Comparison of the intracellular trafficking of two alternatively spliced isoforms of pp120, a substrate of the insulin receptor tyrosine kinase

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein in the hepatocyte. It is expressed as two spliced isoforms differing by the presence (full length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Because the two isoforms differ by their ability to regulate receptor-mediated insulin endocytosis and degradation, we aimed to investigate the cellular basis for this functional difference by comparing their intracellular trafficking. During its intracellular assembly, pp120 is transported from the trans-Golgi network to the sinusoidal domain of the plasma membrane before its final transcytosis to the bile canalicular domain. Because both isoforms are expressed in hepatocytes, we examined their intracellular trafficking in NIH 3T3 fibroblasts individually transfected with each isoform. Pulse-chase experiments demonstrated that most of the newly synthesized full-length isoform reached complete maturation at about 60 min of chase. By contrast, only about 40% of the newly synthesized truncated isoform underwent complete maturation, even at more prolonged chase. Moreover, a significant portion of the truncated isoform appeared to be targeted to lysosomes. Abolishing basal phosphorylation on Ser(503) by cAMP-dependent serine kinase by mutating this residue to alanine was correlated with incomplete maturation of full length pp120 in NIH 3T3 cells and hepatocytes. This finding suggests that the intracellular domain of pp120 contains information that regulates its vectorial sorting from the trans-Golgi network to the plasma membrane.     

AGD5 is a GTPase-activating protein at the trans-Golgi network

ARF‐GTPases are important proteins that control membrane trafficking events. Their activity is largely influenced by the interplay between guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs), which facilitate the activation or inactivation of ARF‐GTPases, respectively. There are 15 predicted proteins that contain an ARF‐GAP domain within the Arabidopsis thaliana genome, and these are classified as ARF‐GAP domain (AGD) proteins. The function and subcellular distribution of AGDs, including the ability to activate ARF‐GTPases in vivo, that remain largely uncharacterized to date. Here we show that AGD5 is localised to the trans‐Golgi network (TGN), where it co‐localises with ARF1, a crucial GTPase that is involved in membrane trafficking and which was previously shown to be distributed on Golgi and post‐Golgi structures of unknown nature. Taking advantage of the in vivo AGD5–ARF1 interaction at the TGN, we show that mutation of an arginine residue that is critical for ARF‐GAP activity of AGD5 leads to longer residence of ARF1 on the membranes, as expected if GTP hydrolysis on ARF1 was impaired due to a defective GAP. Our results establish the nature of the post‐Golgi compartments in which ARF1 localises, as well as identifying the role of AGD5 in vivo as a TGN‐localised GAP. Furthermore, in vitro experiments established the promiscuous interaction between AGD5 and the plasma membrane‐localised ADP ribosylation factor B (ARFB), confirming that ARF‐GAP specificity for ARF‐GTPases within the cell environment may be spatially regulated.     

Intradermal DNA Immunization: Antisera Specific for the Membrane Lectin MR60/ERGIC-53

The trafficking of intracellular membrane proteins in Golgi apparatus, endoplasmic reticulum or intermediate compartment has not yet been fully elucidated. The human MR60/ERGIC-53 and the rat p58 proteins are one such protein; and to study them in cell-free and in situ systems, high quality monospecific antisera are required. Highly specific antisera have been obtained after immunization of mice with plasmids containing a gene encoding either the full length or a truncated protein. The best results were obtained after intradermal injections of a plasmid encoding a truncated protein comprising both the luminal carbohydrate recognition domain and the stem down to a cysteine residue close to the C-terminal end, but neither the transmembrane nor the cytosolic domains. Such antisera have a very high titer and are very efficient tools to visualize the MR60 protein in situ or to selectively precipitate the MR60 proteins from a whole cell lysate.     

The localization of FGFR3 mutations causing thanatophoric dysplasia type I differentially affects phosphorylation, processing and ubiquitylation of the receptor

