Human Erythrocyte Protein Research Articles

Plasmodium Induces Swelling-activated ClC-2 Anion Channels in the Host Erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.

Fructosamine 3-kinase-related protein and deglycation in human erythrocytes.

Fructosamine 3-kinase (FN3K), an enzyme initially identified in erythrocytes, catalyses the phosphorylation of fructosamines on their third carbon, leading to their destabilization and their removal from protein. We show that human erythrocytes also contain FN3K-related protein (FN3K-RP), an enzyme that phosphorylates psicosamines and ribulosamines, but not fructosamines, on the third carbon of their sugar moiety. Protein-bound psicosamine 3-phosphates and ribulosamine 3-phosphates are unstable, decomposing at pH 7.1 and 37 degrees C with half-lives of 8.8 h and 25 min respectively, as compared with 7 h for fructosamine 3-phosphates. NMR analysis indicated that 1-deoxy-1-morpholinopsicose (DMP, a substrate for FN3K and FN3K-RP), like 1-deoxy-1-morpholinofructose (DMF, a substrate of FN3K), penetrated erythrocytes and was converted into the corresponding 3-phospho-derivative. Incubation of erythrocytes with 50 mM allose, 200 mM glucose or 10 mM ribose for 24 h resulted in the accumulation of glycated haemoglobin, and this accumulation was approx. 1.9-2.6-fold higher if DMP, a competitive inhibitor of both FN3K and FN3K-RP, was present in the incubation medium. Incubation with 50 mM allose or 200 mM glucose also caused the accumulation of ketoamine 3-phosphates, which was inhibited by DMP. By contrast, DMF, a specific inhibitor of FN3K, only affected the glucose-dependent accumulation of glycated haemoglobin and ketoamine 3-phosphates. These data indicate that FN3K-RP can phosphorylate intracellular, protein-bound psicosamines and ribulosamines, thus leading to deglycation.

The Affinity of GXXXG Motifs in Transmembrane Helix-Helix Interactions Is Modulated by Long-range Communication

Sequence motifs are responsible for ensuring the proper assembly of transmembrane (TM) helices in the lipid bilayer. To understand the mechanism by which the affinity of a common TM-TM interactive motif is controlled at the sequence level, we compared two well characterized GXXXG motif-containing homodimers, those formed by human erythrocyte protein glycophorin A (GpA, high-affinity dimer) and those formed by bacteriophage M13 major coat protein (MCP, low affinity dimer). In both constructs, the GXXXG motif is necessary for TM-TM association. Although the remaining interfacial residues (underlined) in GpA (LIXXGVXXGVXXT) differ from those in MCP (VVXXGAXXGIXXF), molecular modeling performed here indicated that GpA and MCP dimers possess the same overall fold. Thus, we could introduce GpA interfacial residues, alone and in combination, into the MCP sequence to help decrypt the determinants of dimer affinity. Using both in vivo TOXCAT assays and SDS-PAGE gel migration rates of synthetic peptides derived from TM regions of the proteins, we found that the most distal interfacial sites, 12 residues apart (and approximately 18 A in structural space), work in concert to control TM-TM affinity synergistically.

Reconstitution of ATP-dependent cGMP transport into proteoliposomes by membrane proteins from human erythrocytes

Boadu E, Sager G. Reconstitution of ATP-dependent cGMP transport into proteoliposomes by membrane proteins from human erythrocytes. 2004; 64: 41-48.The cellular efflux of cGMP from human erythrocytes has previously been characterized in functional studies. The purpose of the present study was to find membrane proteins with the ability to restore ATP-dependent uptake of cGMP into proteoliposomes. Human erythrocyte membranes were solubilized with CHAPS (3-([3-cholamidopropyl]dimethylammonio)-1-propanesulfonate) and gel filtration gave three protein fractions with the ability to restore active transport. Only two of these fractions were retained on a lentil lectin column. By using these two purification steps, active transport was 11 times higher in the first fraction compared to the original material and SDS-PAGE showed the presence of proteins with sizes of 145 kDa and 165 kDa. The second fraction gave 20 times higher active transport after purification and comprised proteins with sizes of 145 kDa and 180 kDa. At present three members of the MRP (multi-resistance associated protein) family have been detected in human erythrocytes: MRP1, MRP4 and MRP5. The last two proteins have been shown to transport cyclic nucleotides. The present findings are compatible with MRP4 as the 145 kDa protein, MRP5 as the 165 kDa protein and MRP1 as the 180 kDa protein. However, the 145 kDa protein could also be SMRP (short multi-resistance protein), the gene splice variant of MRP5. lmmunoprecipitation of MRP5 from CHAPS-solubilized extract reduced active transport and specific binding by about 45% and 40%, respectively. This shows that MRP5 is an important cGMP-transporting protein in human erythrocytes.

