Human Membrane Proteins Research Articles

Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping

Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.

DNA Methylation in Promoter Regions of Red Cell Membrane Protein Genes in Healthy Individuals and Patients with Hereditary Membrane Disorders

The methylation state of 5'-CG-3' sites is known to be linked to the regulation of promoter function by modulating DNA-protein interactions and to the structure of chromatin. As part of a project to determine methylation patterns in the human genome, we examined the methylation profiles of several genes for human erythroid membrane proteins: ELB42 (protein 4.2), EPB3 (band 3), SPTB gene (beta-spectrin), and ANK1 (ankyrin). The bisulfite protocol of the genomic sequencing method was applied. The number of 5'-CG-3' dinucleotides was the most abundant in SPTB and ANK1, much less in EPB3, and the least in ELB42. In the DNA of peripheral blood mononuclear cells from healthy individuals, the promoter regions of EPB3 and ELB42 were extensively methylated, but the SPTB and ANK1 promoters were totally unmethylated. We also investigated methylation profiles in peripheral blood mononuclear cells from patients with red cell membrane diseases, such as complete protein 4.2 deficiency due to ELB42 mutations, hereditary spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. The DNA methylation states in these genes of erythroid cells, which we obtained at the second phase of the 2-phase liquid culture of erythroid precursor cells in the peripheral blood, were essentially identical or very similar to those of peripheral blood mononuclear cells. In disease states, the DNA methylation profiles of these red cell membrane protein genes were essentially not different from those in healthy individuals (statistically not significant).

Function of the PEX19-binding Site of Human Adrenoleukodystrophy Protein as Targeting Motif in Man and Yeast: PMP TARGETING IS EVOLUTIONARILY CONSERVED

We predicted in human peroxisomal membrane proteins (PMPs) the binding sites for PEX19, a key player in the topogenesis of PMPs, by virtue of an algorithm developed for yeast PMPs. The best scoring PEX19-binding site was found in the adrenoleukodystrophy protein (ALDP). The identified site was indeed bound by human PEX19 and was also recognized by the orthologous yeast PEX19 protein. Likewise, both human and yeast PEX19 bound with comparable affinities to the PEX19-binding site of the yeast PMP Pex13p. Interestingly, the identified PEX19-binding site of ALDP coincided with its previously determined targeting motif. We corroborated the requirement of the ALDP PEX19-binding site for peroxisomal targeting in human fibroblasts and showed that the minimal ALDP fragment targets correctly also in yeast, again in a PEX19-binding site-dependent manner. Furthermore, the human PEX19-binding site of ALDP proved interchangeable with that of yeast Pex13p in an in vivo targeting assay. Finally, we showed in vitro that most of the predicted binding sequences of human PMPs represent true binding sites for human PEX19, indicating that human PMPs harbor common PEX19-binding sites that do resemble those of yeast. Our data clearly revealed a role for PEX19-binding sites as PMP-targeting motifs across species, thereby demonstrating the evolutionary conservation of PMP signal sequences from yeast to man.

Analysis of leukocyte membrane protein interactions using protein microarrays

BackgroundProtein microarrays represent an emerging class of proteomic tools to investigate multiple protein-protein interactions in parallel. A sufficient proportion of immobilized proteins must maintain an active conformation and an orientation that allows for the sensitive and specific detection of antibody and ligand binding. In order to establish protein array technology for the characterization of the weak interactions between leukocyte membrane proteins, we selected the human leukocyte membrane protein CD200 (OX2) and its cell surface receptor (hCD200R) as a model system. As antibody-antigen reactions are generally of higher affinity than receptor-ligand binding, we first analyzed the reactivity of monoclonal antibodies (mAb) to normal and mutant forms of immobilized CD200R.ResultsFluorescently labelled mAb DX147, DX136 and OX108 were specifically reactive with immobilized recombinant hCD200R extracellular region, over a range of 0.1–40 μg ml-1 corresponding to a limit of sensitivity of 0.01–0.05 femtomol per spot. Orientating hCD200R using capture antibodies, showed that DX147 reacts with an epitope spatially distinct from the more closely related DX136 and OX108 epitopes. A panel of soluble recombinant proteins with mutations in hCD200R domain 1 produced by transiently transfected cells, was arrayed directly without purification and screened for binding to the three mAb. Several showed decreased binding to the blocking mAb DX136 and OX108, suggesting close proximity of these epitopes to the CD200 binding site. Binding of hCD200 to directly immobilized rat, mouse, and hCD200R was achieved with multimeric ligands, in the form of biotinylated-hCD200 coupled to FITC-labelled avidin coated beads.ConclusionWe have achieved sensitive, specific and reproducible detection of immobilized CD200R with different antibodies and mapped antigenic epitopes for two mAb in the vicinity of the ligand binding site using protein microarrays. We also detected CD200 binding to its receptor, a low affinity interaction, using beads presenting multivalent ligands. Our results demonstrate the quantitative aspects of protein arrays and their potential use in detecting simultaneously multiple protein-protein interactions and in particular the weak interactions found between leukocyte membrane proteins.

