The EMBO Journal

The selective lysosomal turnover of endoplasmic reticulum (ER) portions, termed ERphagy, removes aged, excess or damaged ER domains. The intermediates forming between receptor activation and lysosomal delivery are short-lived and remain poorly resolved. Here we identify a reversible temperature block at 25{degree sign}C that arrests ERphagy before lysosomal delivery. At this temperature, nutrient restriction still drives focal accumulation of the endogenously Halo-tagged ERphagy receptor TEX264 (puncta formation), segregation of ER cargo, remodelling of the ER membrane, whereas delivery of this material to degradative endolysosomes is strongly reduced. Rewarming above 27{degrees} restores lysosomal delivery, showing that the accumulated eTEX264-Halo puncta correspond to pre-lysosomal intermediates of the ERphagy program. Their formation requires ULK1/2 activity, the ATG7-dependent conjugation machinery, ATG9A, ATG13 and FIP200, coupling receptor clustering to autophagosome biogenesis. Consistently, expansion microscopy and electron tomography reveal curved, cradle-like TEX264-positive ER domains containing ER cargo, co-localizing with autophagy markers and lying adjacent to p62-cargo condensates.

The translocase of the outer membrane (TOM complex) imports more than 1,000 proteins into mitochondria. Clogging of the TOM pore with a precursor protein causes proteotoxic stress and eventually cell death. Two quality control pathways remove translocation-stalled precursor proteins. In the mitochondrial protein translocation-associated degradation (mitoTAD), Ubx2 recruits the cytosolic AAA-ATPase Cdc48 to clear precursor proteins from the TOM complex. In the mitochondrial compromised protein import response (mitoCPR), the stress-induced Cis1 recruits the AAA-ATPase Msp1 to Tom70. The role of Msp1 for the removal of mitochondrial precursor proteins remains unknown. Here, we demonstrate that parallel loss of Msp1 and Ubx2 strongly affects removal of precursor proteins and cell viability. Msp1 and Ubx2 bind independently of import stress and Cis1 to the TOM complex to remove a large variety of precursor proteins. Msp1-dependent extraction promotes ubiquitylation of precursor proteins, which in turn allows Ubx2-recruited Cdc48 to transfer the substrates to proteasomal degradation. We conclude that two AAA-ATPases cooperate in mitochondrial precursor quality control. Msp1-dependent extraction from the TOM complex facilitates precursor ubiquitylation and Cdc48-mediated transfer to proteasomal degradation.

Phagocytosis, a central process in innate immunity, depends on dynamic lipid remodelling, yet how cholesterol accumulates on late phagosomes remains unresolved. Here, we identify lysosomal acid lipase (LIPA) as a key cholesterol ester (CE) hydrolase driving cholesterol mobilization during phagosomal maturation. Using integrated lipidomics, chemoproteomics, and biochemical assays, we show that LIPA exhibits acidic CE hydrolase activity enriched on late phagosomes, generating free cholesterol essential for lipid raft formation. Pharmacologically inhibiting LIPA disrupts cholesterol-rich lipid raft assembly, impairs phagosomal trafficking, and alters pathogen fate - enhancing Staphylococcus aureus persistence, while restricting Mycobacterium tuberculosis survival. These findings reveal that LIPA couples cholesterol metabolism to phagocytosis, defining a mechanistic link between lipid catabolism and antimicrobial defence. By positioning CE hydrolysis as a critical determinant of phagosomal dynamics, our work uncovers a metabolic checkpoint in innate immunity and identifies LIPA as a potential therapeutic node in infection and inflammation.

