Vacuolar protein sorting-associated protein 4 (VPS4), which occurs in A and B isoforms in humans, is the only enzyme in the core endosomal sorting complex required for transport (ESCRT) machinery. Human VPS4 is considered a potential therapeutic target for activation in neurodegeneration, and for inhibition in cancer and HIV-1 infection. VPS4 assembles transiently into a hexamer, which then removes ESCRT-III subunits from polymeric assemblies by unfolding them and threading them through its central pore. The N-terminal MIT domain of VPS4 binds to C-terminal MIM motifs of ESCRT-III. Here, we determined the cryo-electron microscopy structure of the full-length human VPS4B hexamer in six-membered helical “spiral staircase” states. In one of these states, two of the six MIT domains are ordered and stabilize the hexamer by bridging the seam of the spiral staircase. Residues involved in seam-bridging contacts were found to be important for biochemical and cellular activity. The structures also revealed two modes for polypeptide occupancy of the central pore, one of which involves the MIT-AAA linker peptide. These observations suggest a mechanism for substrate-dependent hexamerization and priming of VPS4 for its ESCRT-III remodeling activity.

Selective autophagy is a lysosomal degradation pathway that is critical for maintaining cellular homeostasis by disposing of harmful cellular material. Although the mechanisms by which soluble cargo receptors recruit the autophagy machinery are becoming increasingly clear, the principles governing how organelle-localized transmembrane cargo receptors initiate selective autophagy remain poorly understood. Here we demonstrate that the human transmembrane cargo receptors can initiate autophagosome biogenesis not only by recruiting the upstream FIP200/ULK1 complex but also via a WIPI-ATG13 complex. This latter pathway is employed by the BNIP3/NIX receptors to trigger mitophagy. Additionally, other transmembrane mitophagy receptors, including FUNDC1 and BCL2L13, exclusively use the FIP200/ULK1 complex, whereas FKBP8 and the ER-phagy receptor TEX264 are capable of utilizing both pathways to initiate autophagy. Our study defines the molecular rules for initiation by transmembrane cargo receptors, revealing remarkable flexibility in the assembly and activation of the autophagy machinery, with important implications for therapeutic interventions.

Defective mitochondrial quality control in response to loss of mitochondrial membrane polarization is implicated in Parkinson's disease by mutations in PINK1 and PRKN. Parkin-expressing U2 osteosarcoma (U2OS) cells were treated with the depolarizing agents oligomycin and antimycin A (OA) and subjected to cryo-focused ion beam milling and in situ cryo-electron tomography. Mitochondria were fragmented and devoid of matrix calcium phosphate crystals. Phagophores were visualized, with bridge-like lipid transporter densities connected to mitophagic phagophores. A subpopulation of ATP synthases relocalized from cristae to the inner boundary membrane. The structure of the dome-shaped prohibitin complex, a dodecamer of PHB1-PHB2 dimers, was determined in situ by subtomogram averaging in untreated and treated cells and found to exist in open and closed conformations, with the closed conformation being enriched by OA treatment. These findings provide a set of native snapshots of the manifold nano-structural consequences of mitochondrial depolarization and provide a baseline for future in situ dissection of Parkin-dependent mitophagy.

Mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-responsive master regulator of metabolism. Amino acids control the recruitment and activation of mTORC1 at the lysosome through the nucleotide loading state of the heterodimeric Rag GTPases. Under low nutrients, including arginine, the GTPase-activating protein complex GATOR1 promotes GTP hydrolysis on RagA/B, inactivating mTORC1. GATOR1 is regulated by the cage-like GATOR2 complex and cytosolic amino acid sensors. To understand how the arginine sensor CASTOR1 binds to GATOR2 to disinhibit GATOR1 under low cytosolic arginine, we determined the cryo-electron microscopy structure of human GATOR2 bound to CASTOR1 in the absence of arginine. Two MIOS WD40 domain β-propellers of the GATOR2 cage engage with both subunits of a single CASTOR1 homodimer. Each propeller binds to a negatively charged MIOS-binding interface on CASTOR1 that is distal to the arginine pocket. The structure shows how arginine-triggered loop ordering in CASTOR1 blocks the MIOS-binding interface, switches off its binding to GATOR2 and, thus, communicates to downstream mTORC1 activation.