The interaction of haemoglobin, magnesium, organic phosphates and band 3 protein in nucleated and anucleated erythrocytes.

The anion influx was measured in order to study the interaction among organic phosphates, magnesium, haemoglobin and the N-terminal of the cytoplasmic domain of band 3 protein in human, chicken and trout erythrocytes. The rate constant for SO(4)(2-) influx in human and trout erythrocytes increased significantly when it was measured with an increased concentration of intracellular Mg(2+). The SO(4)(2-) influx was also measured in human erythrocyte ghosts in the presence and absence of Mg(2+). The smaller activation provoked by Mg(2+) in ghosts could be caused by the presence of a small quantity of haemoglobin which remained inside. The SO(4)(2-) uptake in chicken erythrocytes in the presence and in absence of Mg(2+) was characterized by very similar rate constants. The results suggest that the small increase in intracellular Mg(2+) in the erythrocytes involves an increase in the formation of Mg(2+)-ATP and Mg(2+)-2,3 BPG complexes reducing the affinity of the organic phosphates for Hb. This new situation may influence the functions of the anion transporter with consequent variations of SO(4)(2-) influx throughout the erythrocyte membrane in human and in trout erythrocytes, whereas in chicken RBCs this function cannot occur and, in fact, no increase in sulphate influx was noticeable. The measurement of Hb/O(2) affinity by the use of alternating fixed and variable concentrations of organic phosphates and Mg(2+), confirms the interactions between these elements and their effect on the mechanism of the affinity. When we measured the sulphate influx in the presence of DIDS we found some differences in the three types of cells.

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia

Human skeletal muscle and erythrocyte proteins involved in acid-base homeostasis: adaptations to chronic hypoxia

Chronic hypoxia is accompanied by changes in blood and skeletal muscle acid-base control. We hypothesized that the underlying mechanisms include altered protein expression of transport systems and the enzymes involved in lactate, HCO3- and H+ fluxes in skeletal muscle and erythrocytes. Immunoblotting was used to quantify densities of the transport systems and enzymes. Muscle and erythrocyte samples were obtained from eight Danish lowlanders at sea level and after 2 and 8 weeks at 4100 m (Bolivia). For comparison, samples were obtained from eight Bolivian natives. In muscle membranes there were no changes in fibre-type distribution, lactate dehydrogenase isoforms, Na+,K+-pump subunits or in the lactate-H+ co-transporters MCT1 and MCT4. The Na+-H+ exchanger protein NHE1 was elevated by 39 % in natives compared to lowlanders. The Na+-HCO3- co-transporter density in muscle was elevated by 47-69 % after 2 and 8 weeks at altitude. The membrane-bound carbonic anhydrase (CA) IV in muscle increased in the lowlanders by 39 %, whereas CA XIV decreased by 23-47 %. Levels of cytosolic CA II and III in muscle and CA I and II in erythrocytes were unchanged. The erythrocyte lactate-H+ co-transporter MCT1 increased by 230-405 % in lowlanders and was 324 % higher in natives. The erythrocyte inorganic anion exchanger (Cl--HCO3- exchanger AE1) was increased by 149-228 %. In conclusion, chronic hypoxia induces dramatic changes in erythrocyte proteins, but only moderate changes in muscle proteins involved in acid-base control. Together, these changes suggest a hypoxia-induced increase in the capacity for lactate, HCO3- and H+ fluxes from muscle to blood and from blood to erythrocytes.

Copper-binding proteins in human erythrocytes: searching for potential biomarkers of copper over-exposure.

