Plasma Membrane Of Human Cells Research Articles

Crosstalk of growth factor receptors at plasma membrane clathrin-coated sites.

Cellular communication is regulated at the plasma membrane by the interactions of receptor, adhesion, signaling, exocytic, and endocytic proteins. Yet, the composition and control of these nanoscale complexes in response to external cues remain unclear. Here, we use high-resolution and high-throughput fluorescence imaging to map the localization of growth factor receptors and related proteins at single clathrin-coated structures across the plasma membrane of human squamous HSC3 cells. We find distinct protein signatures between control cells and cells stimulated with ligands. Clathrin sites at the plasma membrane are preloaded with some receptors but not others. Stimulation with epidermal growth factor induces a capture and concentration of epidermal growth factor-, fibroblast growth factor-, and low-density lipoprotein-receptors (EGFR, FGFR, and LDLR). Regulatory proteins including ubiquitin ligase Cbl, the scaffold Grb2, and the mechanoenzyme dynamin2 are also recruited. Disrupting FGFR or EGFR individually with drugs prevents the recruitment of both EGFR and FGFR. Our data reveals novel crosstalk between multiple unrelated receptors and regulatory factors at clathrin-coated sites in response to stimulation by a single growth factor, EGF. This behavior integrates growth factor signaling and allows for complex responses to extracellular cues and drugs at the plasma membrane of human cells.

ER-Golgi-localized proteins TMED2 and TMED10 control the formation of plasma membrane lipid nanodomains.

To promote infections, pathogens exploit host cell machineries such as structural elements of the plasma membrane. Studying these interactions and identifying molecular players are ideal for gaining insights into the fundamental biology of the host cell. Here, we used the anthrax toxin to screen a library of 1,500 regulatory, cell-surface, and membrane trafficking genes for their involvement in the intoxication process. We found that endoplasmic reticulum (ER)-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the plasma membrane of human cells, an essential step dependent on localization to cholesterol-rich lipid nanodomains. Biochemical, morphological, and mechanistic analyses showed that TMED2 and TMED10 are essential components of a supercomplex that operates the exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface.

Analysis of Mycobacterium tuberculosis Uptake by Alveolar Macrophages after Ex vivo Expansion Indicates Processing Host Cells with Pathogen Actually from Lung Tissue of Patients with Pulmonary Tuberculosis.

Previously, the ex vivo cultures of alveolar macrophages were developed from the surgical samples of the lungs in patients with pulmonary tuberculosis (TB) to establish the unique features of Mycobacterium tuberculosis (Mtb) lifestyle in host cells, but the question has remained whether Mtb-infected cells are isolated from the human lungs or they may be the result of Mtb phagocytosis in ex vivo culture. The study was aimed to investigate Mtb uptake by TB patients' cells after ex vivo expansion. Alveolar macrophages were infected with the Mtb clinical isolates in ex vivo culture, and the acid-fast Mtb loads in the cells were analyzed. Immunofluorescent staining and the examination of cytological and histological preparations by confocal microscopy were applied to detect Mtb ligands and macrophage surface markers. The studies shown the lack of Mtb uptake by patients' alveolar macrophages during experimental infection with highly virulent Mtb clinical isolates containing pathogen-associated molecular patterns lipoarabinomannan and Ag38 at all used multiplicity of infection including a very high dose of infection. This fact was probably determined by the absence of pattern recognition receptors CD14, TLR2, and CD11b on the plasma membrane of human cells, likely, as a result of cellular processing from the resected lung tissues of patients. The findings indicate that alveolar macrophages with single Mtb or Mtb in colonies, including those with cord-morphology, found in the ex vivo cell cultures of all TB patients examined, were isolated from the lungs, and they characterize the Mtb infection in patients at the time of surgery.

A Chlamydia pneumoniae adhesin induces phosphatidylserine exposure on host cells

In mammalian cells, the internal and external leaflets of the plasma membrane (PM) possess different phospholipids. Phosphatidylserine (PS) is normally confined to the inner (cytoplasmic) membrane leaflet. Here we report that the adhesin CPn0473 of the human pathogenic bacterium Chlamydia pneumoniae (Cpn) binds to the PM of human cells and induces PS externalization but unexpectedly not apoptosis. PS externalization is increased in human cells exposed to infectious Cpn cells expressing increased CPn0473 and reduced in exposure to Cpn expressing decreased CPn0473. CPn0473 binds specifically to synthetic membranes carrying PS and stimulates pore formation. Asymmetric giant unilamellar vesicles (GUVs) in which PS is restricted to the inner leaflet reveal that CPn0473 induces PS externalization in the absence of other proteins. Thus our identification of CPn0473 as a bacterial PS translocator capable of specific and apoptosis-independent PS externalization during infection extends the spectrum of mechanisms intracellular pathogens use to enter host cells.