In a recent publication, Lange et al.[1][1] proposed an 11-spoked wheel model for the mitochondrial prohibitin complex consisting of six PHB1 and five PHB2 subunits built into low resolution density obtained from 11-fold symmetry averaging. The proposed unequal stoichiometry is inherently incompatible with symmetry. The building of an asymmetric molecular model into density obtained by 11-fold symmetry averaging is inherently self-contradictory. This contradiction alone calls the validity of the derived structure into question. Our own data revealed that the complex primarily adopts an asymmetric open conformation in situ. We show that it was the incorrect symmetry imposition on the open conformation that led to the spoked wheel density reported by Lange et al.. Further validation by re-analysis of crosslinking-mass spectrometry data, and a test of in situ template matching all support a larger prohibitin complex architecture. These findings underscore the need for great care in the imposition of symmetry cryo-ET at less than atomistic resolution. ### Competing Interest Statement J.H.H. is a cofounder of Casma Therapeutics and receives research funding from Hoffmann-La Roche. The other authors declare that they have no competing interests. This research was funded by Aligning Science Across Parkinson’s [ASAP-000350] through the Michael J. Fox Foundation for Parkinson’s Research (MJFF) (J.H.H.) and by the Alexander von Humboldt Foundation (E.H.)., ASAP-000350 [1]: #ref-1

The mechanistic target of rapamycin complex 1 (mTORC1) integrates growth factor (GF) and nutrient signals to stimulate anabolic processes connected to cell growth and inhibit catabolic processes such as autophagy1,2. GF signalling through the tuberous sclerosis complex regulates the lysosomally localized small GTPase RAS homologue enriched in brain (RHEB)3. Direct binding of RHEB-GTP to the mTOR kinase subunit of mTORC1 allosterically activates the kinase by inducing a large-scale conformational change4. Here we reconstituted mTORC1 activation on membranes by RHEB, RAGs and Ragulator. Cryo-electron microscopy showed that RAPTOR and mTOR interact directly with the membrane. Full engagement of the membrane anchors is required for optimal alignment of the catalytic residues in the mTOR kinase active site. Converging signals from GFs and nutrients drive mTORC1 recruitment to and activation on lysosomal membrane in a four-step process, consisting of (1) RAG-Ragulator-driven recruitment to within ~100 Å of the lysosomal membrane; (2) RHEB-driven recruitment to within ~40 Å; (3) RAPTOR-membrane engagement and intermediate enzyme activation; and (4) mTOR-membrane engagement and full enzyme activation. RHEB and membrane engagement combined leads to full catalytic activation and structurally explains GF and nutrient signal integration at the lysosome.

The unc-51-like kinase complex (ULK1C) and the class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) are the key regulators of macroautophagy initiation. Understanding the assembly and coordination of these two complexes is essential for deciphering their cellular regulation and targeting them for therapeutic enhancement. This review highlights recent advances in our understanding of the structural organization and activation mechanisms of ULK1C and PI3KC3-C1 at the molecular level and discusses their roles within the protein interaction network governing autophagy initiation.

The class III phosphatidylinositol-3 kinase complexes I and II (PI3KC3-C1 and PI3KC3-C2) have vital roles in macroautophagy and endosomal maturation, respectively. We elucidated a structural pathway of enzyme activation through cryo-electron microscopy analysis of PI3KC3-C1. The inactive conformation of the VPS15 pseudokinase stabilizes the inactive conformation, sequestering its N-myristate in the N-lobe of the pseudokinase. Upon activation, the myristate is liberated such that the VPS34 lipid kinase catalyzes phosphatidylinositol-3 phosphate production on membranes. The VPS15 pseudokinase domain binds tightly to guanosine triphosphate and stabilizes a web of interactions to autoinhibit the cytosolic complex and promote activation upon membrane binding. These findings show in atomistic detail how the VPS34 lipid kinase is activated in the context of a complete PI3K complex.

The endosomal sorting complex required for transport (ESCRT) is a multicomplex machinery comprising proteins that are conserved from bacteria to humans and has diverse roles in regulating the dynamics of cellular membranes. ESCRT functions have far-reaching consequences for cell biological processes such as intracellular traffic, membrane repair, cell signalling, metabolic regulation, cell division and genome maintenance. Here we review recent insights that emphasize the pathophysiological consequences of ESCRT dysfunctions, including infections, immune disorders, cancers and neurological diseases. We highlight the possibilities of using our knowledge about ESCRT structures and functions for drug discovery.

Selective autophagy is a lysosomal degradation pathway that is critical for maintaining cellular homeostasis by disposing of harmful cellular material. While the mechanisms by which soluble cargo receptors recruit the autophagy machinery are becoming increasingly clear, the principles governing how organelle-localized transmembrane cargo receptors initiate selective autophagy remain poorly understood. Here, we demonstrate that transmembrane cargo receptors can initiate autophagosome biogenesis not only by recruiting the upstream FIP200/ULK1 complex but also via a WIPI-ATG13 complex. This latter pathway is employed by the BNIP3/NIX receptors to trigger mitophagy. Additionally, other transmembrane mitophagy receptors, including FUNDC1 and BCL2L13, exclusively use the FIP200/ULK1 complex, while FKBP8 and the ER-phagy receptor TEX264 are capable of utilizing both pathways to initiate autophagy. Our study defines the molecular rules for initiation by transmembrane cargo receptors, revealing remarkable flexibility in the assembly and activation of the autophagy machinery, with significant implications for therapeutic interventions.