Characterization of presenilin-amyloid precursor interaction using bacterial expression and two-hybrid systems for human membrane proteins

An Escherichia coli system was used to produce the human membrane proteins presenilin 1 and amyloid precursor protein and to analyse their interaction. Our data indicate that the main binding site for amyloid precursor protein is located in the N-terminal three-transmembrane segments of presenilin and not in the proposed active site containing the two conserved aspartate residues. The data also suggest the presence of an additional segment of sufficient hydrophobicity at the C-terminus of PS1 to act potentially as a transmembrane segment. The implications of these findings for the function of gamma-secretase are discussed.

Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase

In higher organisms the formation of the steroid scaffold is catalysed exclusively by the membrane-bound oxidosqualene cyclase (OSC; lanosterol synthase). In a highly selective cyclization reaction OSC forms lanosterol with seven chiral centres starting from the linear substrate 2,3-oxidosqualene. Valuable data on the mechanism of the complex cyclization cascade have been collected during the past 50 years using suicide inhibitors, mutagenesis studies and homology modelling. Nevertheless it is still not fully understood how the enzyme catalyses the reaction. Because of the decisive role of OSC in cholesterol biosynthesis it represents a target for the discovery of novel anticholesteraemic drugs that could complement the widely used statins. Here we present two crystal structures of the human membrane protein OSC: the target protein with an inhibitor that showed cholesterol lowering in vivo opens the way for the structure-based design of new OSC inhibitors. The complex with the reaction product lanosterol gives a clear picture of the way in which the enzyme achieves product specificity in this highly exothermic cyclization reaction.

Protein aggregation during overexpression limited by peptide extensions with large net negative charge

Folding of the human coxsackie and adenovirus receptor immunoglobulin (Ig) variable-type domain (CAR D1) during overexpression in the Escherichia coli cytoplasm was shown previously to be partially rescued by fusion to a 22-residue C-terminal peptide. Here, peptide sequence features required for solubilization and folding of CAR D1 and similar Ig variable-type domains from two other human membrane proteins were investigated. Peptide extensions with net negative charge >−6 fully solubilized CAR D1, and approximately half of the peptide-solubilized protein was correctly folded. The Ig variable-type domains from human A33 antigen and myelin P-zero proteins were only partially solubilized by peptide extensions with net charge of −12, however, and only the solubilized P-zero domain appeared to fold correctly whereas the A33 domain formed soluble microaggregates of misfolded protein. Our results suggest a model where the large net charge of peptide extensions increases electrostatic repulsion between nascent polypeptides. The resulting decrease in aggregation rate can enable some polypeptides to fold spontaneously into their native protein conformations. Analysis of the solubility and folding status of sets of structurally homologous proteins, such as the Ig variable-type domains described here, during overexpression could provide insights into how amino acid and gene sequences influence the efficiency of spontaneous protein folding.