Peptide translocation through the plasma membrane of human cells: Can oxidative stress be exploited to gain better intracellular access?

ABSTRACTCell-penetrating peptides (CPPs) enter cells primarily by escaping from endosomal compartments or by directly translocating across the plasma membrane. Due to their capability of permeating into the cytosolic space of the cell, CPPs are utilized for the delivery of cell-impermeable molecules. However, the fundamental mechanisms and parameters associated with the penetration of CPPs and their cargos through the lipid bilayer have not been fully determined. This in turn has hampered their usage in biotechnological or therapeutic applications. We have recently reported that the cell penetration activity of poly-arginine CPPs (PACPPs) is dependent on the oxidation status of the plasma membrane of cells. Our data support a model where the positively-charged PACPP binds negatively-charged lipids exposed on the cell surface as a result of oxidative damage. The PACPP then crosses the membrane via formation of inverted micelles with these anionic lipids. This model provides a plausible explanation for the high variability in the cell delivery efficiency of a PACPP often observed in different settings. Notably, taking into account the current literature describing the effects of lipid oxidation, our data point to a highly complex and underappreciated interplay between PACPPs and oxidized membrane species. Overall, a better understanding of oxidation-dependent cell penetration might provide a fundamental basis for development of optimal cell permeable peptides (including cyclic peptides, stapled peptides, peptoids, etc…) and of robust delivery protocols.

The Ebola Virus: From Basic Research to a Global Health Crisis.

For me, interest in Ebola virus (EBOV) started in the late ‘90s, as an undergraduate biochemistry major on a fishing trip with the book The Hot Zone in hand. Richard Preston’s ability to communicate how EBOV ravages the human body and yet could be lurking in an animal reservoir was both frightening and thought-provoking. I went on to graduate school, decided to pursue a PhD in lipid–protein interactions, and stayed clear of viruses for the time being. My postdoctoral training then centered on lipid–protein interactions in live human cells and mostly focused on proteins involved in cancer signaling. When I started my own lab in 2006, I had a niche in biochemistry and biophysics of lipid–protein interactions and was particularly interested in discovery of new lipid-binding proteins. I once again picked up my reading of virology and realized my tools and expertise could be utilized to study how viruses replicated in human cells. In order to sustain the infection and be transmitted from a person exhibiting symptoms, EBOV needs to enter, replicate, and exit a human cell. This is an impressive cycle given that EBOV only harbors seven genes in its genome compared to approximately 4,000 in some bacteria and approximately 19,000 in humans. How the virus moves through this cycle was the question that drove my interest in studying EBOV. The EBOV replication cycle had been shown to finalize with the exit from the human cell plasma membrane, the outer membrane being so important for human cell shape and stability. This meant the virus stole its lipid coat from the human cell it infected, and this fact could be a means of understanding how the virus replicates, perhaps a process that could be targeted with new therapeutics. However, EBOV is such a dangerous virus, its work is restricted to high-containment (biosafety level 4) facilities, thus limiting my research options. Fortunately, more than a decade ago, several labs discovered that just one of the seven EBOV proteins, VP40 (viral protein 40), is able to form virus-like particles (VLPs) when expressed in human cells. These VLPs are nearly indistinguishable from authentic virions and have served as an appropriate model of understanding EBOV exit from the plasma membrane. This model led us to several fundamental discoveries of how the EBOV VP40 protein alone interacts with negatively charged lipids in human cells, induces filamentous alterations in membrane shape, and ultimately mediates the pinching-off of the human cell membrane to form a new EBOV particle. The EBOV lipid coat also provides the virus a way to enter human cells, as EBOV mimics damaged cells that expose the lipid phosphatidylserine (PS) on their outer coat. Damaged cells exposing PS are normally cleared by the immune system when human cells take up viruses, thereby exposing PS on their outer coat. The presence of the viral genome means the virus can then replicate in cells that have engulfed the virus. In essence, the virus is using our own lipids against us. Failure to recognize the role of basic science in studying EBOV and other viruses may endanger the future enterprise and training of researchers interested in these pathogens. Admittedly, each project on EBOV doesn’t lead to a new drug, vaccine, or diagnostic test, but overall our basic approaches have led to drug screening assays against the EBOV human cell exit pathway. Additionally, viruses are great tools to study and understand human cell biology, since viruses will often encode their proteins to hijack human cell pathways. These models should transform our understanding of how human cells work, such as movement and trafficking of membranes, overcoming energetic barriers to change membrane shape, and mediating a multitude of signaling events. The current Ebola virus (EBOV) outbreak in western Africa has caused more than 26,000 infections and has claimed at least 11,000 lives as of June 15, 2015. This is by far the largest outbreak of the virus in history, but despite some promising drugs, antibodies, and vaccines being developed, we still lack much fundamental knowledge of how this virus replicates inside human cells. Future basic science funding could help to better understand how EBOV spreads from cell to cell and why some patients survive and others don’t, and it could provide more rapid diagnostic assays for field analyses. Image 1 Robert V. Stahelin.