The prion-like spread of protein aggregates is a leading hypothesis for the propagation of neurofibrillary lesions in the brain, including the spread of tau inclusions associated with Alzheimer's disease. The mechanisms of cellular uptake of tau seeds and subsequent nucleated polymerization of cytosolic tau are major questions in the field, and the potential for coupling between the entry and nucleation mechanisms has been little explored. We found that in primary astrocytes and neurons, endocytosis of tau seeds leads to their accumulation in lysosomes. This in turn leads to lysosomal swelling, deacidification, and recruitment of ESCRT proteins, but not Galectin-3, to the lysosomal membrane. These observations are consistent with nanoscale damage of the lysosomal membrane. Live cell imaging and STORM superresolution microscopy further show that the nucleation of cytosolic tau occurs primarily at the lysosome membrane under these conditions. These data suggest that tau seeds escape from lysosomes via nanoscale damage rather than wholesale rupture and that nucleation of cytosolic tau commences as soon as tau fibril ends emerge from the lysosomal membrane.

The Unc-51-like kinase protein kinase complex (ULK1C) is the most upstream and central player in the initiation of macroautophagy in mammals. Here, we determined the cryo-electron microscopy structure of the human ULK1C core at amino-acid-level resolution. We also determined a moderate-resolution structure of the ULK1C core in complex with another autophagy core complex, the class III phosphatidylinositol 3-OH kinase complex I (PI3KC3-C1). We show that the two complexes coassemble through extensive contacts between the FIP200 scaffold subunit of ULK1C and the VPS15, ATG14 and BECN1 subunits of PI3KC3-C1. The FIP200:ATG13:ULK1 core of ULK1C undergoes a rearrangement from 2:1:1 to 2:2:2 stoichiometry in the presence of PI3KC3-C1. This suggests a structural mechanism for the initiation of autophagy through formation of a ULK1C:PI3KC3-C1 supercomplex and dimerization of ULK1 on the FIP200 scaffold.

Apoptosis linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining giant unilamellar vesicle-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

Rough endoplasmic reticulum (ER) sheets are a fundamental domain of the ER and the gateway into the secretory pathway. Although reticulon proteins stabilize high-curvature ER tubules, it is unclear whether other proteins scaffold the flat membranes of rough ER sheets. Through a proteomics screen using ER sheet-localized RNA-binding proteins as bait, we identify the sigma-1 receptor (SigmaR1) as an ER sheet- shaping factor. High-resolution live cell imaging and electron tomography assign SigmaR1 as an ER sheet-localized factor whose levels determine the amount of rough ER sheets in cells. Structure-guided mutagenesis and in vitro reconstitution on giant unilamellar vesicles further support a mechanism whereby SigmaR1 oligomers use their extended arrays of amphipathic helices to bind and flatten the lumenal leaflet of ER membranes to oppose membrane curvature and stabilize rough ER sheets.

Activation of PINK1 and Parkin in response to mitochondrial damage initiates a response that includes phosphorylation of RAB7A at Ser72. Rubicon is a RAB7A binding negative regulator of autophagy. The structure of the Rubicon:RAB7A complex suggests that phosphorylation of RAB7A at Ser72 would block Rubicon binding. Indeed, in vitro phosphorylation of RAB7A by TBK1 abrogates Rubicon:RAB7A binding. Pacer, a positive regulator of autophagy, has an RH domain with a basic triad predicted to bind an introduced phosphate. Consistent with this, Pacer-RH binds to phosho-RAB7A but not to unphosphorylated RAB7A. In cells, mitochondrial depolarization reduces Rubicon:RAB7A colocalization whilst recruiting Pacer to phospho-RAB7A–positive puncta. Pacer knockout reduces Parkin mitophagy with little effect on bulk autophagy or Parkin-independent mitophagy. Rescue of Parkin-dependent mitophagy requires the intact pRAB7A phosphate-binding basic triad of Pacer. Together these structural and functional data support a model in which the TBK1-dependent phosphorylation of RAB7A serves as a switch, promoting mitophagy by relieving Rubicon inhibition and favoring Pacer activation.

