The acylation of megakaryocyte proteins: glycoprotein IX is primarily myristoylated while glycoprotein Ib is palmitoylated

The acylation of megakaryocyte proteins was studied with special emphasis on the myristoylation and palmitoylation of the glycoprotein (GP) Ib complex. Guinea pig megakaryocytes were purified and separated into subpopulations at different phases of maturation. Cells were incubated with [3H]myristate, [3H]palmitate, or [3H]acetate to study endogenous protein acylation. Cycloheximide was used to distinguish between cotranslational and posttranslational acylation and hydroxylamine to distinguish between thioester and amide linkages. After incubations, delipidated proteins or GPIb complex subunits, immunoprecipitated with PG-1, AN-51 or FMC-25 monoclonal antibody, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and assessed by fluorography. Radiolabeled fatty acids bound to GPIX and GPIb were also analyzed by high pressure liquid chromatography (HPLC) and scintillation spectrometry. With [3H]myristic acid and [3H]acetate, GPIX was found to be a major myristoylated protein in megakaryocytes and CHRF-288 cells. Myristic acid was linked to GPIX by an amide bond, and this process occurred cotranslationally. With [3H]acetate, GPIb was primarily palmitoylated, but with [3H]myristate, GPIb was acylated with about equal mounts of myristic acid and palmitic acids. Both fatty acids were linked to GPIb by thioester bonds, and acylation was posttranslational. The myristoylation of GPIX while the palmitoylation of GPIb occurred throughout megakaryocyte maturation. Myristoylation and palmitoylation may have different functions relevant to the assembly of the GPIb complex in megakaryocytes.     

Altered membrane association of p60v-src and a murine 63-kDa N-myristoyl protein after incorporation of an oxygen-substituted analog of myristic acid

A number of viral and cellular proteins contain covalently bound lipid. In a subset of these acyl proteins, the 14-carbon saturated fatty acid myristic acid is attached through an amide linkage to an NH2-terminal glycine residue. Myristoyl-CoA:protein N-myristoyltransferase (NMT) transfers the myristoyl moiety from myristoyl-CoA to these nascent proteins and is highly selective for fatty acid chain length. We have found that 10-(propoxy)decanoyl-CoA (11-oxymyristoyl-CoA), an analog of myristic acid with reduced hydrophobicity, acts as a substrate for NMT both in vitro and in vivo. Comparison of the in vitro kinetic properties of a number of synthetic octapeptide substrates of NMT using myristoyl-CoA or 11-oxymyristoyl-CoA indicated that there is an interaction between the acyl-CoA and peptide binding sites of this acyltransferase. Peptide catalytic efficiency with 11-oxymyristoyl-CoA was reduced relative to that with myristoyl-CoA, but the extent of the reduction varied widely among the octapeptides tested. These in vitro data accurately predicted that only a subset of myristoyl proteins synthesized in Saccharomyces cerevisiae and a murine myocyte-like cell line (BC3H1) would incorporate 11-oxy[3H]myristate. Substitution of the myristoyl moiety by the 11-oxymyristoyl moiety does not significantly affect the membrane association of most N-myristoyl proteins. However, for the tyrosine kinase p60v-src and a 63-kDa N-myristoyl protein in BC3H1 cells, analog incorporation results in marked redistribution from the membrane to the cytosolic fraction. These studies demonstrate the utility of heteroatom-containing analogs for analysis of the role of myristate in acyl protein targeting. The sequence-specific nature of analog incorporation and the protein-specific effects on membrane association suggests that these compounds may represent a useful class of antiviral and antitumor agents.     

Evidence that a glycolipid tail anchors antigen 117 to the plasma membrane of Dictyostelium discoideum cells.