Tim50, a Component of the Mitochondrial Translocator, Regulates Mitochondrial Integrity and Cell Death

In yeast, Tim50 along with Tim23 regulate translocation of presequence-containing proteins across the mitochondrial inner membrane. Here, we describe the identification and characterization of a novel human mitochondrial inner membrane protein homologous to the yeast Tim50. We demonstrate that human Tim50 possesses phosphatase activity and is present in a complex with human Tim23. Down-regulation of human Tim50 expression by RNA interference increases the sensitivity of human cell lines to death stimuli by accelerating the release of cytochrome c from the mitochondria. Furthermore, injection of Tim50-specific morpholino antisense oligonucleotides during early zebrafish embryonic development causes neurodegeneration, dysmorphic hearts, and reduced motility as a result of increased cell death. These observations indicate that loss of Tim50 in vertebrates causes mitochondrial membrane permeabilization and dysfunction followed by cytoplasmic release of cytochrome c along with other mitochondrial inducers of cell death. Thus Tim50 is important for both mitochondrial function and early neuronal development.

Human dendritic cell lysosome-associated membrane protein expressed in lung type II pneumocytes

Human dendritic cell LAMP (hDC-LAMP) is a unique member of the lysosome-associated membrane protein (LAMP) family with a tissue distribution initially described as restricted to major histocompatibility class II (MHC II) compartments of activated DC before the translocation of MHC II to the cell surface [Immunity 9 (1998) 325]. In this report, we show that hDC-LAMP is also expressed by lung type II pneumocytes, another cell type with constitutive expression of MHC II. A recombinant hDC-LAMP protein and a monospecific anti-hDC-LAMP polyclonal antibody were prepared. The antibody reacted specifically with hDC-LAMP sequences of hDC-LAMP protein expressed in transfected cells and with a 54 kDa protein of normal human lung tissue with properties corresponding to those of transgene expressed hDC-LAMP. Immunohistochemical analysis of hDC-LAMP in human lung showed its presence in alveolar type II epithelial cells (type II pneumocytes) as well as in cells in the interfollicular area of bronchus-associated lymph nodes, where interdigitating DCs are concentrated, and with lesser staining of alveolar macrophages. The native protein contained approximately 16% carbohydrates, most of which are sialyl N-linked oligosaccharides, with an acidic isoelectric point (p I 4.8). The restricted localization of this protein to lung type II pneumocytes and DCs is in contrast to hLAMP-1, which was present in many cell types of the lung and lymph node. Type II pneumocytes are known to express MHC II and the abundant expression of hDC-LAMP in these cells as well as in DCs suggests its possible relationship to specific MHC II related function(s) of DC and type II pneumocytes.

Sperm membrane protein (hSMP-1) and RanBPM complex in the microtubule-organizing centre.

hSMP-1 is a human sperm membrane protein expressed during development. It is a testis-specific component produced during male germ cell differentiation. Proteins that interact with hSMP-1 were identified by the application of the yeast two-hybrid system. One of the components, RanBPM, was found to be associated with hSMP-1 under both in vitro and in vivo conditions. In the human testis, RanBPM is produced in spermatogonia and primary spermatocytes, suggesting expression during the early stages of spermatogenesis; whereas in the rat testis, it is located in round and elongated spermatids, similar to hSMP-1, suggesting expression of both components during spermiogenesis. Images obtained by immunofluorescence and confocal scanning microscopy of CHO-K1 cells co-transfected with pEGFP-C1-hSMP-1 and pDsRed1-Nl-RanBPM revealed that RanBPM and hSMP-1 are distributed in discrete loci throughout the cytoplasm. When superimposed, the stained spots appeared as congruent yellow areas, indicative of co-localization and probable complex formation of these two components. This interaction between hSMP-1 and RanBPM may be involved in the process of male germ cell differentiation. In CHO-Kl cells transfected with pEGFP-Cl-hSMP-1, the exogenously expressed hSMP-1 was found to co-localize with alpha-tubulin. Depolymerization of microtubules can be induced in CHO-Kl cells by cold treatment. In cells transfected with the pEGFP-Cl vector, the dispersed tubulins promptly reassembled upon warming. However, in cells transfected with pEGFP-Cl-hSMP-1, reassembly of the dispersed tubulins was blocked even upon rewarming of the cells. These findings suggest that hSMP-1 interacts with tubulins and thereby may modulate microtubule assembly and/or activity.