Recurrent missense fibroblast growth factor receptor 3 (FGFR3) mutations have been ascribed to skeletal dysplasias of variable severity including the lethal neonatal thanatophoric dysplasia types I (TDI) and II (TDII). To elucidate the role of activating mutations causing TDI on receptor trafficking and endocytosis, a series of four mutants located in different domains of the receptor were generated and transiently expressed. The putatively elongated X807R receptor was identified as three isoforms. The fully glycosylated mature isoform was constitutively but mildly phosphorylated. Similarly, mutations affecting the extracellular domain (R248C and Y373C) induced moderate constitutive receptor phosphorylation. By contrast, the K650M mutation affecting the tyrosine kinase 2 (TK2) domain produced heavy phosphorylation of the nonglycosylated and mannose-rich isoforms that impaired receptor trafficking through the Golgi network. This resulted in defective expression of the mature isoform at the cell surface. Normal processing was rescued by tyrosine kinase inhibitor treatment. Internalization of the R248C and Y373C mutant receptors, which form stable disulfide-bonded dimers at the cell surface was less efficient than the wild-type, whereas ubiquitylation was markedly increased but apparently independent of the E3 ubiquitin-ligase casitas B-lineage lymphoma (c-Cbl). Constitutive phosphorylation of c-Cbl by the K650M mutant appeared to be related to the intracellular retention of the receptor. Therefore, although mutation K650M affecting the TK2 domain induces defective targeting of the overphosphorylated receptor, a different mechanism characterized by receptor retention at the plasma membrane, excessive ubiquitylation and reduced degradation results from mutations that affect the extracellular domain and the stop codon.     

The regulatory or phosphorylation domain of p120 catenin controls E-cadherin dynamics at the plasma membrane

In contrast to growth factor-stimulated tyrosine phosphorylation of p120, its relatively constitutive serine/threonine phosphorylation is not well understood. Here we examined the role of serine/threonine phosphorylation of p120 in cadherin function. Expression of cadherins in cadherin-null cells converted them to an epithelial phenotype, induced p120 phosphorylation and localized it to sites of cell contact. Detergent solubility and immunofluorescence confirmed that phosphorylated p120 was at the plasma membrane. E-cadherin constructs incapable of traveling to the plasma membrane did not induce serine/threonine phosphorylation of p120, nor did cadherins constructs incapable of binding p120. However, an E-cadherin cytoplasmic domain construct artificially targeted to the plasma membrane did induce serine/threonine phosphorylation of p120, suggesting phosphorylation occurs independently of signals from cadherin dimerization and trafficking through the ER/Golgi. Solubility assays following calcium switch showed that p120 isoform 3A was more effective at stabilizing E-cadherin at the plasma membrane relative to isoform 4A. Since the major phosphorylation domain of p120 is included in isoform 3A but not 4A, we tested p120 mutated in the known phosphorylation sites in this domain and found that it was even less effective at stabilizing E-cadherin. These data suggest that serine/threonine phosphorylation of p120 influences the dynamics of E-cadherin in junctions.     

Sphingolipid metabolic flow controls phosphoinositide turnover at the trans‐Golgi network

Sphingolipids are membrane lipids globally required for eukaryotic life. The sphingolipid content varies among endomembranes with pre‐ and post‐Golgi compartments being poor and rich in sphingolipids, respectively. Due to this different sphingolipid content, pre‐ and post‐Golgi membranes serve different cellular functions. The basis for maintaining distinct subcellular sphingolipid levels in the presence of membrane trafficking and metabolic fluxes is only partially understood. Here, we describe a homeostatic regulatory circuit that controls sphingolipid levels at the trans‐Golgi network (TGN). Specifically, we show that sphingomyelin production at the TGN triggers a signalling pathway leading to PtdIns(4)P dephosphorylation. Since PtdIns(4)P is required for cholesterol and sphingolipid transport to the trans‐Golgi network, PtdIns(4)P consumption interrupts this transport in response to excessive sphingomyelin production. Based on this evidence, we envisage a model where this homeostatic circuit maintains a constant lipid composition in the trans‐Golgi network and post‐Golgi compartments, thus counteracting fluctuations in the sphingolipid biosynthetic flow.     

The exomer cargo adaptor structure reveals a novel GTPase‐binding domain

Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans‐Golgi network (TGN)‐derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (F N3–B RCT of e xomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes.     

A distinct class of vesicles derived from the trans‐Golgi mediates secretion of xylogalacturonan in the root border cell

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three‐dimensional structures and macromolecular compositions of these Golgi stacks, we examined high‐pressure frozen/freeze‐substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans‐Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi‐associated vesicles. Margins of trans‐Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan‐I (PGA/RG‐I) are detected in the trans‐most cisternae and TGN compartments. LVs produced from TGN compartments (TGN‐LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN‐LVs containing the XG and PGA/RG‐I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans‐Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans‐cisternae accompanying polysaccharide synthesis with a mathematical model.     