We describe the biochemical features of the putative cell cohesion molecule antigen 117, indicating that it is anchored to the plasma membrane by a glycolipid tail. Antigen 117 can be radiolabeled with [3H]myristate, [3H]palmitate, and [14C]ethanolamine. The fatty acid label is removed by periodate oxidation and nitrous acid deamination, indicating that the fatty acid is attached to the protein by a structure containing carbohydrate and an unsubstituted glucosamine. As cells develop aggregation competence, the antigen is released from the cell surface in a soluble form that can still be radiolabeled with [14C]ethanolamine but not with [3H]myristate or [3H]palmitate. The molecular weight of the released antigen is similar to that found in the plasma membrane, but it preferentially partitions in Triton X-114 as a hydrophilic, as opposed to a hydrophobic, protein. Plasma membranes contain the enzyme activity responsible for the release of the antigen in a soluble form.     

Bacterial lipopolysaccharides, phorbol myristate acetate, and zymosan induce the myristoylation of specific macrophage proteins.

We demonstrate stimulus-dependent incorporation of exogenously added [3H]myristic acid into specific macrophage proteins. In control unstimulated cells an 18-kDa protein is the major acylated species. In cells incubated with bacterial lipopolysaccharide (LPS), or its monoacyl glucosamine phosphate derivative, fatty acid is incorporated into proteins with molecular mass of 68 kDa and a doublet of approximately 42-45 kDa. Phorbol 12-myristate 13-acetate (PMA) or a phagocytic stimulus (zymosan) promotes the acylation of a similar array of proteins. However, PMA and zymosan also promote the myristoylation of unique proteins of 92 and 50 kDa. The fatty acid associated with each of the acylated proteins is myristic acid. The myristate is probably linked to the proteins through amide bonds, since it is not released by treatment with hydroxylamine. Palmitate and arachidonate are not incorporated into proteins in the same manner. Temporal analysis revealed that LPS-induced proteins are myristoylated by 30 min, while the 50-kDa protein myristoylated in response to PMA is labeled later. Most myristoylated proteins appear to be associated with the membrane fraction. Macrophages from C3H/HeJ mice, which do not respond to LPS, do not show any LPS-dependent protein acylation. Interestingly, zymosan and PMA induce the myristoylation of the 50-kDa protein in C3H/HeJ macrophages, but not the acylation of the 68-kDa and 42-kDa doublet species. We suggest that myristoylation of specific proteins is an intermediary in the capacity of LPS, PMA, and zymosan to alter macrophage functions such as arachidonic acid metabolism.     

Differential diagnosis of hydroxydicarboxylic aciduria based on release of3H2O from [9,10-3H]myristic and [9,10-3H]palmitic acids by intact cultured fibroblasts

Summary Intact cultured fibroblasts from patients with deficiency of long-chain 3-hydroxyacyl-CoA dehydrogenase release³H₂O from [9,10-³H]myristic acid and [9,10-³H]palmitic acid more slowly than normal. The ratio of activity (palmitate/myristate) is also low and the expression (rate with palmitate)²/(rate with myristate) gives good differentiation between affected and unaffected cells. In some patients who have shown hydroxydicarboxylic aciduria when unwell there is reduced³H₂O production from [9,10-³H]myristic and [9,10-³H]palmitic acids by intact cultured fibroblasts but normal 3-hydroxyacyl-CoA dehydrogenase activities in disrupted cells. The palmitate/myristate ratio is higher than in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. The basic defect in these patients is still unknown but it is suggested that caution be used over the administration of medium-chain triglyceride.     

Cytochrome c oxidase in Neurospora crassa contains myristic acid covalently linked to subunit 1.

Radiolabel from [3H]myristic acid was incorporated by Neurospora crassa into the core catalytic subunit 1 of cytochrome c oxidase (EC 1.9.3.1), as indicated by immunoprecipitation. This modification of the subunit, which was specific for myristic acid, represents an uncommon type of myristoylation through an amide linkage at an internal lysine, rather than an N-terminal glycine. The [3H]myristate, which was chemically recovered from the radiolabeled subunit peptide, modified an invariant Lys-324, based upon analyses of proteolysis products. This myristoylated lysine is found within one of the predicted transmembrane helices of subunit 1 and could contribute to the environment of the active site of the enzyme. The myristate was identified by mass spectrometry as a component of mature subunit 1 of a catalytically active, purified enzyme. To our knowledge, fatty acylation of a mitochondrially synthesized inner-membrane protein has not been reported previously.     