Evaluation of Cell Membrane-Modulating Properties of Non-Ionic Surfactants with the use of Atomic Force Spectroscopy

Analytical possibilities of atomic force spectroscopy (AFS) in liquid were studied upon interaction of membranotropic polymers with the plasma membrane of human cells. Topographical visualization of tightly adherent dermal fibroblasts, but not relatively soft prostate cancer (PC-3) cells, was achieved using a conventional triangular cantilever. A microsphere-based probe has been developed and applied for AFS analysis of micromechanical properties of PC-3 cells. Non-ionic block copolymers of ethylene oxide and propylene oxide, bi-functional Pluronic® L61, and glycerol-based tri-functional copolymer (TFC) were studied as potential modulators of cellular membranes and drug delivery systems as reported by Bondar et al. (Int. J. Pharm. 461(97), 104, 2014). As indicated by dynamic light scattering and fluorescent techniques, Pluronic® L61 and TFC were adsorbed onto the cell surface and inserted into the plasma membrane in different extent. Analysis of AFS curves for surfactant-treated PC-3 cells showed that both Pluronic® L61 and TFC decreased the Young’s modulus of cellular surface by almost 1.6 and 2 times, respectively. This is in accordance with the ability of amphiphilic polymers of decreasing the microviscosity of cellular membrane and promoting intracellular drug uptake as shown previously by Bondar et al. (Int. J. Pharm. 461(97), 104, 2014). Our results are of particular interest for the characterization of interaction of living cells with amphiphilic polymer-based nanocarriers and drug formulations using AFS and other surface-sensitive techniques.

Phosphoinositides: lipidic essential actors in the intracellular traffic

Phosphoinositides (PPIn) are lipids involved in the vesicular transport of proteins between the different intracellular compartments. They act by recruiting and/or activating effector proteins and are thus involved in crucial cellular functions including vesicle budding, fusion and dynamics of membranes and regulation of the cytoskeleton. Although they are present in low concentrations in membranes, their activity is essential for cell survival and needs to be tightly controlled. Therefore, phosphatases and kinases specific of the various cellular membranes can phosphorylate/dephosphorylate their inositol ring on the positions D3, D4 and/or D5. The differential phosphorylation determines the intracellular localisation and the activity of the PPIn. Indeed, non-phosphorylated phosphatidylinositol (PtdIns) is the basic component of the PPIn and can be found in all eukaryotic cells at the cytoplasmic face of the ER, the Golgi, mitochondria and microsomes. It can get phosphorylated on position D4 to obtain PtdIns4P, a PPIn enriched in the Golgi compartment and involved in the maintenance of this organelle as well as anterograde and retrograde transport to and from the Golgi. PtdIns phosphorylation on position D3 results in PtdIns3P that is required for endosomal transport and multivesicular body (MVB) formation and sorting. These monophosphorylated PtdIns can be further phosphorylated to produce bisphophorylated PtdIns. Thus, PtdIns(4,5)P2, mainly produced by PtdIns4P phosphorylation, is enriched in the plasma membrane and involved in the regulation of actin cytoskeleton and endocytosis. PtdIns(3,5)P2, mainly produced by PtdIns3P phosphorylation, is enriched in late endosomes, MVBs and the lysosome/vacuole and plays a role in endosome to vacuole transport. PtdIns(3,4)P2 is absent in yeast, cells and mainly produced by PtdIns4P phosphorylation in human cells; PtdIns(3,4)P2 is localised in the plasma membrane and plays an important role as a second messenger by recruiting specific protein kinases (Akt and PDK1). Finally the triple phosphorylated PPIn, PtdIns(3,4,5)P3 also absent in yeast, is produced by the phosphorylation of PtdIns(3,4)P2 and localized at the plasma membrane of human cells where it binds proteins via their PH domain. Interaction partners include members of the Arf (ADP-ribosylation factors) family, PDK1 (Phosphoinositide Dependent Kinase 1) and Akt. Therefore this last PPIn is essential for the control of cell proliferation and its deregulation leads to the development of numerous cancers. In conclusion, the regulation of PPIn phosphorylation/dephosphorylation is complex and needs to be very precisely regulated. Indeed phosphatases and kinases allow the maintenance of the equilibrium between the different forms. PPIn play a crucial role in numerous cellular functions and a loss in their synthesis or regulation results in severe genetic diseases.