The monotopic membrane protein human oxidosqualene cyclase is active as monomer

The monotopic integral membrane protein 2,3-oxidosqualene cyclase (OSC) catalyzes the formation of lanosterol the first sterol precursor of cholesterol in mammals. Therefore, it is an important target for the development of new hypocholesterolemic drugs. Here, we report the overexpression and purification of functional human OSC (hOSC) in Pichia pastoris. The obtained IC 50 for the reference inhibitor Ro 48-8071 is nearly identical for the recombinant hOSC compared to OSC from human liver microsomes. The correlation of analytical ultracentrifugation data and activity measurements showed the highest enzymatic activity for the monomeric hOSC indicating that this would be the natural form. Furthermore, these data helped us to identify the detergent for a successful crystallization of the protein. The availability of this active recombinant human membrane protein is a very important step on the way to a more detailed functional and structural characterization of OSCs.

Transport of physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs by recombinant Escherichia coli nucleoside-H(+) cotransporter (NupC) produced in Xenopus laevis oocytes.

The recently identified human and rodent plasma membrane proteins CNT1, CNT2 and CNT3 belong to a gene family (CNT) that also includes the bacterial nucleoside transport protein NupC. Heterologous expression in Xenopus oocytes has established that CNT1-3 correspond functionally to the three major concentrative nucleoside transport processes found in human and other mammalian cells (systems cit, cif and cib, respectively) and mediate Na(+) - linked uptake of both physiological nucleosides and anti-viral and anti-neoplastic nucleoside drugs. Here, one describes a complementary Xenopus oocyte transport study of Escherichia coli NupC using the plasmid vector pGEM-HE in which the coding region of NupC was flanked by 5'- and 3'-untranslated sequences from a Xenopus beta-globin gene. Recombinant NupC resembled human (h) and rat (r) CNT1 in nucleoside selectivity, including an ability to transport adenosine and the chemotherapeutic drugs 3'-azido-3'-deoxythymidine (AZT), 2',3'- dideoxycytidine (ddC) and 2'-deoxy-2',2'-difluorocytidine (gemcitabine), but also interacted with inosine and 2',3'- dideoxyinosine (ddl). Apparent affinities were higher than for hCNT1, with apparent K(m) values of 1.5-6.3 microM for adenosine, uridine and gemcitabine, and 112 and 130 microM, respectively, for AZT and ddC. Unlike the relatively low translocation capacity of hCNT1 and rCNT1 for adenosine, NupC exhibited broadly similar apparent V(max) values for adenosine, uridine and nucleoside drugs. NupC did not require Na(+) for activity and was H(+) - dependent. The kinetics of uridine transport measured as a function of external pH were consistent with an ordered transport model in which H(+) binds to the transporter first followed by the nucleoside. These experiments establish the NupC-pGEM-HE/oocyte system as a useful tool for characterization of NupC-mediated transport of physiological nucleosides and clinically relevant nucleoside therapeutic drugs.

3SD04 ヒト赤血球膜タンパク質Band3の機能と構造(構造から見た膜輸送)

3SD04 ヒト赤血球膜タンパク質Band3の機能と構造(構造から見た膜輸送)

Profiling of generic anti-phosphopeptide antibodies and kinases with peptide microarrays using radioactive and fluorescence-based assays.

Kinases represent one of the largest enzyme families and key regulatory proteins in the cell. Only a small subset of these enzymes has been characterised so far. We have prepared different types of phosphopeptide and peptide microarrays displaying peptides deduced from annotated human phosphorylation sites and cytoplasmic domains of all annotated human membrane proteins. This approach was enabled by fully-automated high throughput micro-scale synthesis of peptides by the SPOT technology combined with chemo-selective immobilisation on modified glass slides. The phosphopeptide microarrays displaying 2923 peptides in total have been used for the characterisation of commercially available generic anti-phosphopeptide antibodies. This enabled us to detect Abl kinase activity on a microarray with anti-phosphotyrosine antibodies yielding results comparable to those obtained from a radioactive assay. More than 13 000 peptides deposited on six glass slides were used to profile casein kinase 2 (CK2) using a radioactive assay, since no generic antibody for the reliable detection of serine or threonine phosphorylation could be identified. All previously identified substrates were detected in the microarray experiment. In order to confirm whether substrates on the microarray are substrates in solution phase assays, more than 700 peptides were synthesised and tested with CK2 in a solution phase assay. All substrates identified in the solution phase assay were also detected on the microarray.

Improved biocompatibility of small intestinal submucosa (SIS) following conditioning by human endothelial cells

Small intestinal submucosa (SIS) is a naturally occurring tissue matrix composed of extracellular matrix proteins and various growth factors. SIS is derived from the porcine jejunum and functions as a remodeling scaffold for tissue repair. While SIS has proven to be a useful biomaterial for implants in vivo, problems associated with endothelialization and thrombogenicity of SIS implants may limit its vascular utility. The goal of this study was to determine if the biological properties of SIS could be improved by growing human umbilical vein endothelial cells (HUVEC) on SIS and allowing these cells to deposit human basement membrane proteins on the porcine substrate to create what we have called “conditioned” SIS (c-SIS). Using an approach in which HUVEC were grown for 2 weeks on SIS and then removed via a technique that leaves behind an intact basement membrane, we hypothesized that the surface properties of SIS might be improved. We found that when re-seeded on c-SIS, HUVEC exhibited enhanced organization of cell junctions and had increased metabolic activity compared to cells on native SIS (n-SIS). Furthermore, HUVEC grown on c-SIS released lower amounts of the pro-inflammatory prostaglandin PGI 2 into the media compared to cells grown on n-SIS. Additionally, we found that adhesion of resting or activated human platelets to c-SIS was significantly decreased compared to n-SIS suggesting that, in addition to improved cell growth characteristics, conditioning SIS with human basement membrane proteins might decrease its thrombogenic potential. In summary, conditioning of porcine SIS by human endothelial cells improves key biological properties of the material that may improve its usefulness as remodeling scaffold for tissue repair. Identification of critical modifications of SIS by human endothelial cells should help guide future efforts to develop more biocompatible vascular grafts.

Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.

Autosomal recessive polycystic kidney disease (ARPKD) is an infantile form of PKD characterized by fusiform dilation of collecting ducts and congenital hepatic fibrosis. The ARPKD gene, PKHD1, is large (approximately 470 kb; 67 exons) with a 12222 bp longest open reading frame, although multiple different splice forms may be generated. The predicted full-length ARPKD protein, fibrocystin, is membrane bound with 4074 amino acids (447 kDa molecular weight). To characterize the pattern of fibrocystin expression we have generated four monoclonal antibodies (mAb) to the cytoplasmic tail of the protein. Western analysis of human kidney membrane protein showed an identical pattern with each mAb; a strongly expressing large product (>450 kDa), consistent with the predicted protein size, and a weaker approximately 220 kDa band. The same large product was detected in rat and mouse kidney with lower level expression in liver. To further show that these mAbs recognize fibrocystin, tissue from ARPKD patients was analyzed and no fibrocystin products were detected. Immunohistochemical analysis of the developing kidney showed expression in the branching ureteric bud and collecting ducts, expression that persisted into adulthood. Biliary duct staining was found in the liver, plus staining in the pancreas and developing testis. Immunofluorescence analysis of MDCK cells showed a major site of expression in the primary cilia. Recent studies have associated the disease protein in various human and animal forms of PKD with cilia. The localization of fibrocystin to cilia further strengthens that correlation and indicates that the primary defect in ARPKD may be linked to ciliary dysfunction.

Targeting of the human adrenoleukodystrophy protein to the peroxisomal membrane by an internal region containing a highly conserved motif