Covalently bound myristate in a lymphoma tyrosine protein kinase.

The murine lymphoma cell line LSTRA expresses high levels of a membrane-associated tyrosine protein kinase, which we now show to be acylated by [3H]myristate in vivo. This [3H]myristate-labeled tyrosine protein kinase is immunoprecipitated from detergent extracts of postnuclear particulate fractions with an antibody directed against its single site of tyrosine phosphorylation. This site has an amino acid sequence also found in the transforming proteins of the Rous sarcoma and Y73 viruses. Preincubation of the antibody with a peptide containing the same sequence completely blocks this immunoprecipitation. The [3H]myristate linkage to the protein is stable in boiling 2% NaDodSO4/0.125 M Tris Cl, pH 6.7/5% 2-mercaptoethanol, which suggests an amide rather than an ester linkage. Analogous attempts to label with [3H]palmitate show negligible incorporation into either nonnuclear particulate proteins or immunoprecipitated proteins. Chemical characterization of the immunoprecipitated protein isolated by NaDodSO4/PAGE verifies that the 3H label is in protein-associated myristate. Sonicated 5% NaDodSO4 extracts of LSTRA and YAC-1 (another murine lymphoma line) cells contain quite different distributions of myristoylated proteins.     

Myristate exchange on the Trypanosoma brucei variant surface glycoprotein.

The glycosyl-phosphatidylinositol (GPI) anchor of the Trypanosoma brucei variant surface glycoprotein (VSG) is unique in having exclusively myristate as its fatty acid component. We previously demonstrated that the myristate specificity is the result of two independent pathways. First, the newly synthesized free GPI, which is not myristoylated, undergoes fatty acid remodeling to replace both its fatty acids with myristate. Second, the myristoylated precursor, glycolipid A, undergoes a myristate exchange reaction, detected by the replacement of unlabeled myristate by [3H]myristate. Remodeling and exchange have different enzymatic properties and apparently occur in different subcellular compartments. We now demonstrate that the GPI anchor linked to VSG is the major substrate for myristate exchange. VSG can be efficiently labeled with [3H]myristate by exchange in the presence of cycloheximide, an inhibitor that prevents new VSG synthesis and thus anchor addition to protein. Not only is newly synthesized VSG subject to exchange, but mature VSG, possibly recycling from the cell surface, also undergoes myristate exchange.     

Myristoylated alpha subunits of guanine nucleotide-binding regulatory proteins.

Antisera directed against specific subunits of guanine nucleotide-binding regulatory proteins (G proteins) were used to immunoprecipitate these polypeptides from metabolically labeled cells. This technique detects, in extracts of a human astrocytoma cell line, the alpha subunits of Gs (stimulatory) (alpha 45 and alpha 52), a 41-kDa subunit of Gi (inhibitory) (alpha 41), a 40-kDa protein (alpha 40), and the 36-kDa beta subunit. No protein that comigrated with the alpha subunit of Go (unknown function) (alpha 39) was detected. In cells grown in the presence of [3H]myristic acid, alpha 41 and alpha 40 contained 3H label, while the beta subunit did not. Chemical analysis of lipids attached covalently to purified alpha 41 and alpha 39 from bovine brain also revealed myristic acid. Similar analysis of brain G protein beta and gamma subunits and of Gt (transducin) subunits (alpha, beta, and gamma) failed to reveal fatty acids. The fatty acid associated with alpha 41, alpha 40, and alpha 39 was stable to treatment with base, suggesting that the lipid is linked to the polypeptide via an amide bond. These GTP binding proteins are thus identified as members of a select group of proteins that contains myristic acid covalently attached to the peptide backbone. Myristate may play an important role in stabilizing interactions of G proteins with phospholipid or with membrane-bound proteins.     

Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria.

