Spatially Distinct Pools of TORC1 Balance Protein Homeostasis.

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.

IST1 regulates select recycling pathways.

ESCRTs (Endosomal Sorting Complex Required for Transports) are a modular set of protein complexes with membrane remodeling activities that include the formation and release of intraluminal vesicles (ILVs) to generate multivesicular endosomes. While most of the 12 ESCRT-III proteins are known to play roles in ILV formation, IST1 has been associated with a wider range of endosomal remodeling events. Here, we extend previous studies of IST1 function in endosomal trafficking and confirm that IST1, along with its binding partner CHMP1B, contributes to scission of early endosomal carriers. Functionally, depleting IST1 impaired delivery of transferrin receptor from early/sorting endosomes to the endocytic recycling compartment and instead increased its rapid recycling to the plasma membrane via peripheral endosomes enriched in the clathrin adaptor AP-1. IST1 is also important for export of mannose 6-phosphate receptor from early/sorting endosomes. Examination of IST1 binding partners on endosomes revealed that IST1 interacts with the MIT domain-containing sorting nexin SNX15, a protein previously reported to regulate endosomal recycling. Our kinetic and spatial analyses establish that SNX15 and IST1 occupy a clathrin-containing subdomain on the endosomal perimeter distinct from those previously implicated in cargo retrieval or degradation. Using live-cell microscopy, we see that SNX15 and CHMP1B alternately recruit IST1 to this subdomain or the base of endosomal tubules. These findings indicate that IST1 contributes to a subset of recycling pathways from the early/sorting endosome.

O-GlcNAcylation regulates epidermal growth factor receptor intracellular trafficking and signaling.

SignificanceEpidermal growth factor receptor (EGFR) is one of the most important membrane receptors that transduce growth signals into cells to sustain cell growth, proliferation, and survival. EGFR signal termination is initiated by EGFR internalization, followed by trafficking through endosomes, and degradation in lysosomes. How this process is regulated is still poorly understood. Here, we show that hepatocyte growth factor regulated tyrosine kinase substrate (HGS), a key protein in the EGFR trafficking pathway, is dynamically modified by a single sugar N-acetylglucosamine. This modification inhibits EGFR trafficking from endosomes to lysosomes, leading to the accumulation of EGFR and prolonged signaling. This study provides an important insight into diseases with aberrant growth factor signaling, such as cancer, obesity, and diabetes.

Structure and dynamics of ESCRT-III membrane remodeling proteins by high-speed atomic force microscopy.

Endosomal sorting complex required for transport (ESCRT) proteins assemble on the cytoplasmic leaflet of membranes and remodel them. ESCRT is involved in biological processes where membranes are bent away from the cytosol, constricted, and finally severed, such as in multivesicular body formation (in the endosomal pathway for protein sorting) or abscission during cell division. The ESCRT system is hijacked by enveloped viruses to allow buds of nascent virions to be constricted, severed, and released. ESCRT-III proteins, the most downstream components of the ESCRT system, are monomeric and cytosolic in their autoinhibited conformation. They share a common architecture, a four-helix bundle with a fifth helix that interacts with this bundle to prevent polymerizing. Upon binding to negatively charged membranes, the ESCRT-III components adopt an activated state that allows them to polymerize into filaments and spirals and to interact with the AAA-ATPase Vps4 for polymer remodeling. ESCRT-III has been studied with electron microscopy and fluorescence microscopy; these methods provided invaluable information about ESCRT assembly structures or their dynamics, respectively, but neither approach provides detailed insights into both aspects simultaneously. High-speed atomic force microscopy (HS-AFM) has overcome this shortcoming, providing movies at high spatiotemporal resolution of biomolecular processes, significantly increasing our understanding of ESCRT-III structure and dynamics. Here, we review the contributions of HS-AFM in the analysis of ESCRT-III, focusing on recent developments of nonplanar and deformable HS-AFM supports. We divide the HS-AFM observations into four sequential steps in the ESCRT-III lifecycle: (1) polymerization, (2) morphology, (3) dynamics, and (4) depolymerization.

Regulation of Hook1-mediated endosomal sorting of clathrin-independent cargo by γ-taxilin.