A loop region in the N-terminal domain of Ebola virus VP40 is important in viral assembly, budding, and egress.

Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys127, Thr129, and Asn130) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and number and brightness analysis we demonstrate these mutations greatly reduce VP40 oligomerization. Lastly, VLP assays demonstrate these mutations significantly reduce VLP release from cells. Taken together, these studies identify an important loop region in VP40 that may be essential to viral egress.

Molecular Basis of the Assembly and Budding of the Ebola Virus from the Plasma Membrane of Human Cells

Lipid enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. The Ebola virus, which buds from the plasma membrane of the host cell causes viral hemorrhagic fever and has a high fatality rate. To date little is known about how the plasma membrane mediates budding and egress of the Ebola virus. My lab is investigating the molecular basis of the plasma membrane assembly, budding and egress of this virus, which is regulated by the matrix protein, VP40. We use biochemical and biophysical tools along with cellular imaging and viral replication assays to investigate how VP40 interacts with the plasma membrane of human cells to regulate viral replication. This presentation will outline the molecular basis of VP40 association with plasma membrane lipids and how lipid-protein interactions regulate VP40 oligomerization and plasma membrane bending. Furthermore, VP40 plasma membrane binding displays sensitivity to the lipid composition in the plasma membrane, which can be altered to inhibit Ebola assembly and egress.

Lipid-like trifunctional block copolymers of ethylene oxide and propylene oxide: Effective and cytocompatible modulators of intracellular drug delivery

A new glycerol-based trifunctional block copolymer (TBC) of propylene oxide and ethylene oxide and its conjugate with succinic acid (TBC-SA) were studied as a drug delivery system and compared with Pluronic L61. TBCs have multiple effects on the plasma membrane of human cells, e.g. increasing its fluidity and ion permeability, inhibiting ATPase activity of efflux transporter P-glycoprotein through reversible membrane destabilization. Such membrane-modulating properties attributed to the unimer form of copolymers increase in the order Pluronic L61≪TBC<TBC-SA and correlate with an ability of TBCs to promote the accumulation of P-glycoprotein substrates in lung cancer A549 cells. Furthermore, TBC, and especially TBC-SA, exhibit substantially lower hemolytic, cytotoxic and proapoptotic activities in comparison with Pluronic L61. Our results demonstrate that TBCs are promising analogs of bifunctional Pluronics in anticancer drug delivery.

Conjugation of succinic acid to non-ionogenic amphiphilic polymers modulates their interaction with cell plasma membrane and reduces cytotoxic activity

Pluronic block copolymers L61 and L121 were reacted with succinic anhydride to produce, respectively, their mono- and bisderivatives with succinic acid. The critical micelle concentration of Pluronics decreased after modification. The modification of Pluronic L61 promoted its association with the plasma membrane of human cells and increased membrane damage, while the membranotropic activity of modified Pluronic L121 reduced compared to the initial copolymer. Modified Pluronics interfered with the viability, apoptosis induction and metabolism of A549 cells and skin fibroblasts to a much lesser extent presumably due to the introduction of succinic acid residue inhibited intracellular penetration of copolymers. Modified Pluronic L121 promoted the cellular uptake of doxorubicin and rhodamine 123 in A549 cells attributed to the inhibition of membrane P-glycoprotein. Our study provides an approach to assessing the mechanism of interaction of amphiphilic polymers with living cells and demonstrates that Pluronic–succinic acid conjugates can be used as safe and efficient modulators of intracellular drug delivery.