The rice dynamin‐related protein DRP2B mediates membrane trafficking,and thereby plays a critical role in secondary cell wall cellulose biosynthesis

Membrane trafficking between the plasma membrane (PM) and intracellular compartments is an important process that regulates the deposition and metabolism of cell wall polysaccharides. Dynamin‐related proteins (DRPs), which function in membrane tubulation and vesiculation are closely associated with cell wall biogenesis. However, the molecular mechanisms by which DRPs participate in cell wall formation are poorly understood. Here, we report the functional characterization of Brittle Culm3 (BC3), a gene encoding OsDRP2B. Consistent with the expression of BC3 in mechanical tissues, the bc3 mutation reduces mechanical strength, which results from decreased cellulose content and altered secondary wall structure. OsDRP2B, one of three members of the DRP2 subfamily in rice (Oryza sativa L.), was identified as an authentic membrane‐associated dynamin via in vitro biochemical analyses. Subcellular localization of fluorescence‐tagged OsDRP2B and several compartment markers in protoplast cells showed that this protein not only lies at the PM and the clathrin‐mediated vesicles, but also is targeted to the trans‐Golgi network (TGN). An FM4‐64 uptake assay in transgenic plants that express green fluorescent protein‐tagged OsDRP2B verified its involvement in an endocytic pathway. BC3 mutation and overexpression altered the abundance of cellulose synthase catalytic subunit 4 (OsCESA4) in the PM and in the endomembrane systems. All of these findings lead us to conclude that OsDRP2B participates in the endocytic pathway, probably as well as in post‐Golgi membrane trafficking. Mutation of OsDRP2B disturbs the membrane trafficking that is essential for normal cellulose biosynthesis of the secondary cell wall, thereby leading to inferior mechanical properties in rice plants.     

Sequential Depletion and Acquisition of Proteins during Golgi Stack Disassembly and Reformation

Herein, we report the stepwise transport of multiple plant Golgi membrane markers during disassembly of the Golgi apparatus in tobacco leaf epidermal cells in response to the induced expression of the GTP‐locked Sar1p or Brefeldin A (BFA), and reassembly on BFA washout. The distribution of fluorescent Golgi‐resident N‐glycan processing enzymes and matrix proteins (golgins) with specific cis–trans‐Golgi sub‐locations was followed by confocal microscopy during disassembly and reassembly. The first event during Golgi disassembly was the loss of trans‐Golgi enzymes and golgins from Golgi membranes, followed by a sequential redistribution of medial and cis‐Golgi enzymes into the endoplasmic reticulum (ER), whilst golgins were relocated to the ER or cytoplasm. This event was confirmed by fractionation and immuno‐blotting. The sequential redistribution of Golgi components in a trans–cis sequence may highlight a novel retrograde trafficking pathway between the trans‐Golgi and the ER in plants. Release of Golgi markers from the ER upon BFA washout occurred in the opposite sequence, with cis‐matrix proteins labelling Golgi‐like structures before cis/medial enzymes. Trans‐enzyme location was preceded by trans‐matrix proteins being recruited back to Golgi membranes. Our results show that Golgi disassembly and reassembly occur in a highly ordered fashion in plants.     

Visualization of the post‐Golgi vesicle‐mediated transportation of TGF‐β receptor II by quasi‐TIRFM

Transforming growth factor β receptor II (Tβ RII) is synthesized in the cytoplasm and then transported to the plasma membrane of cells to fulfil its signalling duty. Here, we applied live‐cell fluorescence imaging techniques, in particular quasi‐total internal reflection fluorescence microscopy, to imaging fluorescent protein‐tagged Tβ RII and monitoring its secretion process. We observed punctuate‐like Tβ RII‐containing post‐Golgi vesicles formed in MCF7 cells. Single‐particle tracking showed that these vesicles travelled along the microtubules at an average speed of 0.51 μm/s. When stimulated by TGF‐β ligand, these receptor‐containing vesicles intended to move towards the plasma membrane. We also identified several factors that could inhibit the formation of such post‐Golgi vesicles. Although the inhibitory mechanisms still remain unknown, the observed characteristics of Tβ RII‐containing vesicles provide new information on intracellular Tβ RII transportation. It also renders Tβ RII a good model system for studying post‐Golgi vesicle‐trafficking and protein transportation. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High‐Throughput Quantitation of Intracellular Trafficking and Organelle Disruption by Flow Cytometry

