Search

Vacuolar H + -ATPases (V-ATPases) are highly conserved multisubunit enzymes that maintain the distinct pH of eukaryotic organelles. The integral membrane a-subunit is encoded by tissue- and organelle-specific isoforms, and its cytosolic N-terminal domain (aNT) modulates organelle-specific regulation and targeting of V-ATPases. Organelle membranes have specific phosphatidylinositol phosphate (PIP) lipid enrichment linked to maintenance of organelle pH. In yeast, the aNT domains of the two a-subunit isoforms bind PIP lipids enriched in the organelle membranes where they reside; these interactions affect activity and regulatory properties of the V-ATPases containing each isoform. Humans have four a-subunit isoforms, and we hypothesize that the aNT domains of these isoforms will also bind to specific PIP lipids. The a1 and a2 isoforms of human V-ATPase a-subunits are localized to endolysosomes and Golgi, respectively. We determined that bacterially expressed Hua1NT and Hua2NT bind specifically to endolysosomal PIP lipids PI(3)P and PI(3,5)P 2 and Golgi enriched PI(4)P, respectively. Despite the lack of canonical PIP-binding sites, we identified potential binding sites in the HuaNT domains by sequence comparisons and existing subunit structures and models. We found that mutations at a similar location in the distal loops of both HuaNT isoforms compromise binding to their cognate PIP lipids, suggesting that these loops encode PIP specificity of the a-subunit isoforms. These data suggest a mechanism through which PIP lipid binding could stabilize and activate V-ATPases in distinct organelles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Exosomes are endosome-derived extracellular vesicles involved in intercellular communication. They are generated as intraluminal vesicles within endosomal compartments that fuse with the plasma membrane (PM). The molecular events that generate secretory endosomes and lead to the release of exosomes are not well understood. We identified a subclass of non-proteolytic endosomes at prelysosomal stage as the compartment of origin of CD63 positive exosomes. These compartments undergo a Rab7a/Arl8b/Rab27a GTPase cascade to fuse with the PM. Dynamic endoplasmic reticulum (ER)-late endosome (LE) membrane contact sites (MCS) through ORP1L have the distinct capacity to modulate this process by affecting LE motility, maturation state, and small GTPase association. Thus, exosome secretion is a multi-step process regulated by GTPase switching and MCS, highlighting the ER as a new player in exosome-mediated intercellular communication., (© 2022 Verweij et al.)

G-protein-coupled receptors (GPCRs), the largest family of signalling receptors, as well as important drug targets, are known to activate extracellular-signal-regulated kinase (ERK)-a master regulator of cell proliferation and survival 1 . However, the precise mechanisms that underlie GPCR-mediated ERK activation are not clearly understood 2-4 . Here we investigated how spatially organized β 2 -adrenergic receptor (β 2 AR) signalling controls ERK. Using subcellularly targeted ERK activity biosensors 5 , we show that β 2 AR signalling induces ERK activity at endosomes, but not at the plasma membrane. This pool of ERK activity depends on active, endosome-localized Gα s and requires ligand-stimulated β 2 AR endocytosis. We further identify an endosomally localized non-canonical signalling axis comprising Gα s , RAF and mitogen-activated protein kinase kinase, resulting in endosomal ERK activity that propagates into the nucleus. Selective inhibition of endosomal β 2 AR and Gα s signalling blunted nuclear ERK activity, MYC gene expression and cell proliferation. These results reveal a non-canonical mechanism for the spatial regulation of ERK through GPCR signalling and identify a functionally important endosomal signalling axis., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Rab22 and Rab31 belong to the Rab5 subfamily of GTPases that regulates endocytic traffic and endosomal sorting. Rab22 and Rab31 (a.k.a. Rab22b) are closely related and share 87% amino acid sequence similarity, but they show distinct intracellular localization and function in the cell. Rab22 is localized to early endosomes and regulates early endosomal recycling, while Rab31 is mostly localized to the Golgi complex with only a small fraction in the endosomes at steady state. The specific determinants that affect this differential localization, however, are unclear. In this study, we identify a novel membrane targeting domain (MTD) consisting of the C-terminal hypervariable domain (HVD), interswitch loop (ISL), and N-terminal domain as a major determinant of endosomal localization for Rab22 and Rab31, as well as Rab5. Rab22 and Rab31 share the same N-terminal domain, but we find Rab22 chimeras with Rab31 HVD exhibit phenotypic Rab31 localization to the Golgi complex, while Rab31 chimeras with the Rab22 HVD localize to early endosomes, similar to wildtype Rab22. We also find that the Rab22 HVD favors interaction with the early endosomal effector protein Rabenosyn-5, which may stabilize the Rab localization to the endosomes. The importance of effector interaction in endosomal localization is further demonstrated by the disruption of Rab22 endosomal localization in Rabenosyn-5 knockout cells and by the shift of Rab31 to the endosomes in Rabenosyn-5-overexpressing cells. Taken together, we have identified a novel MTD that mediates localization of Rab5 subfamily members to early endosomes via interaction with an effector such as Rabenosyn-5., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Lemur tail kinase 1 (LMTK1), previously called apoptosis-associated tyrosine kinase (AATYK), is an endosomal Ser/Thr kinase. We recently reported that LMTK1 regulates axon outgrowth, dendrite arborization and spine formation via Rab11-mediated vesicle transport. Rab11, a small GTPase regulating recycling endosome trafficking, is shown to be associated with late-onset Alzheimer's disease (LOAD). In fact, genome-wide association studies identified many proteins regulating vesicle transport as risk factors for LOAD. Furthermore, LMTK1 has been reported to be a risk factor for frontotemporal dementia. Then, we hypothesized that LMTK1 contributes to AD development through vesicle transport and examined the effect of LMTK1 on the cellular localization of AD-related proteins, amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 (BACE1). The β-cleavage of APP by BACE1 is the initial and rate-limiting step in Aβ generation. We found that LMTK1 accumulated BACE1, but not APP, to the perinuclear endosomal compartment, whereas the kinase-negative(kn) mutant of LMTK1A did not. The β-C-terminal fragment was prone to increase under overexpression of LMTK1A kn. Moreover, the expression level of LMTK1A was reduced in AD brains. These results suggest the possibility that LMTK1 is involved in AD development through the regulation of the proper endosomal localization of BACE1., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Objective: Maturation of megakaryocytes culminates with extensive membrane rearrangements necessary for proplatelet formation. Mechanisms required for proplatelet extension and origin of membranes are still poorly understood. GTPase Rab5 (Ras-related protein in brain 5) regulates endocytic uptake and homotypic fusion of early endosomes and regulates phosphatidylinositol 3-monophosphate production important for binding of effector proteins during early-to-late endosomal/lysosomal maturation. Approach and Results: To investigate the role of Rab5 in megakaryocytes, we expressed GFP (green fluorescent protein)-coupled Rab5 wild type and its point mutants Q79L (active) and N133L (inactive) in primary murine fetal liver-derived megakaryocytes. Active Rab5 Q79L induced the formation of enlarged early endosomes, while inactive Rab5 N133L caused endosomal fragmentation. Consistently, an increased amount of transferrin internalization in Rab5 Q79L was impaired in Rab5 N133L expressing megakaryocytes, when compared with GFP or Rab5 wild type. Moreover, trafficking of GPIbβ (glycoprotein Ib subunit beta), a subunit of major megakaryocytes receptor and membrane marker, was found to be mediated by Rab5 activity. While GPIbβ was mostly present along the plasma membrane, and within cytoplasmic vesicles in Rab5 wild type megakaryocytes, it accumulated in the majority of Rab5 Q79L enlarged endosomes. Conversely, Rab5 N133L caused mostly GPIbβ plasma membrane retention. Furthermore, Rab5 Q79L expression increased incorporation of the membrane dye (PKH26), indicating higher membrane content. Finally, while Rab5 Q79L increased proplatelet production, inactive Rab5 N133L strongly inhibited it and was coupled with a decrease in late endosomes/lysosomes. Localization of GPIbβ in enlarged endosomes was phosphatidylinositol 3-monophosphate dependent., Conclusions: Taken together, our results demonstrate that Rab5-dependent endocytosis plays an important role in megakaryocytes receptor trafficking, membrane formation, and thrombopoiesis.

Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Transient receptor potential mucolipin 2 and 3 (TRPML2 and TRPML3), as key channels in the endosomal-lysosomal system, are associated with many different cellular processes, including ion release, membrane trafficking and autophagy. In particular, they can also facilitate viral entry into host cells and enhance viral infection. We previously identified that two selective TRPML agonists, ML-SA1 and SN-2, that showed antiviral activities against dengue virus type 2 (DENV2) and Zika virus (ZIKV) in vitro, but their antiviral mechanisms are still elusive. Here, we reported that ML-SA1 could inhibit DENV2 replication by downregulating the expression of both TRPML2 and TRPML3, while the other TRPML activator, SN-2, suppressed DENV2 infection by reducing only TRPML3 expression. Consistently, the channel activities of both TRPML2 and TRPML3 were also found to be associated with the antiviral activity of ML-SA1 on DENV2 and ZIKV, but SN-2 relied only on TRPML3 channel activity. Further mechanistic experiments revealed that ML-SA1 and SN-2 decreased the expression of the late endosomal marker Rab7, dependent on TRPML2 and TRPML3, indicating that these two compounds likely inhibit viral infection by promoting vesicular trafficking from late endosomes to lysosomes and then accelerating lysosomal degradation of the virus. As expected, neither ML-SA1 nor SN-2 inhibited herpes simplex virus type I (HSV-1), whose entry is independent of the endolysosomal network. Together, our work reveals the antiviral mechanisms of ML-SA1 and SN-2 in targeting TRPML channels, possibly leading to the discovery of new drug candidates to inhibit endocytosed viruses., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Membrane protein recycling systems are essential for maintenance of the endosome-lysosome system. In yeast, retromer and Snx4 coat complexes are recruited to the endosomal surface, where they recognize cargos. They sort cargo and deform the membrane into recycling tubules that bud from the endosome and target to the Golgi. Here, we reveal that the SNX-BAR protein, Mvp1, mediates an endosomal recycling pathway that is mechanistically distinct from the retromer and Snx4 pathways. Mvp1 deforms the endosomal membrane and sorts cargos containing a specific sorting motif into a membrane tubule. Subsequently, Mvp1 recruits the dynamin-like GTPase Vps1 to catalyze membrane scission and release of the recycling tubule. Similarly, SNX8, the human homolog of Mvp1, which has been also implicated in Alzheimer's disease, mediates formation of an endosomal recycling tubule. Thus, we present evidence for a novel endosomal retrieval pathway that is conserved from yeast to humans., Competing Interests: SS, AO, NN, JJ, MB, SE No competing interests declared, (© 2021, Suzuki et al.)

The canonical model of agonist-stimulated phosphatidylinositol-3-OH kinase (PI3K)-Akt signalling proposes that PI3K is activated at the plasma membrane, where receptors are activated and phosphatidylinositol-4,5-bisphosphate is concentrated. Here we show that phosphatidylinositol-3,4,5-trisphosphate generation and activated Akt are instead largely confined to intracellular membranes upon receptor tyrosine kinase activation. Microtubule-associated protein 4 (MAP4) interacts with and controls localization of membrane vesicle-associated PI3Kα to microtubules. The microtubule-binding domain of MAP4 binds directly to the C2 domain of the p110α catalytic subunit. MAP4 controls the interaction of PI3Kα with activated receptors at endosomal compartments along microtubules. Loss of MAP4 results in the loss of PI3Kα targeting and loss of PI3K-Akt signalling downstream of multiple agonists. The MAP4-PI3Kα assembly defines a mechanism for spatial control of agonist-stimulated PI3K-Akt signalling at internal membrane compartments linked to the microtubule network.