In clathrin-independent endocytosis, Hook1, a microtubule- and cargo-tethering protein, participates in sorting of cargo proteins such as CD98 (encoded by SLC3A2) and CD147 (encoded by BSG) into recycling endosomes. However, the molecular mechanism that regulates Hook1-mediated endosomal sorting is not fully understood. In the present study, we found that γ-taxilin is a novel regulator of Hook1-mediated endosomal sorting. γ-Taxilin depletion promoted both CD98-positive tubular formation and CD98 recycling. Conversely, overexpression of γ-taxilin inhibited the CD98-positive tubular formation. Depletion of Hook1, or Rab10 or Rab22a (which are both involved in Hook1-mediated endosomal sorting), attenuated the effect of γ-taxilin depletion on the CD98-positive tubular formation. γ-Taxilin depletion promoted CD147-mediated spreading of HeLa cells, suggesting that γ-taxilin might be a pivotal player in various cellular functions in which Hook1-mediated cargo proteins are involved. γ-Taxilin bound to the C-terminal region of Hook1 and inhibited its interaction with CD98; the latter interaction is necessary for sorting CD98. We suggest that γ-taxilin negatively regulates the sorting of Hook1-mediated cargo proteins into recycling endosomes by interfering with the interactions between Hook1 and the cargo proteins.

SNX27-FERM-SNX1 complex structure rationalizes divergent trafficking pathways by SNX17 and SNX27.

The molecular events that determine the recycling versus degradation fates of internalized membrane proteins remain poorly understood. Two of the three members of the SNX-FERM family, SNX17 and SNX31, utilize their FERM domain to mediate endocytic trafficking of cargo proteins harboring the NPxY/NxxY motif. In contrast, SNX27 does not recycle NPxY/NxxY-containing cargo but instead recycles cargo containing PDZ-binding motifs via its PDZ domain. The underlying mechanism governing this divergence in FERM domain binding is poorly understood. Here, we report that the FERM domain of SNX27 is functionally distinct from SNX17 and interacts with a novel DLF motif localized within the N terminus of SNX1/2 instead of the NPxY/NxxY motif in cargo proteins. The SNX27-FERM-SNX1 complex structure reveals that the DLF motif of SNX1 binds to a hydrophobic cave surrounded by positively charged residues on the surface of SNX27. The interaction between SNX27 and SNX1/2 is critical for efficient SNX27 recruitment to endosomes and endocytic recycling of multiple cargoes. Finally, we show that the interaction between SNX27 and SNX1/2 is critical for brain development in zebrafish. Altogether, our study solves a long-standing puzzle in the field and suggests that SNX27 and SNX17 mediate endocytic recycling through fundamentally distinct mechanisms.

Cellular abundance of sodium phosphate cotransporter SLC20A1/PiT1 and phosphate uptake are controlled post-transcriptionally by ESCRT.

Inorganic phosphate is essential for human life. The widely expressed mammalian sodium/phosphate cotransporter SLC20A1/PiT1 mediates phosphate uptake into most cell types; however, while SLC20A1 is required for development, and elevated SLC20A1 expression is associated with vascular calcification and aggressive tumor growth, the mechanisms regulating SLC20A1 protein abundance are unknown. Here, we found that SLC20A1 protein expression is low in phosphate-replete cultured cells but is strikingly induced following phosphate starvation, whereas mRNA expression is high in phosphate-replete cells and only mildly increased by phosphate starvation. To identify regulators of SLC20A1 protein levels, we performed a genome-wide CRISPR-based loss-of-function genetic screen in phosphate-replete cells using SLC20A1 protein induction as readout. Our screen revealed that endosomal sorting complexes required for transport (ESCRT) machinery was essential for proper SLC20A1 protein downregulation in phosphate-replete cells. We show that SLC20A1 colocalizes with ESCRT and that ESCRT deficiency increases SLC20A1 protein and phosphate uptake into cells. We also found numerous additional candidate regulators of mammalian phosphate homeostasis, including genes modifying protein ubiquitination and the Krebs cycle and oxidative phosphorylation pathways. Many of these targets have not been previously implicated in this process. We present here a model in which SLC20A1 protein abundance and phosphate uptake are tonically negatively regulated post-transcriptionally in phosphate-replete cells through direct ESCRT-mediated SLC20A1 degradation. Moreover, our screening results provide a comprehensive resource for future studies to elucidate the mechanisms governing cellular phosphate homeostasis. We conclude that genome-wide CRISPR-based genetic screening is a powerful tool to discover proteins and pathways relevant to physiological processes.