Current methods for the quantitation of membrane protein trafficking rely heavily on microscopy, which has limited quantitative capacity for analyses of cell populations and is cumbersome to perform. Here we describe a simple flow cytometry‐based method that circumvents these limitations. The method utilizes fluorescent pulse‐width measurements as a highly sensitive indicator to monitor the changes in intracellular distributions of a fluorescently labelled molecule in a cell. Pulse‐width analysis enabled us to discriminate cells with target proteins in different intracellular locations including Golgi, lyso‐endosomal network and the plasma membrane, as well as detecting morphological changes in organelles such as Golgi perturbation. The movement of endogenous and exogenous retrograde cargo was tracked from the plasma membrane‐to‐endosomes‐to‐Golgi, by decreasing pulse‐width values. A block in transport upon RNAi‐mediated ablation of transport machinery was readily quantified, demonstrating the versatility of this technique to identify pathway inhibitors. We also showed that pulse‐width can be exploited to sort and recover cells based on different intracellular staining patterns, e.g. early endosomes and Golgi, opening up novel downstream applications. Overall, the method provides new capabilities for viewing membrane transport in thousands of cells per minute, unbiased analysis of the trafficking of cargo, and the potential for rapid screening of inhibitors of trafficking pathways.      

Targeting and signaling of Rho of plants guanosine triphosphatases require synergistic interaction between guanine nucleotide inhibitor and vesicular trafficking

Most eukaryotic cells are polarized. Common toolbox regulating cell polarization includes Rho guanosine triphosphatases (GTPases), in which spatiotemporal activation is regulated by a plethora of regulators. Rho of plants (ROPs) are the only Rho GTPases in plants. Although vesicular trafficking was hinted in the regulation of ROPs, it was unclear where vesicle‐carried ROP starts, whether it is dynamically regulated, and which components participate in vesicle‐mediated ROP targeting. In addition, although vesicle trafficking and guanine nucleotide inhibitor (GDI) pathways in Rho signaling have been extensively studied in yeast, it is unknown whether the two pathways interplay. Unclear are also cellular and developmental consequences of their interaction in multicellular organisms. Here, we show that the dynamic targeting of ROP through vesicles requires coat protein complex II and ADP‐ribosylation factor 1‐mediated post‐Golgi trafficking. Trafficking of vesicle‐carried ROPs between the plasma membrane and the trans‐Golgi network is mediated through adaptor protein 1 and sterol‐mediated endocytosis. Finally, we show that GDI and vesicle trafficking synergistically regulate cell polarization and ROP targeting, suggesting that the establishment and maintenance of cell polarity is regulated by an evolutionarily conserved mechanism.     

BFA‐induced compartments from the Golgi apparatus and trans‐Golgi network/early endosome are distinct in plant cells

Brefeldin A (BFA) is a useful tool for studying protein trafficking and identifying organelles in the plant secretory and endocytic pathways. At low concentrations (5–10 μg ml^?1), BFA caused both the Golgi apparatus and trans‐Golgi network (TGN), an early endosome (EE) equivalent in plant cells, to form visible aggregates in transgenic tobacco BY‐2 cells. Here we show that these BFA‐induced aggregates from the Golgi apparatus and TGN are morphologically and functionally distinct in plant cells. Confocal immunofluorescent and immunogold electron microscope (EM) studies demonstrated that BFA‐induced Golgi‐ and TGN‐derived aggregates are physically distinct from each other. In addition, the internalized endosomal marker FM4‐64 co‐localized with the TGN‐derived aggregates but not with the Golgi aggregates. In the presence of the endocytosis inhibitor tyrphostin A23, which acts in a dose‐ and time‐dependent manner, SCAMP1 (secretory carrier membrane protein 1) and FM4‐64 are mostly excluded from the SYP61‐positive BFA‐induced TGN aggregates, indicating that homotypic fusion of the TGN rather than de novo endocytic trafficking is important for the formation of TGN/EE‐derived BFA‐induced aggregates. As the TGN also serves as an EE, continuously receiving materials from the plasma membrane, our data support the notion that the secretory Golgi organelle is distinct from the endocytic TGN/EE in terms of its response to BFA treatment in plant cells. Thus, the Golgi and TGN are probably functionally distinct organelles in plants.     

Chemistry‐based protein modification strategy for endocytic pathway analysis

Background information. The integrated analysis of intracellular trafficking pathways is one of the current challenges in the field of cell biology, and functional proteomics has become a powerful technique for the large‐scale identification of proteins or lipids and the elucidation of biological processes in their natural contexts. For this, new dynamic strategies must be devised to trace proteins that follow a specific pathway such that their initial and final destinations can be detected by automated means. Results. Here, we report a novel vectorial strategy for trafficking pathway analysis. This strategy is based on a chemical modification of plasma membrane proteins with a bSuPeR (biotinylated sulfation site peptide reagent) and metabolic labelling in the Golgi apparatus, such that plasma membrane proteins that traffic via the retrograde route become detectable in complex mixtures. Efficient synthesis schemes are presented for tailor‐made chemical tools that are then applied to the step‐by‐step validation of the strategy, using a known retrograde cargo protein: the STxB (Shiga toxin B‐subunit). bSuPeR modification at the plasma membrane does not affect STxB transport to the Golgi apparatus, where the protein is metabolically labelled, allowing its detection in cell lysates. Conclusions. Our vectorial concept proposes a new chemical approach for traffic‐based profiling of proteins that may prove to be applicable to the analysis of diverse endocytic pathways.     