The recognition that copper is essential but also potentially toxic to humans has prompted the search for biomarkers of copper excess. The experimental approach followed here involves the isolation and subsequent characterization of copper-binding molecules (CuBP) from human erythrocytes. Incubation (0-60 min) of freshly obtained erythrocytes in the presence of increasing concentrations of copper (10-50 microM; as 64Cu-histidine) led to time- and concentration-dependent uptake of the radioisotope. A near-maximal incorporation was attained after 20 min, with 45-55% of the radioactivity being recovered in 20,000 x g hemolysate supernatants (S-20). 64CuBP from S-20 were separated by size exclusion and metal-affinity chromatography. Most radioactivity loaded into a Sephadex G-75 column was recovered in association with molecules of MMr greater than 60 KDa (largely accounted for by hemoglobin; Hb). Only negligible amounts of radioactive Cu were associated with metallothionein. With further purification, the higher MMr 64Cu-binding fractions were resolved by Sephadex G-200 into two major peaks. The cpm/microg protein ratios of the first peak (high MMr) were proportional to the concentrations of copper presented to the erythrocytes. The second one contained mostly Hb molecules. Proteins from the first peak were concentrated in an affinity chromatography mini-column, suited to trap CuBP. The higher-affinity CuBP were eluted as a single peak which comprised around 60% of the load. An SDS-PAGE analysis of such peak reveals the presence of three bands, of which two are non-hemoglobin Cu-binding proteins. The latter, whose identity remains to be established, had MMr of approximately 30 and 40 KDa, respectively. Preliminary data indicate that the two bands bind 64Cu within a range of concentrations, relevant to those expected to occur during copper over-exposure conditions.

Construction and use of Plasmodium falciparum phage display libraries to identify host parasite interactions

BackgroundThe development of Plasmodium falciparum within human erythrocytes induces a wide array of changes in the ultrastructure, function and antigenic properties of the host cell. Numerous proteins encoded by the parasite have been shown to interact with the erythrocyte membrane. The identification of new interactions between human erythrocyte and P. falciparum proteins has formed a key area of malaria research. To circumvent the difficulties provided by conventional protein techniques, a novel application of the phage display technology was utilised.MethodsP. falciparum phage display libraries were created and biopanned against purified erythrocyte membrane proteins. The identification of interacting and in-frame amino acid sequences was achieved by sequencing parasite cDNA inserts and performing bioinformatic analyses in the PlasmoDB database.ResultsFollowing four rounds of biopanning, sequencing and bioinformatic investigations, seven P. falciparum proteins with significant binding specificity toward human erythrocyte spectrin and protein 4.1 were identified. The specificity of these P. falciparum proteins were demonstrated by the marked enrichment of the respective in-frame binding sequences from a fourth round phage display library.ConclusionThe construction and biopanning of P. falciparum phage display expression libraries provide a novel approach for the identification of new interactions between the parasite and the erythrocyte membrane.

Polar residue tagging of transmembrane peptides.

Studies that focus on packing interactions between transmembrane (TM) helices in membrane proteins would greatly benefit from the ability to investigate their association and packing interactions in multi-spanning TM domains. However, the production, purification, and characterization of such units have been impeded by their high intrinsic hydrophobicity. We describe the polar tagging approach to biophysical analysis of TM segment peptides, where incorporation of polar residues of suitable type and number at one or both peptide N- and C-termini can serve to counterbalance the apolar nature of a native TM segment, and render it aqueous-soluble. Using the native TM sequences of the human erythrocyte protein glycophorin A (GpA) and bacteriophage M13 major coat protein (MCP), properties of tags such as Lys, His, Asp, sarcosine, and Pro-Gly are evaluated, and general procedures for tagging a given TM segment are presented. Gel-shift assays on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) establish that various tagged GpA TM segments spontaneously insert into micellar membranes, and exhibit native TM dimeric states. Sedimentation equilibrium analytical centrifugation is used to confirm that Lys-tagged GpA peptides retain the native dimer state. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy studies on Lys-tagged TM MCP peptides selectively enriched with N-15 illustrate the usefulness of this system for evaluating monomer-dimer equilibria in micelle environments. The overall results suggest that polar-tagging of hydrophobic (TM) peptides approach constitutes a valuable tool for the study of protein-protein interactions in membranes.