De Novo VPS4A Mutations Cause Multisystem Disease with Abnormal Neurodevelopment.

The endosomal sorting complexes required for transport (ESCRTs) are essential for multiple membrane modeling and membrane-independent cellular processes. Here we describe six unrelated individuals with de novo missense variants affecting the ATPase domain of VPS4A, a critical enzyme regulating ESCRT function. Probands had structural brain abnormalities, severe neurodevelopmental delay, cataracts, growth impairment, and anemia. In cultured cells, overexpression of VPS4A mutants caused enlarged endosomal vacuoles resembling those induced by expression of known dominant-negative ATPase-defective forms of VPS4A. Proband-derived fibroblasts had enlarged endosomal structures with abnormal accumulation of the ESCRT protein IST1 on the limiting membrane. VPS4A function was also required for normal endosomal morphology and IST1 localization in iPSC-derived human neurons. Mutations affected other ESCRT-dependent cellular processes, including regulation of centrosome number, primary cilium morphology, nuclear membrane morphology, chromosome segregation, mitotic spindle formation, and cell cycle progression. We thus characterize a distinct multisystem disorder caused by mutations affecting VPS4A and demonstrate that its normal function is required for multiple human developmental and cellular processes.

Ykt6-dependent endosomal recycling is required for Wnt secretion in the Drosophila wing epithelium.

Morphogens are important signalling molecules for tissue development and their secretion requires tight regulation. In the wing imaginal disc of flies, the morphogen Wnt/Wingless is apically presented by the secreting cell and re-internalized before final long-range secretion. Why Wnt molecules undergo these trafficking steps and the nature of the regulatory control within the endosomal compartment remain unclear. Here, we have investigated how Wnts are sorted at the level of endosomes by the versatile v-SNARE Ykt6. Using in vivo genetics, proximity-dependent proteomics and in vitro biochemical analyses, we show that most Ykt6 is present in the cytosol, but can be recruited to de-acidified compartments and recycle Wnts to the plasma membrane via Rab4-positive recycling endosomes. Thus, we propose a molecular mechanism by which producing cells integrate and leverage endocytosis and recycling via Ykt6 to coordinate extracellular Wnt levels.

Inhibition of PIKFYVE kinase interferes ESCRT pathway to suppress RNA virus replication.

Endosomal sorting complex required for transport (ESCRT) is essential in the functional operation of endosomal transport in envelopment and budding of enveloped RNA viruses. However, in nonenveloped RNA viruses such as enteroviruses of the Picornaviridae family, the precise function of ESCRT pathway in viral replication remains elusive. Here, we initially evaluated that the ESCRT pathway is important for viral replication upon enterovirus 71 (EV71) infection. Furthermore, we discovered that YM201636, a specific inhibitor of phosphoinositide kinase, FYVE finger containing (PIKFYVE) kinase, significantly suppressed EV71 replication and virus-induced inflammation in vitro and in vivo. Mechanistically, YM201636 inhibits PIKFYVE kinase to block the ESCRT pathway and endosomal transport, leading to the disruption of viral entry and replication complex in subcellular components and ultimately repression of intracellular RNA virus replication and virus-induced inflammatory responses. Further studies found that YM201636 broadly represses the replication of other RNA viruses, including coxsackievirus B3 (CVB3), poliovirus 1 (PV1), echovirus 11 (E11), Zika virus (ZIKV), and vesicular stomatitis virus (VSV), rather than DNA viruses, including adenovirus 3 (ADV3) and hepatitis B virus (HBV). Our findings shed light on the mechanism underlying PIKFYVE-modulated ESCRT pathway involved in RNA virus replication, and also provide a prospective antiviral therapy during RNA viruses infections.

Endosomal Sorting Complex Required for Transport: The Molecular Machine for Ensuring Proper Mitosis.

Mitosis is a key step of eukaryote proliferation.Endosomal sorting complex required for transport, a protein complex closely associated with membrane shearing, is involved in endosome maturation, virus budding, and autophagy.The structural and functional abnormalities of the complex are associated with the occurrence and progression of cancer and other diseases.In this paper, we summarized the roles of the endosomal sorting complex required for transport in different stages of mitosis and reviewed the studies about the role of the complex in regulating mitosis in diseases.

ESCRT-dependent protein sorting is required for the viability of yeast clathrin-mediated endocytosis mutants.

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin-mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin-associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N-terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME-deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony-sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)-dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin-independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild-type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1-dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT-dependent trafficking on endocytic recycling and the PM.

Mechanistic roles of tyrosine phosphorylation in reversible amyloids, autoinhibition, and endosomal membrane association of ALIX.

Human apoptosis-linked gene-2 interacting protein X (ALIX), a versatile adapter protein, regulates essential cellular processes by shuttling between late endosomal membranes and the cytosol, determined by its interactions with Src kinase. Here, we investigate the molecular basis of these transitions and the effects of tyrosine phosphorylation on the interplay between structure, assembly, and intramolecular and intermolecular interactions of ALIX. As evidenced by transmission electron microscopy, fluorescence and circular dichroism spectroscopy, the proline-rich domain of ALIX, which encodes binding epitopes of multiple cellular partners, formed rope-like ß-sheet-rich reversible amyloid fibrils that dissolved upon Src-mediated phosphorylation and were restored on protein-tyrosine phosphatase 1B-mediated dephosphorylation of its conserved tyrosine residues. Analyses of the Bro1 domain of ALIX by solution NMR spectroscopy elucidated the conformational changes originating from its phosphorylation by Src and established that Bro1 binds to hyperphosphorylated proline-rich domain and to analogs of late endosomal membranes via its highly basic surface. These results uncover the autoinhibition mechanism that relocates ALIX to the cytosol and the diverse roles played by tyrosine phosphorylation in cellular and membrane functions of ALIX.

Recycling of SLC38A1 to the plasma membrane by DSCR3 promotes acquired temozolomide resistance in glioblastoma.

PURPOSE: Glioblastoma multiforme (GBM) is a primary brain tumor with devastating prognosis. Although the O6-methylguanine-DNA methyltransferase (MGMT) leads to inherent temozolomide (TMZ) resistance, approximately half of GBMs were sufficient to confer acquired TMZ resistance, which express low levels of MGMT. The purpose of this study was to investigate the underlying mechanisms of the acquired TMZ resistance in MGMT-deficient GBM. METHODS: The function of Down syndrome critical region protein 3 (DSCR3) on MGMT-deficient GBM was investigated in vitro and in an orthotopic brain tumor model in mice. Purification of plasma membrane proteins by membrane-cytoplasmic separation and subsequent label free-based quantitative proteomics were used to identified potential protein partners for DSCR3. Immunofluorescence was performed to show the reverse transport of solute carrier family 38 member 1 (SLC38A1) mediated by DSCR3. RESULTS: DSCR3 is upregulated in MGMT-deficient GBM cells during TMZ treatment. Both DSCR3 and SLC38A1 were highly expressed in recurrent GBM patients. Silencing DSCR3 or SLC38A1 expression can increase TMZ sensitivity in MGMT-deficient GBM cells. Combination of proteomics and in vitro experiments show that DSCR3 directly binds internalized SLC38A1 to mediate its sorting into recycling pathway, which maintains the abundance on plasma membrane and enhances uptake of glutamine in MGMT-deficient GBM cells. CONCLUSIONS: DSCR3 is a crucial regulator of acquired TMZ resistance in MGMT-deficient GBM. The DSCR3-dependent recycling of SLC38A1 maintains its abundance on plasma membrane, leading to tumor progression and acquired TMZ resistance in MGMT-deficient GBM.

RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.

Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.

The ESCRT-III isoforms CHMP2A and CHMP2B display different effects on membranes upon polymerization.