WAVE and WASH polymerize Arp2/3-mediated branched actin at the leading edge of migrating cells and on the surface of endosomes, respectively. These two proteins are regulated within similar multiprotein complexes, the assembly mechanism of which remains poorly understood. Here we found using mass spectrometry that the two smallest subunits, BRK1 of the WAVE complex and CCDC53 of the WASH complex, interact with each other and with the assembly factor HSBP1. HSBP1 promotes WAVE and WASH assemblies and their respective activities, migration persistence and endosomal branched actin. Reduced levels of WAVE and WASH complexes caused by HSBP1 depletion can be corrected by providing cells with an excess of BRK1 or CCDC53 to assemble their specific complex. Unexpectedly, endogenous levels of BRK1 cross-regulate WASH assembly, whereas those of CCDC53 cross-regulate WAVE assembly. We found that various oligomers comprising BRK1, CCDC53 and HSBP1, including a heterotrimer containing one molecule of each, are formed through their promiscuous coiled coils. These oligomers play a coordinating role in the assembly of WAVE and WASH complexes.

Through mitochondrial outer membrane permeabilization (MOMP), mitochondria are central to apoptosis. MOMP is pro-inflammatory, impacting diverse processes including anti-tumor immunity and cellular senescence. Despite this, how damaged mitochondria activate inflammation remains unclear. Recent studies have shown that following MOMP, mitochondria are extensively ubiquitinated activating pro-inflammatory NF-{kappa}B signaling. We identify the E3 ligase, RNF213, as essential for pro-inflammatory mitochondrial ubiquitination following MOMP. RNF213 has an established role in pro- inflammatory, cell-autonomous immunity to bacteria and other pathogens. We discover that RNF213 is required for mitochondrial M1-linked (linear) ubiquitination. Interestingly, this was found to be independent of the canonical M1-ubiquitin ligase complex LUBAC. RNF213 can directly catalyze M1-linked ubiquitination. By promoting mitochondrial M1-linked ubiquitination, RNF213 initiates the recruitment of the essential NF{kappa}B adaptor molecule, NEMO, and subsequently activates pro-inflammatory NF-{kappa}B signaling. Collectively, our findings highlight striking similarities between how cells detect damaged mitochondria and intracellular pathogens, leading to inflammation.

HlyF and CprA, virulence factors from Escherichia coli and Pseudomonas aeruginosa respectively, constitute a previously unrecognized family of cytoplasmic enzymes that drive virulence through the production of outer membrane vesicles (OMVs) enriched in toxic bioactive lipids. Here, we show that OMVs of bacteria producing HlyF/CprA impair host autophagic flux by targeting lysosomal function. Specifically, these OMV-associated lipids disrupt autophagosome-lysosome fusion and induce hallmark features of lysosomal dysfunctions, including defective acidification, cholesterol accumulation, and membrane permeabilization. These lipid-mediated alterations compromise lysosomal integrity and block autophagic degradation. Our findings reveal a novel mechanism by which bacterial lipids subvert lysosomal functions and host clearance pathways. Given the widespread expression of HlyF/CprA homologs across diverse pathogens, this work highlights lipid effectors as key determinants of pathogenesis.

Tunneling nanotubes (TNTs) mediate intercellular communication and contribute to cancer progression. Tumour cells produce excess lactate through increased glycolysis, but whether lactate regulates TNT formation has not been studied. Here, we show that lactate promotes TNT formation in U-87 MG glioblastoma (GBM) cells under both glucose-restricted and glucose-replete conditions, with a similar effect in MDA-MB-231 breast cancer cells. This effect depends on lactate uptake and metabolism rather than receptor-mediated signalling: inhibition of monocarboxylate transporter 1 (MCT1) or lactate dehydrogenase (LDH) reduced TNT formation, while exogenous lactate restored TNT formation during LDH inhibition. 3-chloro-5-hydroxybenzoic acid, an agonist of the plasma membrane-localized lactate receptor (GPR81/HCAR1), did not reproduce the effect of lactate. Lactate also increased intercellular mitochondrial transfer, attenuated temozolomide (TMZ)-induced growth suppression of GBM spheroids, and increased expression of the TNT-associated genes CDC42, MYO10 and FSCN1. Inhibition of p300/CBP-mediated lactylation with A-485 reduced both protein lactylation and TNT formation, implicating lactylation in TNT biogenesis. Lactate increased basal and ATP-linked respiration that persisted during MCT1 inhibition and reduced on LDH inhibition, indicating that the effects of lactate on respiration and on TNT formation are at least partly separable. Together, these findings identify lactate as a regulator of TNT formation and link tumour metabolism to intercellular connectivity and TMZ response, highlighting lactate metabolism and lactylation as potential therapeutic targets.