Amino terminus of Plasmodium falciparum acidic basic repeat antigen interacts with the erythrocyte membrane through band 3 protein

The acidic basic repeat antigen (ABRA) of Plasmodium falciparum is localised in the parasitophorous vacuole, and associates with the merozoite surface at the time of schizont rupture. By virtue of its protease-like activity, it is implicated in the process of merozoite invasion and schizont rupture, and therefore, possibly interacts with erythrocyte membrane proteins to execute its function during these events. In this study, using Escherichia coli expressed recombinant fragments of ABRA, we have demonstrated that ABRA interacts with red blood cells through its N-terminus. Out of the four human erythrocyte proteins tested, namely, band 3, glycophorin A and B and spectrin, ABRA showed dose-dependent and saturable binding with the band 3 protein. This binding was lost on chymotrypsin treatment of erythrocytes or their membrane extract. Studies with the deletion constructs of the N-terminus revealed that the binding domain lies in the cysteine-rich N-proximal region of ABRA. In addition to the recombinant fragments, native ABRA derived from the P. falciparum-infected erythrocytes also showed binding to band 3 protein. Sequencing of the cysteine-rich 528 bp region, amplified from fifteen field isolates of P. falciparum, showed that not only the five cysteines of mature ABRA but also the whole sequence is fully conserved, even at the nucleotide level. This sequence conservation of the N-terminus and its role in RBC binding suggests that this region may be crucial for any putative function of ABRA, therefore emphasising its importance as a vaccine/drug target.

Band-3 protein function in human erythrocytes: effect of oxygenation–deoxygenation

Sulfate transport by band-3 protein in adult human erythrocytes was shown to be modulated by oxygen pressure. In particular, a higher transport activity was measured under high oxygen pressure than at low one (0.0242±0.0073 vs. 0.0074±0.0010 min −1). Other factors, such as magnesium ions and orthovanadate, which can indirectly affect the binding properties of the cytoplasmic domain of band 3 (cdb3), influence significantly the anion exchanger activity. No effect of oxygen pressure on sulfate transport was found in chicken erythrocytes, which may be related to their lacking the cdb3 binding site. These findings are fully consistent with a molecular mechanism where the oxygen-linked transition of hemoglobin (T→R) could play a key role in the regulation of anion exchanger activity.

Localization of the urea transporter UT-B protein in human and rat erythrocytes and tissues.

A new polyclonal antibody to the human erythrocyte urea transporter UT-B detects a broad band between 45 and 65 kDa in human erythrocytes and between 37 and 51 kDa in rat erythrocytes. In human erythrocytes, the UT-B protein is the Kidd (Jk) antigen, and Jk(a+b+) erythrocytes express the 45- to 65-kDa band. However, in Jk null erythrocytes [Jk(a-b-)], only a faint band at 55 kDa is detected. In kidney medulla, a broad band between 41 and 54 kDa, as well as a larger band at 98 kDa, is detected. Human and rat kidney show UT-B staining in nonfenestrated endothelial cells in descending vasa recta. UT-B protein and mRNA are detected in rat brain, colon, heart, liver, lung, and testis. When kidney medulla or liver proteins are analyzed with the use of a native gel, only a single protein band is detected. UT-B protein is detected in cultured bovine endothelial cells. We conclude that UT-B protein is expressed in more rat tissues than previously reported, as well as in erythrocytes.

Comparison of the decameric structure of peroxiredoxin-II by transmission electron microscopy and X-ray crystallography

The decameric human erythrocyte protein torin is identical to the thiol-specific antioxidant protein-II (TSA-II), also termed peroxiredoxin-II (Prx-II). Single particle analysis from electron micrographs of Prx-II molecules homogeneously orientated across holes in the presence of a thin film of ammonium molybdate and trehalose has facilitated the production of a ≥20 Å 3-D reconstruction by angular reconstitution that emphasises the D5 symmetry of the ring-like decamer. The X-ray structure for Prx-II was fitted into the transmission electron microscopic reconstruction by molecular replacement. The surface-rendered transmission electron microscopy (TEM) reconstruction correlates well with the solvent-excluded surface of the X-ray structure of the Prx-II molecule. This provides confirmation that transmission electron microscopy of negatively stained specimens, despite limited resolution, has the potential to reveal a valid representation of surface features of protein molecules. 2-D crystallisation of the Prx-II protein on mica as part of a TEM study resulted in the formation of a p2 crystal form with parallel linear arrays of stacked rings. This latter 2-D form correlates well with that observed from the 2.7 Å X-ray structure of Prx-II solved from a new orthorhombic 3-D crystal form.

A murine monoclonal antibody against Kx protein which reacts also with beta-spectrin.

Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.

Is nitrotyrosine generated in human erythrocytes in circulation?