Intracellular trafficking of lysosomal membrane proteins

Lysosomes are the site of degradation of obsolete intracellular material during autophagy and of extracellular macromolecules following endocytosis and phagocytosis. The membrane of lysosomes and late endosomes is enriched in highly glycosylated transmembrane proteins of largely unknown function. Significant progress has been made in recent years towards elucidating the pathways by which these lysosomal membrane proteins are delivered to late endosomes and lysosomes. While some lysosomal membrane proteins follow the constitutive secretory pathway and reach lysosomes indirectly via the cell surface and endocytosis, others exit the trans-Golgi network in clathrin-coated vesicles for direct delivery to endosomes and lysosomes. Sorting from the Golgi or the plasma membrane into the endosomal system is mediated by signals encoded by the short cytosolic domain of these proteins. This review will discuss the role of lysosomal membrane proteins in the biogenesis of the late endosomal and lysosomal membranes, with particular emphasis on the structural features and molecular mechanisms underlying the intracellular trafficking of these proteins.     

N‐linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H+‐ATPase (V‐ATPase) biosynthesis

The a subunit of the V₀ membrane‐integrated sector of human V‐ATPase has four isoforms, a1‐a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N‐glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N‐glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope‐tagged human a1‐a3 subunits, with or without mutations that convert Asn to Gln at putative N‐glycosylation sites. Expression and N‐glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N‐glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild‐type a subunits after enzymatic deglycosylation. Cycloheximide‐chase experiments showed that unglycosylated subunits were turned over at a higher rate than N‐glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co‐immunoprecipitation studies showed that they were unable to associate with the V‐ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N‐glycosylation is crucial in all four human V‐ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V‐ATPase complexes.     

COOH-terminal signals mediate the trafficking of a peptide processing enzyme in endocrine cells

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH- terminal amidation of bioactive peptides through a two step reaction catalyzed by separate enzymes contained within the PAM precursor. To characterize the trafficking of integral membrane PAM proteins in neuroendocrine cells, we have generated stable AtT-20 cell lines expressing full length and COOH-terminally truncated integral membrane PAM proteins. Full length integral membrane PAM was present on the cell surface in low but detectable amounts and PAM proteins which reached the cell surface were rapidly internalized but not immediately degraded in lysosomes. Internalized PAM complexed with PAM antibody was found in a subcellular compartment which overlapped with internalized transferrin and with structures binding WGA. Thus the punctate juxtanuclear staining of full length PAM represents PAM in endosomes. Endoproteolytic processing of full length PAM-1 and PAM-2 resulted in the secretion of soluble PAM proteins; the secretion of these soluble PAM proteins was stimulus dependent. Although some of the truncated PAM protein was also processed and stored in AtT-20 cells, much of the expressed protein was redistributed to the plasma membrane. Soluble proteins not observed in large amounts in cells expressing full length PAM were released from the surface of cells expressing truncated PAM and little internalization of truncated integral membrane PAM was observed. Thus, the COOH-terminal domain of PAM contains information important for its trafficking within the regulated secretory pathway as well as information necessary for its retrieval from the cell surface.     

Conserved V‐ATPase c subunit plays a role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking

In plant cells, the vacuolar‐type H⁺‐ATPases (V‐ATPase) are localized in the tonoplast, Golgi, trans‐Golgi network and endosome. However, little is known about how V‐ATPase influences plant growth, particularly with regard to the V‐ATPase c subunit (VHA‐c). Here, we characterized the function of a VHA‐c gene from Puccinellia tenuiflora (PutVHA‐c) in plant growth. Compared to the wild‐type, transgenic plants overexpressing PutVHA‐c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V‐ATPase activity. Consistently, the Arabidopsis atvha‐c5 mutant shows reduced V‐ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA‐c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V‐ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA‐c‐GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling‐1 (AtRGS1). These findings suggest that the decrease in V‐ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA‐c plays an important role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking.