Calmodulin-like 5 promotes PEDV replication by regulating late-endosome synthesis and innate immune response

The infection caused by porcine epidemic diarrhea virus(PEDV)is associated with high mortality in piglets worldwide.Host factors involved in the efficient replication of PEDV,however,remain largely unknown.Our recent proteomic study in the virus-host interaction network revealed a significant increase in the accumulation of CALML5(EF-hand protein calmodulin-like 5)following PEDV infection.A further study unveiled a biphasic increase of CALML5 in 2 and 12 h after viral infection.Similar trends were observed in the intestines of piglets in the early and late stages of the PEDV challenge.Moreover,CALML5 depletion reduced PEDV mRNA and protein levels,leading to a one-order-of-magnitude decrease in virus titer.At the early stage of PEDV infection,CALML5 affected the endosomal trafficking pathway by regulating the expression of endosomal sorting complex related cellular proteins.CALML5 depletion also suppressed IFN-βand IL-6 production in the PEDV-infected cells,thereby indicating its involvement in negatively regulating the innate immune response.Our study reveals the biological function of CALML5 in the virology field and offers new insights into the PEDV-host cell interaction.

New aspects of a small GTPase RAB35 in brain development and function

In eukaryotic cells,organelles in the secretory,lysosomal,and endocytic pathways actively exchange biological materials with each other through intracellular membrane trafficking,which is the process of transporting the cargo of proteins,lipids,and other molecules to appropriate compartments via transport vesicles or intermediates.These processes are strictly regulated by various small GTPases such as the RAS-like in rat brain(RAB)protein family,which is the largest subfamily of the RAS superfamily.Dysfunction of membrane trafficking affects tissue homeostasis and leads to a wide range of diseases,including neurological disorders and neurodegenerative diseases.Therefore,it is important to understand the physiological and pathological roles of RAB proteins in brain function.RAB35,a member of the RAB family,is an evolutionarily conserved protein in metazoans.A wide range of studies using cultured mammalian cells and model organisms have revealed that RAB35 mediates various processes such as cytokinesis,endocytic recycling,actin bundling,and cell migration.RAB35 is also involved in neurite outgrowth and turnover of synaptic vesicles.We generated brain-specific Rab35 knockout mice to study the physiological roles of RAB35 in brain development and function.These mice exhibited defects in anxiety-related behaviors and spatial memory.Strikingly,RAB35 is required for the precise positioning of pyramidal neurons during hippocampal development,and thereby for normal hippocampal lamination.In contrast,layer formation in the cerebral cortex occurred superficially,even in the absence of RAB35,suggesting a predominant role for RAB35 in hippocampal development rather than in cerebral cortex development.Recent studies have suggested an association between RAB35 and neurodegenerative diseases,including Parkinson's disease and Alzheimer's disease.In this review,we provide an overview of the current understanding of subcellular functions of RAB35.We also provide insights into the physiological role of RAB35 in mammalian brain development and function,and discuss the involvement of RAB35 dysfunction in neurodegenerative diseases.

The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice

G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice,but how these pathways are coordinated in regulating this process is unknown.Here,we show that Chang Li Geng 1(CLG1),which encodes an E3 ligase,regulates grain size by targeting the Gγprotein GS3,a negative regulator of grain length,for degradation.Overexpression of CLG1 led to increased grain length,while overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length.We found that CLG1 physically interacts with and ubiquitinats GS3which is subsequently degraded through the endosome degradation pathway,leading to increased grain size.Furthermore,we identified a critical SNP in the exon 3 of CLG1 that is significantly associated with grain size variation in a core collection of cultivated rice.This SNP results in an amino acid substitution from Arg to Ser at position 163 of CLG1 that enhances the E3 ligase activity of CLG1 and thus increases rice grain size.Both the expression level of CLG1 and the SNP CLG1163S may be useful variations for manipulating grain size in rice.

Atg5 regulates late endosome and lysosome biogenesis

Autophagy is an evolutionarily conserved lysosome-based degradation process.Atg5 plays a very important role in autophagosome formation.Here we show that Atg5 is required for biogenesis of late endosomes and lysosomes in an autophagy-independent manner.In Atg5 cells,but not in other essential autophagy genes defecting cells,recycling and retrieval of late endosomal components from hybrid organelles are impaired,causing persistent hybrid organelles and defective formation of late endosomes and lysosomes.Defective retrieval of late endosomal components from hybrid organelles resulting from impaired recruitment of a component of V1-ATPase to acidic organelles blocks the pH-dependent retrieval of late endosomal components from hybrid organelles.Lowering the intracellular pH restores late endosome/lysosome biogenesis in Atg5 cells.Our data demonstrate an unexpected role of Atg5 and shed new light on late endosome and lysosome biogenesis.

Adenosine Diphosphate Ribosylation Factor-GTPase-Activating Protein Stimulates the Transport of AUX1 Endosome,Which Relies on Actin Cytoskeletal Organization in Rice Root Development

Polar auxin transport,which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1（AUX1/LAX） influx carriers and PIN-FORMED（PIN） efflux carriers,mediates various processes of plant growth and development.Endosomal recycling of PIN1 is mediated by an adenosine diphosphate（ADP）ribosylation factor（ARF）-GTPase exchange factor protein,GNOM.However,the mediation of auxin influx carrier recycling is poorly understood.Here,we report that overexpression of OsAGAP,an ARF-GTPase-activating protein in rice,stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1.AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption.Furthermore,OsAGAP is involved in actin cytoskeletal organization,and its overexpression tends to reduce the thickness and bundling of actin filaments.Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells,and was only slightly promoted when the actin filaments were completely disrupted by Lat B.Thus,we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.

Endosomes and Microtubles are Required for Productive Infection in Aquareovirus

Grass carp reovirus(GCRV),the genus Aquareovirus in family Reoviridae,is viewed as the most pathogenic aquareovirus.To understand the molecular mechanism of how aquareovirus initiates productive infection,the roles of endosome and microtubule in cell entry of GCRV are investigated by using quantum dots(QDs)-tracking in combination with biochemical approaches.We found that GCRV infection and viral protein synthesis were significantly inhibited by pretreating host cells with endosome acidification inhibitors NH4Cl,chloroquine and bafilomycin A1(Bafi).Confocal images indicated that GCRV particles could colocalize with Rab5,Rab7 and lysosomes in host cells.Further ultrastructural examination validated that viral particle was found in late endosomes.Moreover,disruption of microtubules with nocodazole clearly blocked GCRV entry,while no inhibitory effects were observed with cytochalasin D treated cells in viral infection,hinting that intracellular transportation of endocytic uptake in GCRV infected cells is via microtubules but not actin filament.Notably,viral particles were observed to transport along microtubules by using QD-labeled GCRV.Altogether,our results suggest that GCRV can use endosomes and microtubules to initiate productive infection.

Evaluation of the reducing potential of PSMA-containing endosomes by FRET imaging

Aim:Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues.For many ligand-targeted drug conjugates,a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells.Because disulfide bond reduction is frequently exploited to ensure intracellular drug release,an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design.While the redox properties of folate receptor trafficking endosomes have been previously reported,little is known about the trafficking of prostate-specific membrane antigen(PSMA),a receptor that is experiencing increasing use for drug targeting in humans.Methods:To obtain this information,we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green.Results:We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells,and that reduction of its disulfide bond proceeds steadily but incompletely following internalization.The fact that maximal disulfide reduction reaches only~50%,even after 24 h incubation,suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity.Conclusion:As the level of disulfide reduction differs between PSMA trafficked and previously published folate trafficked conjugates,it also follows that not all internalizing receptors are translocated through similar intracellular compartments.Taken together,these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.

Langerin-mediated internalization of a modified peptide routes antigens to early endosomes and enhances cross-presentation by human Langerhans cells

The potential of the skin immune system to generate immune responses is well established, and the skin is actively exploited as a vaccination site. Human skin contains several antigen-presenting cell subsets with specialized functions. In particular, the capacity to cross-present exogenous antigens to CD8^＋ T cells is of interest for the design of effective immunotherapies against viruses or cancer. Here, we show that primary human Langerhans cells （LCs） were able to cross-present a synthetic long peptide （SLP） to CD8^＋ T cells. In addition, modification of this SLP using antibodies against the receptor langerin, but not dectin-1, further enhanced the cross-presenting capacity of LCs through routing of internalized antigens to less proteolytic early endosome antigen 1 ^＋ early endosomes. The potency of LCs to enhance CD8^＋ T-cell responses could be further increased through activation of LCs with the toll-like receptor 3 ligand polyinosinic：polycytidylic acid （phC）. Altogether, the data provide evidence that human LCs are able to cross-present antigens after langerin-mediated internalization. Furthermore, the potential for antigen modification to target LCs specifically provides a rationale for generating effective anti-tumor or anti-viral cytotoxic T lymphocyte responses.

Interleukin-6 secretion is limited by self-signaling in endosomes

Cells producing cytokines often express the receptor for the same cytokine, which makes them prone to autocrine signaling. How cytokine release and signaling are regulated in the same cell is not understood? In this study, we demonstrate that signaling by exogenous and self-synthesized inflammatory cytokine interleukin-6 (IL-6) within endosomal compartments acts as a cellular brake that limits the synthesis of IL-6. Our data show that IL-6 is internalized by dendritic cells and signals from endosomal compartments containing the IL-6 receptor. Newly synthesized IL-6 also traffics via these endosomal compartments and signals in transit to the plasma membrane. This allows activation of STAT3 which in turn limits toll-like receptor 4 stimulant lipopolysaccharide (LPS) triggered transcription of IL-6. Long-term exposure to LPS removes this brake via inhibition of STAT3 by increased expression of suppressor of cytokine signaling 3 and results in fully fledged IL-6 production. This transient regulation could prevent excessive IL-6 production during early infections.

Converging links between adult-onset neurodegenerative Alzheimer’s disease and early life neurodegenerative neuronal ceroid lipofuscinosis?

Evidence from genetics and from analyzing cellular and animal models have converged to suggest links between neurodegenerative disorders of early and late life.Here,we summarize emerging links between the most common late life neurodegenerative disease,Alzheimer’s disease,and the most common early life neurodegenerative diseases,neuronal ceroid lipofuscinoses.Genetic studies reported an overlap of clinically diagnosed Alzheimer’s disease and mutations in genes known to cause neuronal ceroid lipofuscinoses.Accumulating data strongly suggest dysfunction of intracellular trafficking mechanisms and the autophagy-endolysosome system in both types of neurodegenerative disorders.This suggests shared cytopathological processes underlying these different types of neurodegenerative diseases.A better understanding of the common mechanisms underlying the different diseases is important as this might lead to the identification of novel targets for therapeutic concepts,the transfer of therapeutic strategies from one disease to the other and therapeutic approaches tailored to patients with specific mutations.Here,we review dysfunctions of the endolysosomal autophagy pathway in Alzheimer’s disease and neuronal ceroid lipofuscinoses and summarize emerging etiologic and genetic overlaps.

Endocytic regulation of TGF-β signaling

Transforming growth factor-β （TGF-β） signaling is tightly regulated to ensure its proper physiological functions in different cells and tissues. Like other cell surface receptors, TGF-β receptors are internalized into the cell, and this process plays an important regulatory role in TGF-β signaling. It is well documented that TGF-β receptors are endocytosed via clathrin-coated vesicles as TGF-β endocytosis can be blocked by potassium depletion and the GTPasedeficient dynamin K44A mutant. TGF-β receptors may also enter cells via cholesterol-rich membrane microdomain lipid rafts/caveolae and are found in caveolin-l-positive vesicles. Although receptor endocytosis is not essential for TGF-β signaling, clathrin-mediated endocytosis has been shown to promote TGF-β-induced Smad activation and transcriptional responses. Lipid rafts/caveolae are widely regarded as signaling centers for G protein-coupled recep- tors and tyrosine kinase receptors, but they are indicated to facilitate the degradation of TGF-β receptors and there- fore turnoff of TGF-β signaling. This review summarizes current understanding of TGF-β receptor endocytosis, the possible mechanisms underlying this process, and the role of endocytosis in modulation of TGF-β signaling.

A multi-functional nanoplatform for efficacy tumor theranostic applications

Nanomaterials with multiple functions have become more and more popular in the domain of cancer research. MoS2 has a great potential in photothermal therapy, X-ray/CT imaging and drug delivery. In this study, a water soluble MoS2 nanosystem(MoS2-PEG) was synthesized and explored in drug delivery, photothermal therapy(PTT) and X-ray imaging.Doxorubicin(DOX) was loaded onto MoS2-PEG with a high drug loading efficiency(~69%)and obtained a multifunctional drug delivery system(MoS2-PEG/DOX). As the drug delivery, MoS2-PEG/DOX could efficiently cross the cell membranes, and escape from the endosome via NIR light irradiation, lead to more apoptosis in MCF-7 cells, and afford higher antitumor efficacy without obvious toxic effects to normal organs owing to its prolonged blood circulation and 11.6-fold higher DTX uptake of tumor than DOX. Besides, MoS2-PEG/DOX not only served as a drug delivery system, but also as a powerful PTT agent for thermal ablation of tumor and a strong X-ray contrast agent for tumor diagnosis. In the in vitro and in vivo studies, MoS2-PEG/DOX exhibited excellent tumor-targeting efficacy, outstanding synergistic anti-cancer effect of photothermal and chemotherapy and X-ray imaging property,demonstrating that MoS2-PEG/DOX had a great potential for simultaneous diagnosis and photothermal-chemotherapy in cancer treatment.

Important relationships between Rab and MICAL proteins in endocytic trafficking

The internalization of essential nutrients,lipids and receptors is a crucial process for all eukaryotic cells.Accordingly,endocytosis is highly conserved across cell types and species.Once internalized,small cargocontaining vesicles fuse with early endosomes(also known as sorting endosomes),where they undergo segregation to distinct membrane regions and are sorted and transported on through the endocytic pathway.Although the mechanisms that regulate this sorting are still poorly understood,some receptors are directed to late endosomes and lysosomes for degradation,whereas other receptors are recycled back to the plasma membrane;either directly or through recycling endosomes.The Rab family of small GTP-binding proteins plays crucial roles in regulating these trafficking pathways.Rabs cycle from inactive GDP-bound cytoplasmic proteins to active GTP-bound membraneassociated proteins,as a consequence of the activity of multiple specific GTPase-activating proteins(GAPs) and GTP exchange factors(GEFs).Once bound to GTP,Rabs interact with a multitude of effector proteins that carry out Rab-specific functions.Recent studies have shown that some of these effectors are also interaction partners for the C-terminal Eps15 homology(EHD) proteins,which are also intimately involved in endocytic regulation.A particularly interesting example of common Rab-EHD interaction partners is the MICALlike protein,MICAL-L1.MICAL-L1 and its homolog,MICAL-L2,belong to the larger MICAL family of proteins,and both have been directly implicated in regulating endocytic recycling of cell surface receptors and junctional proteins,as well as controlling cytoskeletal rearrangement and neurite outgrowth.In this review,we summarize the functional roles of MICAL and Rab proteins,and focus on the significance of their interactions and the implications for endocytic transport.

The BLOC Interactomes Form a Network in Endosomal Transport

With the identification of more than a dozen novel Hermansky-Pudlak Syndrome （HPS） proteins in vesicle trafficking in higher eukaryotes, a new class of trafficking pathways has been described. It mainly consists of three newly-defined protein com- plexes, BLOC-l, -2, and -3. Compelling evidence indicates that these complexes together with two other well-known complexes, AP3 and HOPS, play important roles in endosomal transport. The interactions between these complexes form a network in protein trafficking via endosomes and cytoskeleton. Each node of this network has intra-complex and extra-complex interactions. These complexes are connected by direct interactions between the subunits from different complexes or by indirect interactions through coupling nodes that interact with two or more subunits from different complexes. The dissection of this network facilitates the understanding of a dynamic but elaborate transport machinery in protein/membrane trafficking. The disruption of this network may lead to abnormal trafficking or defective organellar development as described in patients with Hermansky-Pudlak syndrome.

CDK11 negatively regulates Wnt/β-catenin signaling in the endosomal compartment by affecting microtubule stability

Objectives:Improper activation of Wnt/β-catenin signaling has been implicated in human diseases.Beyond the well-studied glycogen synthase kinase 3p(GSK3p)and casein kinase 1(CK1),other kinases affecting Wnt/β-catenin signaling remain to be defined.Methods:To identify the kinases that modulate Wnt/β-catenin signaling,we applied a kinase small interfering RNA(siRNA)library screen approach.Luciferase assays,immunoblotting,and real-time polymerase chain reaction(PCR)were performed to confirm the regulation o f the Wnt/β-catenin signaling pathway by cyclin-dependent kinase 11(CDK11)and to investigate the underlying mechanism.Confocal immunofluorescence,coimmunoprecipitation(co-IP),and scratch wound assays were used to demonstrate colocalization,detect protein interactions,and explore the function of CDK11.Results:CDK11 was found to be a significant candidate kinase participating in the negative control of Wnt/P-catenin signaling.Down-regulation of CDK11 led to the accumulation of Wnt/β-catenin signaling receptor complexes,in a manner dependent on intact adenomatosis polyposis coli(APC)protein.Further analysis showed that CDK11 modulation of Wnt/P-catenin signaling engaged the endolysosomal machinery,and CDK11 knockdown enhanced the colocalization of Wnt/β-catenin signaling receptor complexes with early endosomes and decreased colocalization with lysosomes.Mechanistically,CDK11 was found to function in Wnt/β-catenin signaling by regulating microtubule stability.Depletion of CDK11 down-regulated acetyl-a-tubulin.Moreover,co-IP assays demonstrated that CDK11 interacts with the a-tubulin deacetylase SIRT2,whereas SIRT2 down-regulation in CDK11-depleted cells reversed the accumulation of Wnt/(3-catenin signaling receptor complexes.CDK11 was found to suppress cell migration through altered W nt/β-catenin signaling.Conclusions:CDK11 is a negative modulator of Wnt/β-catenin signaling that stabilizes microtubules,thus resulting in the dysregulation of receptor complex trafficking from early endosomes to lysosomes.

The convergent point of the endocytic and autophagicpathways in leydig cells

Endocytic tracers and marker enzyme of lysosomeswere used in the present study to analyze the processesof autophagocytosis and endocytosis, and the convergentpoint of these two pathways in Leydig cells. The endocyticand autophagic compartments call be easily identified inLeydig cells, which makes easier to define the stages of twopathways than was possible before. The evidences indicated that the late endosomes (dense MVBs) deliver theirendocytosed gold tracers together with lysosomal enzymesto the early autophagosomes and they are the convergentpoint of the two pathways. During this convergent process,the early autophagosomes transform into late autophagosomes and the late endosomes transform into mature lysosomes.

pH-responsive magnesium- and carbonate-substituted apatite nano-crystals for efficient and cell-targeted delivery of transgenes

The short half-lives due to the enzymatic degradation in blood, the lack of tissue targetability and the incapability to passively diffuse across the plasma membrane and smoothly traffic across the harsh intracelluar environment are the major shortcomings for nucleic acid-based potential therapeutics, such as recombinant plasmid and antisense oligonucleotides or small interferring RNA (siRNA). Plasmid DNA containing a gene of interest could have immense impact as a promising therapeutic drug for treating genetic as well as acquired human diseases at the molecular level with high level of efficacy and precision. Thus both viral and non-viral synthetic vectors have been developed in the past decades to address the aforementioned challenges of naked DNA. While in the viral particles plasmid DNA is integrated into the viral genome, in most non-viral cases the DNA being anionic in nature is electrostatically associated with a cationic lipid or polymer forming lipoplex or polyplex, respectively, or a cationized inorganic gold, silica or iron oxide particle. Due to the potential immunogenicity and carcinogenicity issues with the viral particles, non-viral vectors have drawn much more attention for the clinical evaluation. However, the main concern of using non-biodegradable particles, specially the inorganic ones, is the adverse effects owing to their long term interactions with body components. We have recently developed biodegradable pH-sensitive inorganic nanoparticles of Mg/CaPi and carbonate apatite for efficient transgene delivery to primary, cancer and embryonic stem cells, by virtue of their high affinity binding with the DNA, ability to contact the cell membrane by ionic or ligand-receptor interactions and fast dissolution kinectis in endosomal acidic pH facilitating release of the DNA from the dissolving particles and also from the endosomes.

Taking out the garbage:cathepsin D and calcineurin in neurodegeneration

Cellular homeostasis requires a tightly controlled balance between protein synthesis, folding and degradation. Especially long-lived, post-mitotic cells such as neurons depend on an efficient proteostasis system to maintain cellular health over decades. Thus, a functional decline of processes contributing to protein degradation such as autophagy and general lysosomal proteolytic capacity is connected to several age-associated neurodegenerative disorders, including Parkinson＇s, Alzheimer＇s and Huntington＇s diseases. These so called proteinopathies are characterized by the accumulation and misfolding of distinct proteins, subsequently driving cellular demise. We recently linked efficient lysosomal protein breakdown via the protease cathep- sin D to the Ca2＋/calmodulin-dependent phosphatase calcineurin. In a yeast model for Parkinson＇s disease, functional calcineurin was required for proper trafficking of cathepsin D to the lysosome and for recycling of its endosomal sorting receptor to allow further rounds of shuttling. Here, we discuss these findings in relation to present knowledge about the involvement of cathepsin D in proteinopathies in general and a possible connection between this protease, calcineurin signalling and endosomal sorting in particular. As dysregulation of Ca2＋ homeostasis as well as lysosomal impairment is connected to a plethora of neurode- generative disorders, this novel interplay might very well impact pathologies beyond Parkinson＇s disease.

Elucidation of the early infection machinery of hepatitis B virus by using bio-nanocapsule

Currently, hepatitis B virus(HBV), upon attaching to human hepatocytes, is considered to interact first with heparan sulfate proteoglycan(HSPG) via an antigenic loop of HBV envelope S protein. Then, it is promptly transferred to the sodium taurocholate cotransporting polypeptide(NTCP) via the myristoylated N-terminal sequence of pre-S1 region(from Gly-2 to Gly-48, HBV genotype D), and it finally enters the cell by endocytosis. However, it is not clear how HSPG passes HBV to NTCP and how NTCP contributes to the cellular entry of HBV. Owing to the poor availability and the difficulty of manipulations, including fluorophore encapsulation, it has been nearly impossible to perform biochemical and cytochemical analyses using a substantial amount of HBV. A bio-nanocapsule(BNC), which is a hollow nanoparticle consisting of HBV envelope L protein, was efficiently synthesized in Saccharomyces cerevisiae. Since BNC could encapsulate payloads(drugs, genes, proteins) and specifically enter human hepatic cells utilizing HBV-derived infection machinery, it could be used as a model of HBV infection to elucidate the early infection machinery. Recently, it was demonstrated that the N-terminal sequence of pre-S1 region(from Asn-9 to Gly-24) possesses low p H-dependent fusogenic activity, which might play a crucial role in the endosomal escape of BNC payloads and in the uncoating process of HBV. In this minireview, we describe a model in which each domain of the HBV L protein contributes to attachment onto human hepatic cells through HSPG, initiation of endocytosis, interaction with NTCP in endosomes, and consequent provocation of membrane fusion followed by endosomal escape.

Functional coupling of V-ATPase and CLC-5

Dent’s disease is an X-linked renal tubulopathy characterized by low molecular weight proteinuria, hypercalciuria and progressive renal failure. Disease aetiology is associated with mutations in the CLCN5 gene coding for the electrogenic 2Cl-/H＋ antiporter chloride channel 5 （CLC-5）, which is expressed in the apical endosomes of renal proximal tubules with the vacuolar type H＋-ATPase （V-ATPase）. Initially identified as a member of the CLC family of Cl- channels, CLC-5 was presumed to provide Cl shunt into the endosomal lumen to dissipate H＋ accumulation by V-ATPase, thereby facilitating efficient endosomal acidification. However, recent findings showing that CLC-5 is in fact not a Cl- channel but a 2Cl-/H＋ antiporter challenged this classical shunt model, leading to a renewed and intense debate on its physiological roles. Cl- accumulation via CLC-5 is predicted to play a critical role in endocytosis, as illustrated in mice carrying an artifcial Cl- channel mutation E211A that developed defective endocytosis but normal endo-somal acidification. Conversely, a recent functional analysis of a newly identifed disease-causing Cl- channel mutation E211Q in a patient with typical Dent’s disease confrmed the functional coupling between V-ATPase and CLC-5 in endosomal acidifcation, lending support to the classical shunt model. In this editorial, we will address the current recognition of the physiological role of CLC-5 with a specific focus on the functional coupling of V-ATPase and CLC-5.