Nitrotyrosine is considered a stable biomarker of reactive nitrogen species, including nitrogen dioxide (NO2) and peroxynitrous acid (ONOOH) in biomaterials. There are inconsistent observations on the detection of free and protein-associated nitrotyrosine in normal human plasma. Human erythrocytes, differentiated from erythrocyte precursor cells in the bone marrow, circulating in the body for an average of 120 d, and finally removed by spleen macrophages, may be exposed to reactive nitrogen species. In the present study, membrane proteins and hemoglobin from the senescent erythrocyte population were compared with those from young erythrocytes separated from the same individuals in their nitrotyrosine presence using newly prepared rabbit polyclonal anti-nitrotyrosine-ribonuclease A and anti-nitro(N-butoxycarbonyl)tyrosine-bovine serum albumin antibodies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the membranes and hemoglobin, and subsequent Western blot analysis, showed that these antibodies only slightly bind to the bands of the proteins from both young and senescent erythrocytes, whereas these antibodies definitely bind to the protein bands of membranes and hemoglobin nitrated by NO2 or ONOOH in vitro. This result indicates that nitrotyrosine is not detected in the membrane proteins and hemoglobin in human normal erythrocytes in circulation. However, this does not conclude that erythrocytes are not exposed to reactive nitrogen species in the circulation.

Studies on functions of the 63-kDa A- and 74-kDa B'(delta)-regulatory subunits in human erythrocyte protein phosphatase 2A: dissociation and reassociation of the subunits.

A heterodimeric form, CA, of protein-serine/threonine phosphatase (PP) 2A purified from human erythrocytes was dissociated into a 34-kDa catalytic subunit C and 63-kDa inactive subunit A by Sephacryl S-200 gel filtration in the presence of 6 M urea. Reassociation of the C- and A-subunits in the absence of urea suppressed the PP activity of the C subunit toward phosphorylase a, P-H2B histone, and P-H1 histone in the presence or absence of 20 mM MnCl(2) or 50 mM Mg(CH(3)COO)(2), but stimulated the PP activity toward P-H1 histone in the presence of 200 mM NaCl and the Mn(2+)-dependent protein-tyrosine phosphatase (PTP) activity toward P-Tyr-Glu copolymers. The 74-kDa inactive B'(delta) subunit was isolated from a heterotrimeric form, CAB'(delta), of PP2A partially purified from human erythrocytes, by heparin-Sepharose column chromatography. The B'(delta) subunit reassociated with CA and suppressed the PP- and PTP-activities of CA. The B'(delta) subunit did not associate with the isolated C subunit directly, and had no effect on the activities of the C subunit, indicating that the A subunit is essential for the association of the B'(delta) subunit with CA and the resulting suppression of the PP- and PTP-activities.

Protein and lipid oxidation of banked human erythrocytes:: Role of glutathione

In banked human erythrocytes (RBCs), biochemical and functional changes are accompanied with vesiculation and reduced in vivo survival. We hypothesized that some of these changes might have resulted from oxidative modification of membrane lipids, proteins, or both as a result of atrophy of the antioxidant defense system(s). In banked RBCs, we observed a time-dependent increase in protein clustering, especially band 3; carbonyl modification of band 4.1; and malondialdehyde, a lipid peroxidation product. Examination of the antioxidative defense system showed a time-dependent decline in glutathione (GSH) concentration and glutathione-peroxidase (GSH-PX) activity, with a concomitant increase in extracellular GSH, cysteine, and homocysteine, and unchanged catalase activity. When subjected to acute oxidant stress by exposure to ferric/ascorbic acid or tert-butylhydroperoxide (tert-BHT), catalase activity showed a steeper decline compared with GSH-PX. The results demonstrate that GSH and GSH-PX appear to provide the primary antioxidant defense in stored RBCs, and their decline, concurrent with an increase in oxidative modifications of membrane lipids and proteins, may destabilize the membrane skeleton, thereby compromising RBC survival.

Molecular analysis of regulation of gene expression of the human erythroid anion exchanger (AE) 1

The anion exchange protein AE1 is the most abundant membrane protein in human erythrocytes mediating the electroneutral chloride/bicarbonate exchange. We identified a promoter region in the 5′ flanking region of the human AE1 gene which controls transcription in a cell type independent manner. In addition a second, distal promoter element mediates gene expression only in erythroid cells and in dependence upon differentiation. Within this distal promoter region we defined a 44 bp sequence containing a novel CT-rich motif with very strong promoter activity whereas a second 28 bp segment suppresses gene expression.

Kx, a Quantitatively Minor Protein from Human Erythrocytes, Is Palmitoylatedin Vivo

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.