Tropomyosins play a central role in the regulation of the actin cytoskeleton by assembling along filaments into rope-like, head-to-tail polymers ("clusters") along filaments that regulate the binding and activity of numerous actin-binding proteins. Importantly, in cells, tropomyosins do not decorate actin filaments uniformly; some filaments are devoid of tropomyosins, whereas others recruit specific isoforms. To explore the diversity of tropomyosin-isoform binding kinetics and identify factors that control their assembly, we designed tropomyosin constructs, bearing a natural N-terminal acetylation and targeted cysteine mutations for fluorescence labelling. We analysed the binding of different tropomyosin isoforms to individual actin filaments reconstituted inside a microfluidic chamber. We show that, despite significant differences in spontaneous nucleation and elongation rates, all tested tropomyosin isoforms can coassemble within the same clusters. In addition, we identified tropomodulin and leiomodin, two pointed-end-binding proteins, to efficiently nucleate the formation of tropomyosin clusters on actin filaments, for all tested tropomyosin isoforms. Notably, while tropomodulin nucleates tropomyosin clusters from the pointed end, leiomodin does so along the entire filament.

The interferon (IFN)-inducible RNA editor ADAR1 p150 catalyzes adenosine-to-inosine (A-to-I) editing of host and viral double-stranded RNAs, limiting aberrant innate immune sensing and regulating gene expression. How ADAR1 p150 activity is regulated during host-virus interactions remains poorly understood. Here, we identify the IFN-stimulated gene OASL as an inhibitor of ADAR1 p150. OASL engages p150 through two distinct interactions: its N-terminal OAS domain associates with the RNA-binding domains in an RNA-dependent manner, whereas its C-terminal ubiquitin-like domains bind the Z domain independently of RNA. Despite their distinct canonical functions, the OAS and ubiquitin-like domains converge to limit ADAR1 p150 RNA binding and suppress A-to-I editing across the transcriptome. OASL also reverses p150-mediated suppression of antiviral and innate immune gene expression and counteracts p150-mediated enhancement of measles virus and vesicular stomatitis virus replication. Thus, OASL restrains ADAR1 p150, linking interferon signaling to the control of RNA editing and antiviral immunity.

Lipid droplets (LDs) are assembled at discrete endoplasmic reticulum (ER) subdomains whose local phospholipid composition, rather than their protein content alone, determines whether a LD nucleates and how large it grows. Phosphatidic acid (PA) is the pivotal lipid of this environment, yet how PA reaches a forming LD has remained unknown. Here we show that the bridge-like lipid transfer protein BLTP1 is required to maintain PA in LD-associated membranes. BLTP1 depletion impaired LD formation and produced fewer but enlarged LDs across mammalian cell types, and lowered LD accumulation during adipogenic differentiation. At the ER, BLTP1 engaged the LD emergence factor FIT2 through its N-terminal transmembrane segment, an interaction that strengthened upon induction of LD formation and was strongly reduced by the disease-associated FIT2 G232R substitution. Co-depletion of FIT2 relieved the LD overgrowth caused by loss of BLTP1. Wild-type BLTP1, but neither a FIT2-binding-defective chimera nor a lipid-transfer-groove mutant, rescued LD formation in BLTP1-depleted cells. Lipidomics of purified LDs showed that BLTP1 loss most strongly lowered PA, most prominently the species PA 36:2, whereas PA and the major phospholipid classes of bulk ER membranes were unchanged. Collectively, we propose that local PA supply by BLTP1 coordinates LD assembly and growth.

Accumulation of aberrant and unfolded proteins induce cellular stresses and contributes in human to diseases and aging. Several cellular pathways combat the formation of these deleterious molecules. Recently, processes limiting the production of unwanted polypeptides through the co-translational degradation of their messenger RNAs (mRNAs) have been identified but remain poorly understood. In Saccharomyces cerevisiae, Caf130 was proposed to regulate the levels of mRNAs encoding some ribosomal proteins, principally Rpl4, when they do not fold properly. We report the functional and structural characterization this process. Biochemical purifications, interaction assays, and in vivo analyses combined with Alphafold modelling define the organization and function of the Caf130 module of the CCR4-NOT complex and associated factors that mediate this process. Our data reveal the critical role of different forms of the Nascent protein Associated Complex (NAC) to dock the CCR4-NOT complex onto translating ribosomes. The CCR4-NOT complex directly recognizes some nascent unfolded proteins emerging from the ribosome tunnel triggering deadenylation of the mRNAs encoding them. Our data further reveal unsuspected evolutionary and functional similarities between yeast and mammalian proteins involved in the quality control of nascent proteins through mRNA degradation. Altogether, our data indicate the existence of a conserved machinery contributing to protein quality control by triggering the decay of mRNAs encoding deleterious polypeptides in eukaryotes.

During apoptosis, the permeabilization of the mitochondrial inner membrane (MIM) through still-unclear mechanisms releases mtDNA into the cytosol, triggering the inflammatory cGAS/STING pathway under low caspase activity. Here, we report that, in apoptosis, the active form of the pore-forming protein Gasdermin E (GSDME-N) damages mitochondria before plasma membrane disruption. We visualize GSDME-N pore-like nano-assemblies in the MIM of apoptotic cells and of isolated mitochondria, which we bridge with the cryo-EM structure of the GSDME-N pore in mitochondria-like membranes. Deep membrane insertion of the anchor domain, which acts as a determinant of GSDME-N cardiolipin binding preferences, supports a role in pore formation. Notably, GSDME depletion results in a reduction in cristae swelling, MIM extrusion and mtDNA release during apoptosis. Subsequently, this decreases STING activation and inflammatory responses under caspase inhibition. Our findings reveal that GSDME mediates MIM permeabilization and mtDNA release during apoptosis and define the mechanism of GSDME-mediated membrane damage.

Ether glycerophospholipids (ether lipids) are a subclass of glycerophospholipids that are synthesized at the peroxisome and constitute nearly 20% of the total phospholipid content in metazoans. Biochemically, ether lipids have an alkyl chain attached by an ether bond at the sn-1 position of the glycerol backbone (alkyl-ether) primarily containing choline or ethanolamine head groups. Some ether lipids contain a cis bond adjacent to the ether linkage (alkenyl-ether) and are commonly referred to as plasmalogens. While the importance of ether lipids in humans is clear, highlighted by the severity of inherited peroxisomal disorders caused by ether lipid deficiency, the molecular roles of these lipids remain poorly understood. Here, we identify a link between ether lipid metabolism, peroxisome degradation, and oxygen availability. We find Fatty acyl-CoA-reductase 1 (FAR1), the rate-limiting enzyme in peroxisomal ether lipid metabolism, acts as a peroxisome receptor necessary for peroxisome degradation when oxygen is limiting. Hypoxia increases choline ether lipid abundance, promotes autophagy activation, and peroxisome degradation. Mechanistically, FAR1 binds ATG8-family protein LC3B and is necessary and sufficient to control peroxisome degradation under hypoxia. FAR1s enzymatic function as well as its capacity to bind LC3B is necessary to degrade peroxisomes under hypoxia. Similarly, loss of multiple components of peroxisomal ether lipid metabolism blocks peroxisome degradation under hypoxia in cells and in vivo. In mouse models of chronic HIF activation, Far1 is a direct HIF target gene and is necessary for peroxisome degradation in this setting. Increasing ether lipids using ether lipid precursors activates autophagy and promotes autophagy-dependent cardioprotection against ischemic injury. This work reveals FAR1 as a critical regulator of peroxisome homeostasis in the setting of oxygen limitation.

Ribosome hibernation mediated by long-form hibernation-promoting factor (HPF) requires coordination of ribosome dimerization and translational repression, but how these functions are integrated remains unclear. Here, we present cryo-EM structures of native 100S from a deep-sea Bacillus isolate, revealing a conformationally heterogeneous 100S population. Beyond the established roles of the HPF N-terminal domain (NTD) in decoding-center occlusion and the C-terminal domain (CTD) in dimerization, our analyses reveal additional contributions from the Linker and a charged CTD surface near the 16S rRNA 3' terminus containing the anti-Shine-Dalgarno (aSD) sequence. Linker substitutions differentially affect translational repression while retaining 100S formation, with distinct effects of the two Linker segments. Mutation of the charged CTD surface enhances translational repression while retaining robust 100S formation, suggesting an additional role beyond CTD-mediated ribosome dimerization. Functional analyses in an E. coli cell-free translation system further show that the inhibitory activity of individual HPF regions varies with translational context. Together, these findings support a modular model in which the NTD, Linker, and CTD make distinct but interconnected contributions to 100S assembly and translational repression.

Microtubule minus ends are central to non-centrosomal microtubule organization, yet how their recognition, protection and molecular organization are integrated remains unclear. Here we show that Drosophila Patronin forms dynamic condensates at microtubule minus ends through a prion-like domain (PrLD). PrLD-mediated condensation cooperates with the CKK domain and a second CKK-independent recognition region to convert weak or broadly distributed microtubule-binding activities into persistent minus-end recognition and tracking. Replacing the PrLD with a heterologous phase-separating HNRNPA1 IDR restores minus-end targeting, indicating that condensation capacity rather than PrLD primary sequence is critical. Patronin condensates further form a protective cap that resists minus-end depolymerization and recruit the cytoskeletal regulators Sas-4, Katanin and Ena. Disrupting PrLD multivalency impairs Patronin localization at multiple developmental sites and causes early organismal lethality. Thus, Patronin condensation creates a physiologically essential microtubule minus-end compartment coupling end recognition, stabilization and cytoskeletal organization.

Previous studies have demonstrated that Telomerase Reverse Transcriptase (TERT) is localized inside mitochondria, where it associates with mitochondrial DNA (mtDNA), thereby influencing replication and transcription. Using a combination of quantitative fluorescence microscopy and biochemistry, we demonstrate that TERT binds indirectly to mtDNA through the interaction with mitochondrial transcription factor A (TFAM) to induce mitochondrial fragmentation. These findings reveal a novel mitochondrial interaction involving TERT and highlight a role for TERT and TFAM in the regulation of auto/mitophagy. This interaction is an important factor for mitochondrial function and contributes to the remodelling of mitochondrial respiratory capacity.

Integral membrane enzymes operate within molecular environments, yet how these are organized remains poorly understood. Using M. smegmatis as a mycobacterial model, we define the membrane environment of the protein O-mannosyltransferase MtPMT through stratified interactomics combining affinity purification-mass spectrometry under four stringencies with a persistence score. This approach resolves a persistent membrane-associated core of 108 proteins from more transient layers, revealing a hierarchically organized landscape enriched in Sec protein translocation, protein biogenesis and envelope proteostasis machineries. Reciprocal affinity purification and in vivo FRET identify YajC and FtsH as neighbors of MtPMT, while structural modelling supports integration within a Sec-associated assembly. Genetic disruption of factors reduces O-mannosylation activity, demonstrating dependence on platform integrity. The MtPMT-associated network is remodelled during growth, indicating an adaptive system. Loss of POM compromises envelope permeability and stress resilience, linking O-mannosylation to fitness. Together, our results establish protein O-mannosylation as an integrated component of a dynamic membrane platform coordinating secretion, membrane protein biogenesis and proteostasis. Our findings support a model in which mycobacterial membranes contain specialized domains dynamically modulated by physiological state.

Crista junctions (CJs) are narrow infoldings of the inner mitochondrial membrane, serving as entry points into the cristae microenvironment. The mitochondrial contact site and cristae organizing system (MICOS) complex is a highly conserved protein machinery controlling CJ biogenesis and architecture, but its assembly mechanism driving CJ formation has remained unclear. By determining a crystal structure of a Chaetomium thermophilum Mic60-Mic19 complex and combining it with modelling and mutagenesis studies, we identify an extended helical region (EHR) of the Mic60 mitofilin domain and a further Mic19 helix as crucial elements of complex formation. In vitro cross-linking data and hydrogen-deuterium exchange mass spectrometry resolve domain assembly and conformational dynamics of the Mic60 Mic19 subcomplex and point to the EHR as a molecular switch controlling Mic60 transition from a closed dimer to a Mic19-bound hetero-octamer. Our data provide a structural model for Mic60 activation and therefore MICOS assembly as a prerequisite for crista junction formation.

Microtubules are dynamic polymers of tubulin subunits that are vital for a variety of eukaryotic cell functions, including intracellular trafficking and cell division. Microtubule-targeting drugs are among the oldest and most widely used chemotherapeutics. Despite their extensive use, the development and optimization of these drugs is hindered by our limited understanding of the molecular impacts on microtubules. Recent high-resolution structures have begun to explain how paclitaxel (Taxol) stabilizes microtubules: it reverses the hydrolysis-driven lateral strain at the microtubule seam and induces an expanded, GTP-like conformational state that promotes polymerization. In this work, we demonstrate that the microtubule-targeting natural product taccalonolide AJ (taccaAJ) stabilizes microtubules in a manner completely distinct from paclitaxel. Using cryo-EM, we show that taccaAJ stabilizes a compacted, post-GTP hydrolysis state, which is usually prone to depolymerization. In vitro assays show that taccaAJ-stabilized microtubules are more rigid than paclitaxel-stabilized microtubules, and reconstituted dynamics with taccaAJ exhibit increased rescue close to the plus-end. Finally, we use fluorescent biosensors for the compacted tubulin conformation to confirm that taccaAJ-stabilized microtubules in cells are compacted yet stable against depolymerization. We propose that taccaAJ preferentially binds a GDP lattice, promotes lateral interactions between protofilaments, and thus creates a rigid, stable microtubule in a compacted, post-GTP hydrolysis state.

The cytosolic ATP-binding cassette F (ABC-F) family modulates ribosomal activity in eukaryotes and prokaryotes. Although they all bind to the E site of the ribosome, some bacterial ABC-Fs confer resistance to ribosome-targeting antibiotics, while housekeeping members have poorly defined roles. Interestingly, in Escherichia coli, which possesses four ABC-F (EttA, Uup, YbiT and YheS), Uup has been rather implicated in DNA recombination. Using an unbiased approach, we reconcile this apparent discrepancy by showing that Uup's involvement in DNA recombination is indirect and is dictated by Uup-mediated regulation of the last steps of translation that rescues problematic translation of genes involved in DNA maintenance such as ssb and dnaX. Accordingly, the elongated cell phenotype observed in uup deletion background is reverted by expressing a ssb variant that no longer depends on Uup. Unlike its paralog EttA, which helps ribosomes in translating proteins with repeats of negatively charged residues in the N-terminal 30 amino acids, Uup instead facilitates the rescue of stalled translation termination events that, in its absence, are handled by the tmRNA pathway.