The small GTPase Rab5 is one of the master regulators of vesicular trafficking that participates in early stages of the endocytic pathway, such as endocytosis and endosome maturation. Three Rab5 isoforms (a, b, and c) share high sequence identity, and exhibit complex functions. However, the role of Rab5c in virus infection and cellular immune responses remains poorly understood. In this study, based on the established virus-cell infection model, Singapore grouper iridovirus (SGIV)-infected grouper spleen (GS) cells, we investigated the role of Rab5c in virus infection and host immune responses. Rab5c was cloned from the orange-spotted grouper, Epinephelus coioides , and termed EcRab5c. EcRab5c encoded a 220-amino-acid polypeptide, showing 99% and 91% identity to Anabas testudineus , and Homo sapiens , respectively. Confocal imaging showed that EcRab5c localized as punctate structures in the cytoplasm. However, a constitutively active (CA) EcRab5c mutant led to enlarged vesicles, while a dominant negative (DN) EcRab5c mutant reduced vesicle structures. EcRab5c expression levels were significantly increased after SGIV infection. EcRab5c knockdown, or CA/DN EcRab5c overexpression significantly inhibited SGIV infection. Using single-particle imaging analysis, we further observed that EcRab5c disruption impaired crucial events at the early stage of SGIV infection, including virus binding, entry, and transport from early to late endosomes, at the single virus level. Furthermore, it is the first time to investigate that EcRab5c is required in autophagy. Equally, EcRab5c positively regulated interferon-related factors and pro-inflammatory cytokines. In summary, these data showed that EcRab5c exerted a bi-functional role on iridovirus infection and host immunity in fish, which furthers our understanding of virus and host immune interactions., (Copyright © 2020 Wang, Li, Zhang, Yang, Wei, Huang, Qin and Wang.)

Cancer cells break tissue barriers by use of small actin-rich membrane protrusions called invadopodia. Complete invadopodia maturation depends on protrusion outgrowth and the targeted delivery of the matrix metalloproteinase MT1-MMP via endosomal transport by mechanisms that are not known. Here, we show that the ER protein Protrudin orchestrates invadopodia maturation and function. Protrudin formed contact sites with MT1-MMP-positive endosomes that contained the RAB7-binding Kinesin-1 adaptor FYCO1, and depletion of RAB7, FYCO1, or Protrudin inhibited MT1-MMP-dependent extracellular matrix degradation and cancer cell invasion by preventing anterograde translocation and exocytosis of MT1-MMP. Moreover, when endosome translocation or exocytosis was inhibited by depletion of Protrudin or Synaptotagmin VII, respectively, invadopodia were unable to expand and elongate. Conversely, when Protrudin was overexpressed, noncancerous cells developed prominent invadopodia-like protrusions and showed increased matrix degradation and invasion. Thus, Protrudin-mediated ER-endosome contact sites promote cell invasion by facilitating translocation of MT1-MMP-laden endosomes to the plasma membrane, enabling both invadopodia outgrowth and MT1-MMP exocytosis., (© 2020 Pedersen et al.)

The high rate of thrombotic complications associated with COVID-19 seems likely to reflect viral infection of vascular endothelial cells, which express the ACE2 protein that enables SARS-CoV-2 to invade cells. Various proinflammatory stimuli can promote thrombosis by inducing luminal endothelial expression of tissue factor (TF), which interacts with circulating coagulation factor VII to trigger extrinsic coagulation. The signalling mechanism whereby these stimuli evoke TF expression entails activation of NADPH oxidase, upstream from activation of the NF-kappaB transcription factor that drives the induced transcription of the TF gene. When single-stranded RNA viruses are taken up into cellular endosomes, they stimulate endosomal formation and activation of NADPH oxidase complexes via RNA-responsive toll-like receptor 7. It is therefore proposed that SARS-CoV-2 infection of endothelial cells evokes the expression of TF which is contingent on endosomal NADPH oxidase activation. If this hypothesis is correct, hydroxychloroquine, spirulina (more specifically, its chromophore phycocyanobilin) and high-dose glycine may have practical potential for mitigating the elevated thrombotic risk associated with COVID-19., Competing Interests: Competing interests: JJD is Director of Scientific Affairs at AIDP. MM is an owner of a nutraceutical company and co-inventor and co-owner of a US patent covering nutraceutical uses of phycocyanobilin oligopeptides derived from cyanobacteria such as spirulina., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Coronaviruses (CoVs), including Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and the novel coronavirus disease-2 (SARS-CoV-2) are a group of enveloped RNA viruses that cause a severe respiratory infection which is associated with a high mortality [...]., Competing Interests: The authors declare no conflict of interest.

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

Background: Over 100 million people worldwide suffer from birch pollen allergy. Bet v 1 has been identified as the major birch pollen allergen. However, the molecular mechanisms of birch allergic sensitization, including the roles of Bet v 1 and other components of the birch pollen extract, remain incompletely understood. Here, we examined how known birch pollen-derived molecules influence the endolysosomal processing of Bet v 1, thereby shaping its allergenicity., Methods: We analyzed the biochemical and immunological interaction of ligands with Bet v 1. We then investigated the proteolytic processing of Bet v 1 by endosomal extracts in the presence and absence of ligands, followed by a detailed kinetic analysis of Bet v 1 processing by individual endolysosomal proteases as well as the T-cell epitope presentation in BMDCs., Results: We identified E 1 phytoprostanes as novel Bet v 1 ligands. Pollen-derived ligands enhanced the proteolytic resistance of Bet v 1, affecting degradation kinetics and preferential cleavage sites of the endolysosomal proteases cathepsin S and legumain. E 1 phytoprostanes exhibited a dual role by stabilizing Bet v 1 and inhibiting cathepsin protease activity., Conclusion: Bet v 1 can serve as a transporter of pollen-derived, bioactive compounds. When carried to the endolysosome, such compounds can modulate the proteolytic activity, including its processing by cysteine cathepsins. We unveil a paradigm shift from an allergen-centered view to a more systemic view that includes the host endolysosomal enzymes., (© 2019 The Authors. Allergy published by John Wiley & Sons Ltd.)

Unesterified cholesterol accumulation in the late endosomal/lysosomal (LE/LY) compartment is the cellular hallmark of Niemann-Pick C (NPC) disease, caused by defects in the genes encoding NPC1 or NPC2. We previously reported the dramatic stimulation of NPC2 cholesterol transport rates to and from model membranes by the LE/LY phospholipid lysobisphosphatidic acid (LBPA). It had been previously shown that enrichment of NPC1-deficient cells with LBPA results in cholesterol clearance. Here we demonstrate that LBPA enrichment in human NPC2-deficient cells, either directly or via its biosynthetic precursor phosphtidylglycerol (PG), is entirely ineffective, indicating an obligate functional interaction between NPC2 and LBPA in cholesterol trafficking. We further demonstrate that NPC2 interacts directly with LBPA and identify the NPC2 hydrophobic knob domain as the site of interaction. Together these studies reveal a heretofore unknown step of intracellular cholesterol trafficking which is critically dependent upon the interaction of LBPA with functional NPC2 protein., Competing Interests: LM, AL, RL, OI, DB, MW, KL, PK, JS No competing interests declared, (© 2019, McCauliff et al.)