Fluorogenic Probe for Constitutive Cellular Endocytosis

Endocytosis is a fundamental process of eukaryotic cells that is critical for nutrient uptake, signal transduction, and growth. We have developed a molecular probe to quantify endocytosis. The probe is a lipid conjugated to a fluorophore that is masked with an enzyme-activatable moiety known as the trimethyl lock. The probe is not fluorescent when incorporated into the plasma membrane of human cells but becomes fluorescent upon internalization into endosomes, where cellular esterases activate the trimethyl lock. Using this probe, we found that human breast cancer cells undergo constitutive endocytosis more rapidly than do matched noncancerous cells. These data reveal a possible phenotypic distinction of cancer cells that could be the basis for chemotherapeutic intervention.

Single-Particle Tracking Demonstrates that Actin Coordinates the Movement of the Ebola Virus Matrix Protein

The Ebola virus causes severe hemorrhagic fever and has a mortality rate that can be as high as 90%, yet no vaccines or approved therapeutics, to our knowledge, are available. To replicate and egress the infected host cell the Ebola virus uses VP40, its major matrix protein to assemble at the inner leaflet of the plasma membrane. The assembly and budding of VP40 from the plasma membrane of host cells seem still poorly understood. We investigated the assembly and egress of VP40 at the plasma membrane of human cells using single-particle tracking. Our results demonstrate that actin coordinates the movement and assembly of VP40, a critical step in viral egress. These findings underscore the ability of single-molecule techniques to investigate the interplay of VP40 and host proteins in viral replication.

Monitoring of the Zeta Potential of Human Cells upon Reduction in Their Viability and Interaction with Polymers

The dynamic light scattering (DLS) technique was applied in order to assess the zeta potential of the plasma membrane of human cells. At pH 7.4, the cell zeta potential for different types of cells showed variations over a wide range and was equal to -19.4 ± 0.8 mV for HeLa cells and -31.8 ± 1.1 mV for erythrocytes. The difference could presumably be attributed to the differences in the biochemical composition of the cell plasma membrane. As a result of the heating of HeLa cells, the zeta potential shifted towards more negative voltages by 4.2 mV. An increase in the zeta potential correlated with an increase in the content of phosphatidylserine on the cell surface, which is considered to be an early marker of apoptosis. The DLS technique was also used to study the interactions between the cells and membranotropic polymers, such as polycations and nonionogenic Pluronic L121.

The Schistosoma mansoni Protein Sm16/SmSLP/SmSPO-1 Assembles into a Nine-Subunit Oligomer with Potential To Inhibit Toll-Like Receptor Signaling

The Sm16/SmSLP/SmSPO-1 (Sm16) protein is secreted by the parasite Schistosoma mansoni during skin penetration and has been ascribed immunosuppressive activities. Here we describe the strategy behind the design of a modified Sm16 protein with a decreased aggregation propensity, thus facilitating the expression and purification of an Sm16 protein that is soluble in physiological buffers. The Stokes radii and sedimentation coefficients of recombinant and native proteins indicate that Sm16 is an approximately nine-subunit oligomer. Analysis of truncated Sm16 derivatives showed that both oligomerization and binding to the plasma membrane of human cells depend on multiple C-terminal regions. For analysis of immunomodulatory activities, Sm16 was expressed in Pichia pastoris to facilitate the preparation of a pyrogen/endotoxin-free purified protein. Recombinant Sm16 was found to have no effect on T-lymphocyte activation, cell proliferation, or the basal level of cytokine production by whole human blood or monocytic cells. However, Sm16 exerts potent inhibition of the cytokine response to the Toll-like receptor (TLR) ligands lipopolysaccharide (LPS) and poly(I:C) while being less efficient at inhibiting the response to the TLR ligand peptidoglycan or a synthetic lipopeptide. Since Sm16 specifically inhibits the degradation of the IRAK1 signaling protein in LPS-stimulated monocytes, our findings indicate that inhibition is exerted proximal to the TLR complex.