The class III phosphatidylinositol (PI) 3-kinase complexes I and II (PI3KC3-C1 and -C2) are central to the initiation of macroautophagy and endosomal maturation, respectively. Through three-dimensional classification of a large cryo-EM dataset of human PI3KC3-C1 bound to the small GTPase RAB1A, we were able to map the structural pathway of enzyme activation. The inactive conformation is stabilized by an N -myristoyl modification of the pseudokinase (PK) subunit VPS15. The N -myristate is sequestered in the N-lobe of the VPS15 PK domain, which stabilizes a series of interactions whereby VPS15 sequesters and blocks the catalytic and membrane binding units of the VPS34 lipid kinase. In the activated conformation, the N -myristate and the VPS34 lipid kinase domain are liberated to interact with membranes and catalyze PI3P formation. The VPS15 PK domain contains a unique Arg at the gatekeeper position and binds tightly to GTP. GTP binding structurally stabilizes the N -myristate “in” conformation, which promotes the inactive conformation. This pathway provides a general mechanism for PI3KC3 activation in autophagy and endosome biogenesis and a roadmap for their pharmacological upregulation.

The covalent attachment of ubiquitin-like LC3 proteins (microtubule-associated proteins 1A/1B light chain 3) prepares the autophagic membrane for cargo recruitment. We resolve key steps in LC3 lipidation by combining molecular dynamics simulations and experiments in vitro and in cellulo. We show how the E3-like ligaseautophagy-related 12 (ATG12)–ATG5-ATG16L1 in complex with the E2-like conjugase ATG3 docks LC3 onto the membrane in three steps by (i) the phosphatidylinositol 3-phosphate effector protein WD repeat domain phosphoinositide-interacting protein 2 (WIPI2), (ii) helix α2 of ATG16L1, and (iii) a membrane-interacting surface of ATG3. Phosphatidylethanolamine (PE) lipids concentrate in a region around the thioester bond between ATG3 and LC3, highlighting residues with a possible role in the catalytic transfer of LC3 to PE, including two conserved histidines. In a near-complete pathway from the initial membrane recruitment to the LC3 lipidation reaction, the three-step targeting of the ATG12–ATG5-ATG16L1 machinery establishes a high level of regulatory control.

Mechanistic target of rapamycin complex 1 (mTORC1), which consists of mTOR, Raptor, and mLST8, receives signaling inputs from growth factor signals and nutrients. These signals are mediated by the Rheb and Rag small GTPases, respectively, which activate mTORC1 on the cytosolic face of the lysosome membrane. We biochemically reconstituted the activation of mTORC1 on membranes by physiological submicromolar concentrations of Rheb, Rags, and Ragulator. We determined the cryo-EM structure and found that Raptor and mTOR directly interact with the membrane at anchor points separated by up to 230 Å across the membrane surface. Full engagement of the membrane anchors is required for maximal activation, which is brought about by alignment of the catalytic residues in the mTOR kinase active site. The observations show at the molecular and atomic scale how converging signals from growth factors and nutrients drive mTORC1 recruitment to and activation on the lysosomal membrane in a three-step process, consisting of (1) Rag-Ragulator-driven recruitment to within ∼100 Å of the lysosomal membrane, (2) Rheb-driven recruitment to within ∼40 Å, and finally (3) direct engagement of mTOR and Raptor with the membrane. The combination of Rheb and membrane engagement leads to full catalytic activation, providing a structural explanation for growth factor and nutrient signal integration at the lysosome.

In the late stages of the HIV-1 life cycle, membrane localization and self-assembly of Gag polyproteins induce membrane deformation and budding. Release of the virion requires direct interaction between immature Gag lattice and upstream ESCRT machinery at the viral budding site, followed by assembly of downstream ESCRT-III factors, culminating in membrane scission. However, molecular details of upstream ESCRT assembly dynamics at the viral budding site remain unclear. In this work, using coarse-grained (CG) molecular dynamics (MD) simulations, we investigated the interactions between Gag, ESCRT-I, ESCRT-II, and membrane to delineate the dynamical mechanisms by which upstream ESCRTs assemble templated by late-stage immature Gag lattice. We first systematically derived "bottom-up" CG molecular models and interactions of upstream ESCRT proteins from experimental structural data and extensive all-atom MD simulations. Using these molecular models, we performed CG MD simulations of ESCRT-I oligomerization and ESCRT-I/II supercomplex formation at the neck of the budding virion. Our simulations demonstrate that ESCRT-I can effectively oligomerize to higher-order complexes templated by the immature Gag lattice both in the absence of ESCRT-II and when multiple copies of ESCRT-II are localized at the bud neck. The ESCRT-I/II supercomplexes formed in our simulations exhibit predominantly columnar structures, which has important implications for the nucleation pathway of downstream ESCRT-III polymers. Importantly, ESCRT-I/II supercomplexes bound to Gag initiate membrane neck constriction by pulling the inner edge of the bud neck closer to the ESCRT-I headpiece ring. Our findings serve to elucidate a network of interactions between upstream ESCRT machinery, immature Gag lattice, and membrane neck that regulate protein assembly dynamics at the HIV-1 budding site.