BACKGROUND: ESCRT-III proteins are involved in many membrane remodeling processes including multivesicular body biogenesis as first discovered in yeast. In humans, ESCRT-III CHMP2 exists as two isoforms, CHMP2A and CHMP2B, but their physical characteristics have not been compared yet. RESULTS: Here, we use a combination of techniques on biomimetic systems and purified proteins to study their affinity and effects on membranes. We establish that CHMP2B binding is enhanced in the presence of PI(4,5)P2 lipids. In contrast, CHMP2A does not display lipid specificity and requires CHMP3 for binding significantly to membranes. On the micrometer scale and at moderate bulk concentrations, CHMP2B forms a reticular structure on membranes whereas CHMP2A (+CHMP3) binds homogeneously. Thus, CHMP2A and CHMP2B unexpectedly induce different mechanical effects to membranes: CHMP2B strongly rigidifies them while CHMP2A (+CHMP3) has no significant effect. CONCLUSIONS: We therefore conclude that CHMP2B and CHMP2A exhibit different mechanical properties and might thus contribute differently to the diverse ESCRT-III-catalyzed membrane remodeling processes.

HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region.

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane-binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.

Host ESCRT factors are recruited during chikungunya virus infection and are required for the intracellular viral replication cycle.

Chikungunya fever is a re-emerging zoonotic disease caused by chikungunya virus (CHIKV), a member of the Alphavirus genus in the Togaviridae family. Only a few studies have reported on the host factors required for intracellular CHIKV trafficking. Here, we conducted an imaging-based siRNA screen to identify human host factors for intracellular trafficking that are involved in CHIKV infection, examined their interactions with CHIKV proteins, and investigated the contributions of these proteins to CHIKV infection. The results of the siRNA screen revealed that host endosomal sorting complexes required for transport (ESCRT) proteins are recruited during CHIKV infection. Co-immunoprecipitation analyses revealed that both structural and nonstructural CHIKV proteins interact with hepatocyte growth factor-regulated tyrosine kinase substrate (HGS), a component of the ESCRT-0 complex. We also observed that HGS co-localizes with the E2 protein of CHIKV and with dsRNA, a marker of the replicated CHIKV genome. Results from gene knockdown analyses indicated that, along with other ESCRT factors, HGS facilitates both genome replication and post-translational steps during CHIKV infection. Moreover, we show that ESCRT factors are also required for infections with other alphaviruses. We conclude that during CHIKV infection, several ESCRT factors are recruited via HGS and are involved in viral genome replication and post-translational processing of viral proteins.

TOR complex 2 (TORC2) signaling and the ESCRT machinery cooperate in the protection of plasma membrane integrity in yeast.

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

The machinery for endocytosis of epidermal growth factor receptor coordinates the transport of incoming hepatitis B virus to the endosomal network.

Sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the surface of human hepatocytes and functions as an entry receptor of hepatitis B virus (HBV). Recently, we have reported that epidermal growth factor receptor (EGFR) is involved in NTCP-mediated viral internalization during the cell entry process. Here, we analyzed which function of EGFR is essential for mediating HBV internalization. In contrast to the reported crucial function of EGFR-downstream signaling for the entry of hepatitis C virus (HCV), blockade of EGFR-downstream signaling proteins, including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K), and signal transducer and activator of transcription (STAT), had no or only minor effects on HBV infection. Instead, deficiency of EGFR endocytosis resulting from either a deleterious mutation in EGFR or genetic knockdown of endocytosis adaptor molecules abrogated internalization of HBV via NTCP and prevented viral infection. EGFR activation triggered a time-dependent relocalization of HBV preS1 to the early and late endosomes and to lysosomes in concert with EGFR transport. Suppression of EGFR ubiquitination by site-directed mutagenesis or by knocking down two EGFR-sorting molecules, signal-transducing adaptor molecule (STAM) and lysosomal protein transmembrane 4ß (LAPTM4B), suggested that EGFR transport to the late endosome is critical for efficient HBV infection. Cumulatively, these results support the idea that the EGFR endocytosis/sorting machinery drives the translocation of NTCP-bound HBV from the cell surface to the endosomal network, which eventually enables productive viral infection.