Protein N-myristoylation refers to the covalent attachment of a myristoyl group (C14:0), via amide linkage, to the NH2-terminal glycine residue of certain cellular and viral proteins. Myristoyl-CoA:protein N-myristoyltransferase (NMT) catalyzes this cotranslational modification. We have developed a system for studying the substrate requirements and biological effects of protein N-myristoylation as well as NMT structure-activity relationships. Expression of the yeast NMT1 gene in Escherichia coli, a bacterium that has no endogenous NMT activity, results in production of the intact 53-kDa NMT polypeptide as well as a truncated polypeptide derived from proteolytic removal of its NH2-terminal 39 amino acids. Each E. coli-synthesized NMT species has fatty acid and peptide substrate specificities that are indistinguishable from those of NMT recovered from Saccharomyces cerevisiae, suggesting that the NH2-terminal domain of this enzyme is not required for its catalytic activity. By using a dual plasmid system, N-myristoylation of a mammalian protein was reconstituted in E. coli by simultaneous expression of the yeast NMT1 gene and a murine cDNA encoding the catalytic (C) subunit of cAMP-dependent protein kinase (PK-A). The fatty acid specificity of N-myristoylation was preserved in this system: [9,10(n)-3H]myristate but not [9,10(n)3H]palmitate was efficiently linked to Gly-1 of the C subunit. [13,14(n)-3H]10-Propoxydecanoic acid, a heteroatom-containing analog of myristic acid with reduced hydrophobicity but similar chain length, was an effective alternative substrate for NMT that also could be incorporated into the C subunit of PK-A. Such analogs have recently been shown to inhibit replication of certain retroviruses that depend upon linkage of a myristoyl group to their gag polyprotein precursors (e.g., the Pr55gag of human immunodeficiency virus type 1). A major advantage of the bacterial system over eukaryotic systems is the absence of endogenous NMT and substrates, providing a more straightforward way of preparing myristoylated, analog-substituted, and nonmyristoylated forms of a given protein for comparison of their structural and functional properties. The system should facilitate screening of enzyme inhibitors as well as alternative NMT fatty acid substrates for their ability to be incorporated into a specific target protein. Our experimental system may prove useful for recapitulating other eukaryotic protein modifications in E. coli so that structure-activity relationships of modifying enzymes and their substrates can be more readily assessed.     

Primary structure of a cerulenin-binding beta-ketoacyl-[acyl carrier protein] synthase from barley chloroplasts.

The radioactively labeled beta-ketoacyl thioester synthase inhibitor [3H] cerulenin was used to tag three dimeric barely chloroplast proteins (alpha alpha, alpha beta, and beta beta) from the stromal fraction. Oligonucleotides corresponding to amino acid sequences obtained from the purified proteins were used to generate with the polymerase chain reaction a probe for cDNAs encoding the beta subunit. cDNA sequencing revealed an open reading frame for 462 residues comprising the mature protein and a 35-amino acid transit peptide. The deduced amino acid sequence of the mature protein is homologous to the beta-ketoacyl-[acyl carrier protein] (ACP) synthase I [3-oxoacyl-ACP synthase; acyl-ACP:malonyl-ACP C-acyltransferase (decarboxylating), EC 2.3.1.41] of Escherichia coli. Under analogous experimental conditions [3H]cerulenin tagged a single dimeric protein from spinach chloroplasts.     

Unconventional myristoylation of large-conductance Ca²⁺-activated K⁺ channel (Slo1) via serine/threonine residues regulates channel surface expression