In this study we addressed the targeting requirements of peroxisomal ABC transporters, in particular the human adrenoleukodystrophy protein. This membrane protein is defective or missing in X-linked adrenoleukodystrophy, a neurodegenerative disorder predominantly presenting in childhood. Using adrenoleukodystrophy protein deletion constructs and green fluorescent protein fusion constructs we identified the amino acid regions 1-110 and 67-164 to be sufficient for peroxisomal targeting. However, the minimal region shared by these constructs (amino acids 67-110) is not sufficient for peroxisomal targeting by itself. Additionally, the NH2-terminal 66 amino acids enhance targeting efficiency. Green fluorescent protein-labeled fragments of human peroxisomal membrane protein 69 and Saccharomyces cerevisiae Pxa1 corresponding to the amino acid 67-164 adrenoleukodystrophy protein region were also directed to the mammalian peroxisome. The required region contains a 14-amino-acid motif (71-84) conserved between the adrenoleukodystrophy protein and human peroxisomal membrane protein 69 and yeast Pxa1. Omission or truncation of this motif in the adrenoleukodystrophy protein abolished peroxisomal targeting. The single amino acid substitution L78F resulted in a significant reduction of targeting efficiency. The in-frame deletion of three amino acids (del78-80LLR) within the proposed targeting motif in two patients suffering from X-linked adrenoleukodystrophy resulted in the mislocalization of a green fluorescent protein fusion protein to nucleus, cytosol and mitochondria. Our data define the targeting region of human adrenoleukodystrophy protein containing a highly conserved 14-amino-acid motif.

Functional asymmetry of the human Na+/glucose transporter (hSGLT1) in bacterial membrane vesicles.

The functional characteristics of the forward and reverse transport modes of the human Na(+)/glucose transporter (hSGLT1) were investigated using plasma membrane vesicles of E. coli expressing the recombinant transporter. Correctly and inverse-oriented vesicles were employed to measure the initial rates of methyl-alpha-D-glucose uptake, under zero-trans conditions, as a function of Na(+), sugar, and phlorizin concentrations and membrane potential. This approach enabled the analysis of the two faces of hSGLT1 in parallel, revealing the reversibility of Na(+)/sugar cotransport. While the key characteristics of secondary active sugar transport were maintained in both modes, namely, Na(+) and voltage dependence, the kinetic properties of the two sides indicated a functional asymmetry of the transporter. That is, the apparent affinity for sugar and driver cation Na(+) exhibited a difference of more than 1 order of magnitude between the two modes. Furthermore, the selectivity pattern of ligands and the interaction of the transporter with the competitive inhibitor phlorizin were different. Whereas the high-affinity substrates, D-glucose and D-galactose, inhibited uptake of radioactive sugar tracer at their physiological concentrations (10 mM) in the forward reaction, they were poor inhibitors even at high concentrations in the reverse transport mode. Taken together, these results confirm the successful employment of E. coli to express and characterize a human membrane protein (hSGLT1), elucidating the functional asymmetry of this cotransporter.

Antisense Down-regulation of Lipocalin-interacting Membrane Receptor Expression Inhibits Cellular Internalization of Lipocalin-1 in Human NT2 Cells

There is increasing experimental evidence demonstrating that many lipocalins bind to specific cell surface receptors. However, whereas the binding of lipocalins to their lipophilic ligands has now been characterized in much detail, there is a lack of knowledge about the nature of lipocalin receptors, the physiological role of receptor binding, and the molecular mechanism of ligand delivery. We previously identified a novel human membrane protein (lipocalin-1-interacting membrane receptor (LIMR)), which interacts with lipocalin-1 (Wojnar, P., Lechner, M., Merschak, P., and Redl, B. (2001) J. Biol. Chem. 276, 20206-20212). In the present study, we investigated the physiological role of LIMR and found this protein to be essential for mediating internalization of lipocalin-1 (Lcn-1) in NT2 cells, leading to its degradation. Whereas control NT2 cells rapidly internalized (125)I-Lcn-1 or fluorescein isothiocyanate-labeled Lcn-1, NT2 cells that were made LIMR deficient by cDNA antisense expression greatly accumulated Lcn-1 in the culture medium but did not internalize it. Because sequence and structure analysis indicated that proteins similar to LIMR are present in several organisms and at least two closely related orthologues are found in human and mouse, we suggest LIMR to be the prototype of a new family of endocytic receptors, which are topographically characterized by nine putative transmembrane domains and a characteristic large central cytoplasmic loop.

The a-Subunit of the V-type H+-ATPase Interacts with Phosphofructokinase-1 in Humans

V-type or H+-ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in alpha-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H+-ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzyme-linked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pull-down assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in alpha-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H+-ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H+-ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.