Structural Basis for Pattern Recognition by the Receptor for Advanced Glycation End Products (RAGE)

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC ', the second interaction surface consists of strand C ', strand F, and loop FG, and the third interaction surface consists of strand A ' and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-micros time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, approximately 10 microm. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nm.

The pattern-recognition molecule Nod1 is localized at the plasma membrane at sites of bacterial interaction

The pattern-recognition molecule Nod1 is a critical sensor for bacterial derived diaminopimelic acid-containing peptidoglycan fragments which induces innate immune responses in epithelial cells. Here we report the subcellular localization of this protein in human epithelial cells. Nod1 is localized in the cytosol and at the plasma membrane in human cells. This membrane association is dependent on the integrity of the protein, on its signalling capacity and on an intact actin cytoskeleton. Signalling-inactive mutants of Nod1 or disruption of the actin cytoskeleton interferes with this localization pattern and impacts on downstream NF-kappaB activation. Moreover, the invasive bacterium Shigella flexneri was used as a model for physiological activation of Nod1. Imaging revealed that Nod1 is recruited to the site of bacterial entry, where it colocalizes with NEMO. Our data provide evidence that membrane association is linked to Nod1 function and, in view of recent findings on Nod2, that this may be a common feature of NLR family members.

Functional Characterization of Novel Human and Mouse Equilibrative Nucleoside Transporters (hENT3 and mENT3) Located in Intracellular Membranes

The first mammalian examples of the equilibrative nucleoside transporter family to be characterized, hENT1 and hENT2, were passive transporters located predominantly in the plasma membranes of human cells. We now report the functional characterization of members of a third subgroup of the family, from human and mouse, which differ profoundly in their properties from previously characterized mammalian nucleoside transporters. The 475-residue human and mouse proteins, designated hENT3 and mENT3, respectively, are 73% identical in amino acid sequence and possess long N-terminal hydrophilic domains that bear typical (DE)XXXL(LI) endosomal/lysosomal targeting motifs. ENT3 transcripts and proteins are widely distributed in human and rodent tissues, with a particular abundance in placenta. However, in contrast to ENT1 and ENT2, the endogenous and green fluorescent protein-tagged forms of the full-length hENT3 protein were found to be predominantly intracellular proteins that co-localized, in part, with lysosomal markers in cultured human cells. Truncation of the hydrophilic N-terminal region or mutation of its dileucine motif to alanine caused the protein to be relocated to the cell surface both in human cells and in Xenopus oocytes, allowing characterization of its transport activity in the latter. The protein proved to be a broad selectivity, low affinity nucleoside transporter that could also transport adenine. Transport activity was relatively insensitive to the classical nucleoside transport inhibitors nitrobenzylthioinosine, dipyridamole, and dilazep and was sodium ion-independent. However, it was strongly dependent upon pH, and the optimum pH value of 5.5 probably reflected the location of the transporter in acidic, intracellular compartments.

Identification of Methionine-rich Clusters That Regulate Copper-stimulated Endocytosis of the Human Ctr1 Copper Transporter

Copper uptake and subsequent delivery to copper-dependent enzymes are essential for many cellular processes. However, the intracellular levels of this nutrient must be controlled because of its potential toxicity. The hCtr1 protein functions in high affinity copper uptake at the plasma membrane of human cells. Recent studies have shown that elevated copper stimulates the endocytosis and degradation of the hCtr1 protein, and this response is likely an important homeostatic mechanism that prevents the overaccumulation of copper. The domains of hCtr1 involved in copper-stimulated endocytosis and degradation are unknown. In this study we examined the importance of potential copper-binding sequences in the extracellular domain and a conserved transmembrane (150)MXXXM(154) motif for copper-stimulated endocytosis and degradation of hCtr1. The endocytic response of hCtr1 to low copper concentrations required an amino-terminal methionine cluster ((40)MMMMPM(45)) closest to the transmembrane region. However, this cluster was not required for the endocytic response to higher copper levels, suggesting this motif may function as a high affinity copper-sensing domain. Moreover, the transmembrane (150)MXXXM(154) motif was absolutely required for copper-stimulated endocytosis and degradation of hCtr1 even under high copper concentrations. Together with previous studies demonstrating a role for these motifs in high affinity copper transport activity, our findings suggest common biochemical mechanisms regulate both transport and trafficking functions of hCtr1.