The primary site for KRAS signaling is the inner leaflet of the plasma membrane (PM). We previously reported that oxanthroquinone G01 (G01) inhibited KRAS PM localization and blocked KRAS signaling. In this study, we identified acylpeptide hydrolase (APEH) as a molecular target of G01. APEH formed a stable complex with biotinylated G01, and the enzymatic activity of APEH was inhibited by G01. APEH knockdown caused profound mislocalization of KRAS and reduced clustering of KRAS that remained PM localized. APEH knockdown also disrupted the PM localization of phosphatidylserine (PtdSer), a lipid critical for KRAS PM binding and clustering. The mislocalization of KRAS was fully rescued by ectopic expression of APEH in knockdown cells. APEH knockdown disrupted the endocytic recycling of epidermal growth factor receptor and transferrin receptor, suggesting that abrogation of recycling endosome function was mechanistically linked to the loss of KRAS and PtdSer from the PM. APEH knockdown abrogated RAS-RAF-MAPK signaling in cells expressing the constitutively active (oncogenic) mutant of KRAS (KRASG12V), and selectively inhibited the proliferation of KRAS-transformed pancreatic cancer cells. Taken together, these results identify APEH as a novel drug target for a potential anti-KRAS therapeutic., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Emerging viruses such as severe fever and thrombocytopenia syndrome virus (SFTSV) and Ebola virus (EBOV) are responsible for significant morbidity and mortality. Host cell proteases that process the glycoproteins of these viruses are potential targets for antiviral intervention. The aspartyl protease signal peptide peptidase (SPP) has recently been shown to be required for processing of the glycoprotein precursor, Gn/Gc, of Bunyamwera virus and for viral infectivity. Here, we investigated whether SPP is also required for infectivity of particles bearing SFTSV-Gn/Gc. Entry driven by the EBOV glycoprotein (GP) and the Lassa virus glycoprotein (LASV-GPC) depends on the cysteine proteases cathepsin B and L (CatB/CatL) and the serine protease subtilisin/kexin-isozyme 1 (SKI-1), respectively, and was examined in parallel for control purposes. We found that inhibition of SPP and SKI-1 did not interfere with SFTSV Gn + Gc-driven entry but, unexpectedly, blocked entry mediated by EBOV-GP. The inhibition occurred at the stage of proteolytic activation and the SPP inhibitor was found to block CatL/CatB activity. In contrast, the SKI-1 inhibitor did not interfere with CatB/CatL activity but disrupted CatB localization in endo/lysosomes, the site of EBOV-GP processing. These results underline the potential of protease inhibitors for antiviral therapy but also show that previously characterized compounds might exert broader specificity than initially appreciated and might block viral entry via diverse mechanisms., Competing Interests: The authors have declared that no competing interests exist.

Cells respond to stress in the ER by initiating the widely conserved unfolded protein response. Activation of the ER transmembrane nuclease IRE1 leads to the degradation of specific mRNAs, but how this pathway affects the ability of cells to recover from stress is not known. Here, we show that degradation of the mRNA encoding biogenesis of lysosome-related organelles 1 subunit 1 ( Blos1 ) leads to the repositioning of late endosomes (LEs)/lysosomes to the microtubule-organizing center in response to stress in mouse cells. Overriding Blos1 degradation led to ER stress sensitivity and the accumulation of ubiquitinated protein aggregates, whose efficient degradation required their independent trafficking to the cell center and the LE-associated endosomal sorting complexes required for transport. We propose that Blos1 regulation by IRE1 promotes LE-mediated microautophagy of protein aggregates and protects cells from their cytotoxic effects., (© 2019 Bae et al.)

The eukaryotic TORC1 kinase is a homeostatic controller of growth that integrates nutritional cues and mediates signals primarily from the surface of lysosomes or vacuoles. Amino acids activate TORC1 via the Rag GTPases that combine into structurally conserved multi-protein complexes such as the EGO complex (EGOC) in yeast. Here we show that Ego1, which mediates membrane-anchoring of EGOC via lipid modifications that it acquires while traveling through the trans-Golgi network, is separately sorted to vacuoles and perivacuolar endosomes. At both surfaces, it assembles EGOCs, which regulate spatially distinct pools of TORC1 that impinge on functionally divergent effectors: vacuolar TORC1 predominantly targets Sch9 to promote protein synthesis, whereas endosomal TORC1 phosphorylates Atg13 and Vps27 to inhibit macroautophagy and ESCRT-driven microautophagy, respectively. Thus, the coordination of three key regulatory nodes in protein synthesis and degradation critically relies on a division of labor between spatially sequestered populations of TORC1., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Eukaryotic cells maintain a highly organized endolysosomal system. This system regulates the protein and lipid content of the plasma membrane, it participates in the intracellular quality control machinery and is needed for the efficient removal of damaged organelles. This complex network comprises an endosomal membrane system that feeds into the lysosomes, yet also allows recycling of membrane proteins, and probably lipids. Moreover, lysosomal degradation provides the cell with macromolecules for further growth. In this review, we focus primarily on the role of the small Rab GTPases Rab5 and Rab7 as organelle markers and interactors of multiple effectors on endosomes and lysosomes and highlight their role in membrane dynamics, particularly fusion along the endolysosomal pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Fatty acid uptake and metabolism are often dysregulated in cancer cells. Fatty acid activation is a critical step that allows these biomolecules to enter cellular metabolic pathways such as mitochondrial β-oxidation for ATP generation or the lipogenic routes that generate bioactive lipids such as the inositol phospholipids. Fatty acid activation by the addition of coenzyme A is catalysed by a family of enzymes called the acyl CoA synthetase ligases (ACSL). Furthermore, enhanced expression of particular ACSL isoforms, such as ACSL4, is a feature of some more aggressive cancers and may contribute to the oncogenic phenotype. This study focuses on ACSL3 and ACSL4, closely related structural homologues that preferentially activate palmitate and arachidonate fatty acids, respectively. In this study, immunohistochemical screening of multiple soft tissue tumour arrays revealed that ACSL3 and ACSL4 were highly, but differentially, expressed in a subset of leiomyosarcomas, fibrosarcomas and rhabdomyosarcomas, with consistent cytoplasmic and granular stainings of tumour cells. The intracellular localisations of endogenously expressed ACSL3 and ACSL4 were further investigated by detailed subcellular fractionation analyses of HT1080 fibrosarcoma and MCF-7 breast cancer cells. ACSL3 distribution closely overlapped with proteins involved in trafficking from the trans-Golgi network and endosomes. In contrast, the ACSL4 localisation pattern more closely followed that of calnexin which is an  endoplasmic reticulum resident chaperone. Confocal immunofluorescence imaging of MCF-7 cells confirmed the intracellular localisations of both enzymes. These observations reveal new information regarding the compartmentation of fatty acid metabolism in cancer cells.

The filovirus, Zaire Ebolavirus (EBOV), infects tissue macrophages (Mϕs) and dendritic cells (DCs) early during infection. Viral infection of both cells types is highly productive, leading to increased viral load. However, virus infection of these two cell types results in different consequences for cellular function. Infection of Mϕs stimulates the production of proinflammatory and immunomodulatory cytokines and chemokines, leading to the production of a cytokine storm, while simultaneously increasing tissue factor production and thus facilitating disseminated intravascular coagulation. In contrast, EBOV infection of DCs blocks DC maturation and antigen presentation rendering these cells unable to communicate with adaptive immune response elements. Details of the known interactions of these cells with EBOV are reviewed here. We also identify a number of unanswered questions that remain about interactions of filoviruses with these cells., (©2018 Society for Leukocyte Biology.)

Rab GTPases are key regulators of intracellular trafficking, and cycle between a GTP-bound active state and a GDP-bound inactive state. This cycle is regulated by guanine-nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Several efforts have been made in connecting the correct GEFs and GAPs to their specific Rab. Here, we aimed to identify GAPs for Rab7b, the small GTPase involved in transport from late endosomes to the trans-Golgi. An siRNA screen targeting proteins containing TBC domains critical for Rab GAPs was performed and coupled to a phenotypic read-out that visualized the distribution of Rab7b. Silencing of TBC1D5 provided the strongest phenotype and this protein was subsequently validated in various in vitro and cell-based assays. TBC1D5 localizes to Rab7b-positive vesicles, interacts with Rab7b and has GAP activity towards Rab7b in vitro , which is further increased by retromer proteins. Similarly to the constitutively active mutant of Rab7b, inactivation of TBC1D5 also reduces the number of CI-MPR- and sortilin-positive vesicles. Together, the results show that TBC1D5 is a GAP for Rab7b in the control of endosomal transport to the trans-Golgi.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Our recent genome-wide association study found that the NELFCD/CTSZ locus was significantly associated with progression of primary biliary cholangitis (PBC) to jaundice stage in the Japanese population. In this study, we investigated the role of cathepsin Z in the etiology and pathology of PBC. Serum cathepsin Z levels were measured using enzyme-linked immunosorbent assay. The expression and localization of cathepsin Z in liver specimens were analyzed by western blotting and immunohistochemistry. In PBC patients, serum cathepsin Z levels were significantly increased with disease progression. In addition, its levels were positively correlated with alanine transaminase, aspartate transaminase and total bilirubin, and were negatively correlated with platelet count and albumin. Cathepsin Z expression was markedly increased in hepatocytes at later stages of PBC, and its localization was altered from the peri-bile canaliculus to the cytoplasm, where a fraction was no longer colocalized with endosomal/lysosomal vesicles. Similar altered expression of cathepsin Z was observed in end-stage of other cholestatic liver diseases including sepsis, obstructive jaundice, and Alagille syndrome. Our results indicate that altered expression and localization of cathepsin Z in hepatocytes are characteristic features of PBC and other cholestatic liver diseases, and are implicated in the progression of PBC.

Excessive fat accumulation is a major risk factor for the development of type 2 diabetes mellitus and other common conditions, including cardiovascular disease and certain types of cancer. Here, we identify a mechanism that regulates adiposity based on the activator of autophagy TP53INP2. We report that TP53INP2 is a negative regulator of adipogenesis in human and mouse preadipocytes. In keeping with this, TP53INP2 ablation in mice caused enhanced adiposity, which was characterized by greater cellularity of subcutaneous adipose tissue and increased expression of master adipogenic genes. TP53INP2 modulates adipogenesis through autophagy-dependent sequestration of GSK3β into late endosomes. GSK3β sequestration was also dependent on ESCRT activity. As a result, TP53INP2 promotes greater β-catenin levels and induces the transcriptional activity of TCF/LEF transcription factors. These results demonstrate a link between autophagy, sequestration of GSK3β into late endosomes and inhibition of adipogenesis in vivo.

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson's disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles., Competing Interests: KY, CW, SS, CM, RK, NN, YH, MK, KT, NM No competing interests declared, WH, RY Reviewing editor, eLife, (© 2018, Yamano et al.)

The yeast Dsc E3 ligase complex has long been recognized as a Golgi-specific protein ubquitination system. It shares a striking sequence similarity to the Hrd1 complex that plays critical roles in the ER-associated degradation pathway. Using biochemical purification and mass spectrometry, we identified two novel Dsc subunits, which we named as Gld1 and Vld1. Surprisingly, Gld1 and Vld1 do not coexist in the same complex. Instead, they compete with each other to form two functionally independent Dsc subcomplexes. The Vld1 subcomplex takes the AP3 pathway to reach the vacuole membrane, whereas the Gld1 subcomplex travels through the VPS pathway and is cycled between Golgi and endosomes by the retromer. Thus, instead of being Golgi-specific, the Dsc complex can regulate protein levels at three distinct organelles, namely Golgi, endosome, and vacuole. Our study provides a novel model of achieving multi-tasking for transmembrane ubiquitin ligases with interchangeable trafficking adaptors., Competing Interests: XY, FA, WZ, ML No competing interests declared, (© 2018, Yang et al.)

G-protein coupled receptors represent the largest family of membrane receptors. G-protein dependent signal of GPCR is classically thought to originate exclusively from the plasma membrane and, until very recently, internalized GPCRs were considered silent. At present, experimental proofs exist showing that GPCR can continue to signal via G proteins after internalization. We demonstrated that, once internalized in early endosomes, Glucose-dependent Insulinotropic Peptide Receptor (GIPR) continues to stimulate production of cAMP and activate PKA. In addition to indirect proofs showing that kinetics of cAMP production and PKA activation depend on internalization and GIPR trafficking, we identified the active form of Gαs on early endosomes containing GIPR and detected a distinct FRET signal accounting for cAMP production at the surface of endosomes containing GIP, relative to endosomes without GIP., (© Société de Biologie, 2018.)

The vacuolar H + -ATPase (V-ATPase) is a ~1 MDa membrane protein complex that couples the hydrolysis of cytosolic ATP to the transmembrane movement of protons. In essentially all eukaryotic cells, this acid pumping function plays critical roles in the acidification of endosomal/lysosomal compartments and hence in transport, recycling and degradative pathways. It is also important in acid extrusion across the plasma membrane of some cells, contributing to homeostatic control of cytoplasmic pH and maintenance of appropriate extracellular acidity. The complex, assembled from up to 30 individual polypeptides, operates as a molecular motor with rotary mechanics. Historically, structural inferences about the eukaryotic V-ATPase and its subunits have been made by comparison to the structures of bacterial homologues. However, more recently, we have developed a much better understanding of the complete structure of the eukaryotic complex, in particular through advances in cryo-electron microscopy. This chapter explores these recent developments, and examines what they now reveal about the catalytic mechanism of this essential proton pump and how its activity might be regulated in response to cellular signals.

The P-type ATPase ATP13A2 protein was originally associated with a form of Parkinson's Disease (PD) known as Kufor Rakeb Syndrome (KRS). However, in the last years it has been found to underlay variants of neuronal ceroid-lipofuscinoses and hereditary spastic paraplegia. These findings expand the clinical and genetic spectrum of ATP13A2-associated disorders, which are commonly characterized by lysosomal dysfunction. Nowadays it is well known that lysosomes are not merely related to the degradation and recycling of cellular waste, but are also involved in fundamental processes such as secretion, plasma membrane repair, signaling, energy metabolism and autophagy. The essential role of lysosomes in these cellular processes has significant implications for health and disease. ATP13A2 is localized in lysosomes and late endosomes and its mutation leads to lysosome dysfunction, diminishes the exosome secretion and impairs autophagic flux. In this review, we first describe ATP13A2-associated disorders and their relation with the endolysosomal pathway. We then describe the ATP13A2-involvement in iron homeostasis and its potential linkage with new pathologies like cancer, and finally, we consider the putative role of ATP13A2 in lipid processing and degradation, opening the interesting possibility of a broader role of this protein providing protection against a variety of disease-associated changes affecting cellular homeostasis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Objective: VEGF (vascular endothelial growth factor-A) signaling to the endothelial cell (EC) through VEGFR2 (VEGF receptor-2) is the principal cue driving new blood vessel formation. FGD5 (faciogenital dysplasia-5)-a Rho-family guanine nucleotide exchange factor-is selectively expressed in EC. Deficiency of FGD5 is embryonically lethal in mice and perturbs angiogenesis and VEGF signal transduction. However, the mechanism of FGD5 regulation of VEGF signaling is poorly understood., Approach and Results: Angiogenic sprouting and EC cytoskeletal remodeling were evaluated in a 3-dimensional in vitro model. We examined the subcellular localization of FGD5 and VEGFR2 in EC by immunofluorescent staining and studied the association by immunoprecipitation. FGD5 deficiency reduced the number of angiogenic sprouts and tip cell filopodia by ≈80% and ≈70%, respectively. These defects were accompanied by downregulation of the expression of tip cell-specific markers. FGD5 inactivation led to a decrease in EC migration and early protrusion (lamellipodia) formation. In resting and VEGF-stimulated EC, FGD5 forms a complex with VEGFR2 and was enriched at the leading edge of the cell and among endosomes. FGD5 loss reduced mTORC2 (mammalian target of rapamycin complex-2)/Akt-dependent cortactin activation downstream of VEGFR2 but did not alter VEGFR2 plasma membrane expression, Y1175 phosphorylation, or endocytosis. However, FGD5 loss decreased endosomal VEGFR2 coupling to phosphoinositide-3 kinase and diverted VEGFR2 to lysosomal degradation., Conclusions: FGD5 regulates VEGFR2 retention in recycling endosomes and coupling to PI3 (phosphoinositide-3) kinase/mTORC2-dependent cytoskeletal remodeling., (© 2017 American Heart Association, Inc.)

Membrane tethering is a fundamental process essential for the compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, are reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans -Rab assemblies on opposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane-tethering reactions., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Isoprenylcysteine carboxylmethyltransferase (ICMT) catalyzes the post-translational modification of RAB GTPases that contain C-terminal CXC motifs. However, the functional impact of this modification on RAB proteins has not been actively explored. We found that inhibition of ICMT significantly reduced cell migration in vitro and cancer invasion and metastasis in vivo. This role of ICMT was found to be mediated by RAB4A, an essential regulator of the fast recycling of integrin β3. Integrin β3 regulates cell polarity and migration when localized appropriately to the plasma membrane, thereby having an essential role in cancer metastasis. ICMT catalyzed carboxylmethylation is critical for RAB4A activation and interaction with effectors, its localization to endosomes and recycling vesicles, and hence important for RAB4A-dependent integrin β3 recycling to plasma membrane. These findings bring attention to the effects of C-terminal carboxylmethylation on RAB GTPases and provide a rationale for targeting ICMT in the treatment of metastatic cancer.

Regulator of the H + -ATPase of the vacuolar and endosomal membranes (RAVE) is essential for the reversible assembly of H + -ATPase. RAVE primarily consists of three subunits: Rav1p, Rav2p and Skp1p. To characterize these subunits, in this study, four strains derived from Saccharomyces cerevisiae BY4742 were constructed with a FLAG tag on the Rav1p and Rav2p subunits. Then, the corresponding RAVE containing complex was isolated by affinity purification. Western blot and MALDI-TOF mass spectrometry analyses showed that the RAVE complex contains not only the known V 1 -ATPase subunits (Vma1p and Vma2p) but also a newly found Leu1p that interacts with the RAVE subunit. Furthermore, we constructed rav1-/rav2-/vma2-/leu1-deficient recombinants by fusion PCR and homologous recombination and demonstrated that leu1 is indispensable in adjusting the microbial cell to adverse environments and that the function is similar to that of rav1/rav2 but significantly differs from that of vma2. Leu1p probably plays an important role in RAVE regulation of V-ATPase activity in conjunction with RAVE.

The PI 3-kinase Vps34 (Pik3c3) synthesizes phosphatidylinositol 3-phosphate (PI3P), a lipid critical for both endosomal membrane traffic and macroautophagy. Human genetics have implicated PI3P dysregulation, and endosomal trafficking in general, as a recurring cause of demyelinating Charcot-Marie-Tooth (CMT) peripheral neuropathy. Here, we investigated the role of Vps34, and PI3P, in mouse Schwann cells by selectively deleting Vps34 in this cell type. Vps34-Schwann cell knockout (Vps34 SCKO ) mice show severe hypomyelination in peripheral nerves. Vps34 -/- Schwann cells interact abnormally with axons, and there is a delay in radial sorting, a process by which large axons are selected for myelination. Upon reaching the promyelinating stage, Vps34 -/- Schwann cells are significantly impaired in the elaboration of myelin. Nerves from Vps34 SCKO mice contain elevated levels of the LC3 and p62 proteins, indicating impaired autophagy. However, in the light of recent demonstrations that autophagy is dispensable for myelination, it is unlikely that hypomyelination in Vps34 SCKO mice is caused by impaired autophagy. Endosomal trafficking is also disturbed in Vps34 -/- Schwann cells. We investigated the activation of the ErbB2/3 receptor tyrosine kinases in Vps34 SCKO nerves, as these proteins, which play essential roles in Schwann cell myelination, are known to traffic through endosomes. In Vps34 SCKO nerves, ErbB3 was hyperphosphorylated on a tyrosine known to be phosphorylated in response to neuregulin 1 exposure. ErbB2 protein levels were also decreased during myelination. Our findings suggest that the loss of Vps34 alters the trafficking of ErbB2/3 through endosomes. Abnormal ErbB2/3 signaling to downstream targets may contribute to the hypomyelination observed in Vps34 SCKO mice., (© 2017 Wiley Periodicals, Inc.)

The iron-regulated metastasis suppressor N-myc downstream-regulated gene 1 (NDRG1) has been shown to inhibit numerous oncogenic signaling pathways in cancer cells. Recent findings have demonstrated that NDRG1 inhibits the ErbB family of receptors, which function as key inducers of carcinogenesis. NDRG1 attenuates ErbB signaling by inhibiting formation of epidermal growth factor receptor (EGFR)/human epidermal growth factor receptor 2 (HER2) and HER2/HER3 heterodimers and by down-regulating EGFR via a mechanism involving its degradation. Understanding the complex interplay between NDRG1, iron, and ErbB signaling is vital for identifying novel, more effective targets for cancer therapy., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes., (© 2017 Lőrincz et al.)

Adaptation to environmental stress is critical for cell survival. Adaptation generally occurs via changes in transcription and translation. However, there is a time lag before changes in gene expression, which suggests that more rapid mechanisms likely exist. In this study, we show that in yeast, the cyclin-dependent kinase Pho85/CDK5 provides protection against hyperosmotic stress and acts before long-term adaptation provided by Hog1. This protection requires the vacuolar/endolysosomal signaling lipid PI3,5P 2 We show that Pho85/CDK5 directly phosphorylates and positively regulates the PI3P-5 kinase Fab1/PIKfyve complex and provide evidence that this regulation is conserved in mammalian cells. Moreover, this regulation is particularly crucial in yeast for the stress-induced transient elevation of PI3,5P 2 Our study reveals a rapid protection mechanism regulated by Pho85/CDK5 via signaling from the vacuole/lysosome, which is distinct temporally and spatially from the previously discovered long-term adaptation Hog1 pathway, which signals from the nucleus., (© 2017 Jin et al.)

The endosomal sorting complex required for transport (ESCRT)-III mediates membrane fission in fundamental cellular processes, including cytokinesis. ESCRT-III is thought to form persistent filaments that over time increase their curvature to constrict membranes. Unexpectedly, we found that ESCRT-III at the midbody of human cells rapidly turns over subunits with cytoplasmic pools while gradually forming larger assemblies. ESCRT-III turnover depended on the ATPase VPS4, which accumulated at the midbody simultaneously with ESCRT-III subunits, and was required for assembly of functional ESCRT-III structures. In vitro, the Vps2/Vps24 subunits of ESCRT-III formed side-by-side filaments with Snf7 and inhibited further polymerization, but the growth inhibition was alleviated by the addition of Vps4 and ATP. High-speed atomic force microscopy further revealed highly dynamic arrays of growing and shrinking ESCRT-III spirals in the presence of Vps4. Continuous ESCRT-III remodelling by subunit turnover might facilitate shape adaptions to variable membrane geometries, with broad implications for diverse cellular processes.

The covalent attachment of ubiquitin onto proteins can elicit a variety of downstream consequences. Attachment is mediated by a large array of E3 ubiquitin ligases, each thought be subject to regulatory control and to have a specific repertoire of substrates. Assessing the biological roles of ligases, and in particular, identifying their biologically relevant substrates has been a persistent yet challenging question. In this study, we describe tools that may help achieve both of these goals. We describe a strategy whereby the activity of a ubiquitin ligase has been enzymatically reversed, accomplished by fusing it to a catalytic domain of an exogenous deubiquitinating enzyme. We present a library of 72 "anti-ligases" that appear to work in a dominant-negative fashion to stabilize their cognate substrates against ubiquitin-dependent proteasomal and lysosomal degradation. We then used the ligase-deubiquitinating enzyme (DUb) library to screen for E3 ligases involved in post-Golgi/endosomal trafficking. We identify ligases previously implicated in these pathways (Rsp5 and Tul1), in addition to ligases previously localized to endosomes (Pib1 and Vps8). We also document an optimized workflow for isolating and analyzing the "ubiquitome" of yeast, which can be used with mass spectrometry to identify substrates perturbed by expression of particular ligase-DUb fusions., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Apoptotic cells generated by programmed cell death are engulfed by phagocytes and enclosed within plasma membrane-derived phagosomes. Maturation of phagosomes involves a series of membrane-remodeling events that are governed by the sequential actions of Rab GTPases and lead to formation of phagolysosomes, where cell corpses are degraded. Here we identified gop-1 as a novel regulator of apoptotic cell clearance in Caenorhabditis elegans Loss of gop-1 affects phagosome maturation through the RAB-5-positive stage, causing defects in phagosome acidification and phagolysosome formation, phenotypes identical to and unaffected by loss of unc-108, the C. elegans Rab2 GOP-1 transiently associates with cell corpse-containing phagosomes, and loss of its function abrogates phagosomal association of UNC-108. GOP-1 interacts with GDP-bound and nucleotide-free UNC-108/Rab2, disrupts GDI-UNC-108 complexes, and promotes activation and membrane recruitment of UNC-108/Rab2 in vitro. Loss of gop-1 also abolishes association of UNC-108 with endosomes, causing defects in endosome and dense core vesicle maturation. Thus, GOP-1 is an activator of UNC-108/Rab2 in multiple processes., (© 2017 Yin et al.)

Nutrient sensing by mechanistic target of rapamycin complex 1 (mTORC1) on lysosomes and late endosomes (LyLEs) regulates cell growth. Many factors stimulate mTORC1 activity, including the production of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P 3 ] by class I phosphatidylinositol 3-kinases (PI3Ks) at the plasma membrane. We investigated mechanisms that repress mTORC1 under conditions of growth factor deprivation. We identified phosphatidylinositol 3,4-bisphosphate [PI(3,4)P 2 ], synthesized by class II PI3K β (PI3KC2β) at LyLEs, as a negative regulator of mTORC1, whereas loss of PI3KC2β hyperactivated mTORC1. Growth factor deprivation induced the association of PI3KC2β with the Raptor subunit of mTORC1. Local PI(3,4)P 2 synthesis triggered repression of mTORC1 activity through association of Raptor with inhibitory 14-3-3 proteins. These results unravel an unexpected function for local PI(3,4)P 2 production in shutting off mTORC1., (Copyright © 2017, American Association for the Advancement of Science.)

Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P 2 in T. brucei. We find a low steady state level of PI(3,5)P 2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P 2 pool leading to rapid cell death. TbFab1 and PI(3,5)P 2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P 2 metabolism, the PI(3,5)P 2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P 2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Recently, intracellular receptor signaling has been identified as a key component mediating cell responses for various receptor tyrosine kinases (RTKs). However, the extent each endocytic compartment (endocytic vesicle, early endosome, recycling endosome, late endosome, lysosome and nucleus) contributes to receptor signaling has not been quantified. Furthermore, our understanding of endocytosis and receptor signaling is complicated by cell- or receptor-specific endocytosis mechanisms. Therefore, towards understanding the differential endocytic compartment signaling roles, and identifying how to achieve signal transduction control for RTKs, we delineate how endocytosis regulates RTK signaling. We achieve this via a meta-analysis across eight RTKs, integrating computational modeling with experimentally derived cell (compartment volume, trafficking kinetics and pH) and ligand-receptor (ligand/receptor concentration and interaction kinetics) physiology. Our simulations predict the abundance of signaling from eight RTKs, identifying the following hierarchy in RTK signaling: PDGFRβ > IGFR1 > EGFR > PDGFRα > VEGFR1 > VEGFR2 > Tie2 > FGFR1. We find that endocytic vesicles are the primary cell signaling compartment; over 43% of total receptor signaling occurs within the endocytic vesicle compartment for these eight RTKs. Mechanistically, we found that high RTK signaling within endocytic vesicles may be attributed to their low volume (5.3 × 10 -19 L) which facilitates an enriched ligand concentration (3.2 μM per ligand molecule within the endocytic vesicle). Under the analyzed physiological conditions, we identified extracellular ligand concentration as the most sensitive parameter to change; hence the most significant one to modify when regulating absolute compartment signaling. We also found that the late endosome and nucleus compartments are important contributors to receptor signaling, where 26% and 18%, respectively, of average receptor signaling occurs across the eight RTKs. Conversely, we found very low membrane-based receptor signaling, exhibiting <1% of the total receptor signaling for these eight RTKs. Moreover, we found that nuclear translocation, mechanistically, requires late endosomal transport; when we blocked receptor trafficking from late endosomes to the nucleus we found a 57% reduction in nuclear translocation. In summary, our research has elucidated the significance of endocytic vesicles, late endosomes and the nucleus in RTK signal propagation.

Phosphatidylinositol-3-phosphate (PI3P) is a key player in membrane dynamics and trafficking regulation. Most PI3P is associated with endosomal membranes and with the autophagosome preassembly machinery, presumably at the endoplasmic reticulum. The enzyme responsible for most PI3P synthesis, VPS34 and proteins such as Beclin1 and ATG14L that regulate PI3P levels are positive modulators of autophagy initiation. It had been assumed that a local PI3P pool was present at autophagosomes and preautophagosomal structures, such as the omegasome and the phagophore. This was recently confirmed by the demonstration that PI3P-binding proteins participate in the complex sequence of signalling that results in autophagosome assembly and activity. Here we summarize the historical discoveries of PI3P lipid kinase involvement in autophagy, and we discuss the proposed role of PI3P during autophagy, notably during the autophagosome biogenesis sequence., (© 2016 Federation of European Biochemical Societies.)

Objectives: Hydroxychloroquine (HCQ) has been used for decades to treat patients with rheumatic diseases, for example, systemic lupus erythematosus (SLE), rheumatoid arthritis or the antiphospholipid syndrome (APS). We hypothesise that HCQ might target endosomal NADPH oxidase (NOX), which is involved in the signal transduction of cytokines as well as antiphospholipid antibodies (aPL)., Methods: For in vitro experiments, monocytic cells were stimulated with tumour necrosis factor α (TNFα), interleukin-1β (IL-1β) or a human monoclonal aPL and the activity of NOX was determined by flow cytometry. The expression of genes known to be induced by these stimuli was quantified by quantitative reverse transcription PCR. Live cell imaging was performed by confocal laser scanning microscopy. Finally, the effects of HCQ on NOX-induced signal transduction were analysed in an in vivo model of venous thrombosis., Results: HCQ strongly reduces or completely prevents the induction of endosomal NOX by TNFα, IL-1β and aPL in human monocytes and MonoMac1 cells. As a consequence, induction of downstream genes by these stimuli is reduced or abrogated. This effect of HCQ is not mediated by direct interference with the agonists but by inhibiting the translocation of the catalytic subunit of NOX2 (gp91phox) into the endosome. In vivo, HCQ protects mice from aPL-induced and NOX2-mediated thrombus formation., Conclusions: We describe here a novel mechanism of action of HCQ, that is, interference with the assembly of endosomal NOX2. Since endosomal NOX2 is involved in many inflammatory and prothrombotic signalling pathways, this activity of HCQ might explain many of its beneficial effects in rheumatic diseases including the APS., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to activate Akt and also demonstrates that M-COPA is efficacious for growth suppression of neoplastic mast cells.