endoplasmic reticulum (ER)-associated protein degradation, ribosome-associated protein quality control at the ER and ER-phagy.

Development of new tools to study membrane-anchored mammalian Atg8 proteins.

A:C autophagic membrane:cytosol; ALS amyotrophic lateral sclerosis; ATG4 autophagy related 4; Atg8 autophagy related 8; BafA1 bafilomycin A; BNIP3L/Nix BCL2 interacting protein 3 like; CALCOCO2/NDP52 calcium binding and coiled-coil domain 2; EBSS Earle's balanced salt solution; GABARAP GABA type A receptor-associated protein; GST glutathione S transferase; HKO hexa knockout; K dissociation constant; LIR LC3-interacting region; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; NLS nuclear localization signal/sequence; PE phosphatidylethanolamine; SpHfl1 organic solute transmembrane transporter; SQSTM1/p62 SQSTM1/p62; TARDBP/TDP-43 TAR DNA binding protein; TKO triple knockout.

Lipid Transport from Endoplasmic Reticulum to Autophagic Membranes.

Autophagy is an intracellular degradation system involving de novo generation of autophagosomes, which function as a transporting vesicle of cytoplasmic components to lysosomes for degradation. Isolation membranes (IMs) or phagophores, the precursor membranes of autophagosomes, require millions of phospholipids to expand and transform into autophagosomes, with the endoplasmic reticulum (ER) being the primary lipid source. Recent advances in structural and biochemical studies of autophagy-related proteins have revealed their lipid transport activities: Atg2 at the interface between IM and ER possesses intermembrane lipid transfer activities, while Atg9 at IM and VMP1 and TMEM41B at ER possess lipid scrambling activities.

Qualitative differences in disease-associated MEK mutants reveal molecular signatures and aberrant signaling-crosstalk in cancer.

Point-mutations of MEK1, a central component of ERK signaling, are present in cancer and RASopathies, but their precise biological effects remain obscure. Here, we report a mutant MEK1 structure that uncovers the mechanisms underlying abnormal activities of cancer- and RASopathy-associated MEK1 mutants. These two classes of MEK1 mutations differentially impact on spatiotemporal dynamics of ERK signaling, cellular transcriptional programs, gene expression profiles, and consequent biological outcomes.