Protein myristoylation is a means by which cells anchor proteins into membranes. The most common type of myristoylation occurs at an N-terminal glycine. However, myristoylation rarely occurs at an internal amino acid residue. Here we tested whether the α-subunit of the human large-conductance voltage- and Ca²⁺-activated K⁺ channel (hSlo1) might undergo internal myristoylation. hSlo1 expressed in HEK293T cells incorporated [³H]myristic acid via a posttranslational mechanism, which is insensitive to cycloheximide, an inhibitor of protein biosynthesis. In-gel hydrolysis of [³H]myristoyl-hSlo1 with alkaline NH₂OH (which cleaves hydroxyesters) but not neutral NH₂OH (which cleaves thioesters) completely removed [³H]myristate from hSlo1, suggesting the involvement of a hydroxyester bond between hSlo1’s hydroxyl-bearing serine, threonine, and/or tyrosine residues and myristic acid; this type of esterification was further confirmed by its resistance to alkaline Tris·HCl. Treatment of cells expressing hSlo1 with 100 μM myristic acid caused alteration of hSlo1 activation kinetics and a 40% decrease in hSlo1 current density from 20 to 12 nA*MΩ. Immunocytochemistry confirmed a decrease in hSlo1 plasmalemma localization by myristic acid. Replacement of the six serines or the seven threonines (but not of the single tyrosine) of hSlo1 intracellular loops 1 and 3 with alanines decreased hSlo1 direct myristoylation by 40–44%, whereas in combination decreased myristoylation by nearly 90% and abolished the myristic acid-induced change in current density. Our data demonstrate that an ion channel, hSlo1, is internally and posttranslationally myristoylated. Myristoylation occurs mainly at hSlo1 intracellular loop 1 or 3, and is an additional mechanism for channel surface expression regulation.     

Capsid protein VP4 of poliovirus is N-myristoylated.

Poliovirus was labeled in vivo with [3H]myristic acid. Analysis of the capsid polypeptides revealed that the [3H]myristic acid residues copurified with VP4, the smallest and internal capsid protein of the virion. Evidence is presented showing unambiguously that the N-terminal glycine residue of VP4 is N-myristoylated. A previous analysis of the tryptic peptides of VP4 [Dorner, A. J., Dorner, L. F., Larsen, G. R., Wimmer, E. & Anderson, C. W. (1982) J. Virol. 42, 1017-1028] had shown that the N-terminal blocking group exists on all VP4 molecules as well as on VP0 and P1, two precursor polypeptides to VP4 in poliovirus. The possible function of the myristic acid residue in VP4 and in its precursor in poliovirus proliferation is discussed.     

The presence of cysteine in the cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition.

The transmembrane glycoprotein (G protein) of vesicular stomatitis virus (VSV) is known to contain 1-2 mol of covalently linked fatty acid (palmitate) per mol of protein. G protein is oriented in cellular membranes such that the carboxyl-terminal 29 amino acids protrude into the cytoplasm. We have obtained expression in eukaryotic cells of mutagenized cDNA clones that encode VSV G proteins lacking portions of this cytoplasmic domain. Labeling of these truncated proteins with [3H]palmitate indicated that the palmitate might be linked to an amino acid residue within the first 14 residues on the carboxyl-terminal side of the transmembrane domain. Using oligonucleotide directed mutagenesis, we changed the single codon specifying cysteine in this domain to a codon specifying serine. Expression of this mutant gene results in synthesis of a G protein lacking palmitate. We suggest that linkage of palmitate to G protein is through the cysteine in the cytoplasmic domain and that such a linkage may occur in many viral and cellular glycoproteins. The G protein lacking palmitate is glycosylated and is transported normally to the cell surface.     

Selective inhibition of synthesis of enzymes for de novo fatty acid biosynthesis by an endotoxin-induced mediator from exudate cells.

An endotoxin-induced mediator from exudate cells markedly suppresses the activities of the key enzymes for de novo fatty acid biosynthesis--acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.2] and fatty acid synthetase--in differentiating 3T3-L1 murine preadipocytes. The loss in activity, at least in part, appears to be due to a specific effect on the synthesis of the enzymes, as determined by a decreased incorporation of [35S]methionine into immunoadsorbable acetyl-CoA carboxylase and fatty acid synthetase when the cells were exposed to the mediator. During this exposure, the radiolabeling of proteins with [35S]methionine in a particulate fraction was decreased by nearly 50% with little change in the soluble protein fraction. Sodium dodecyl sulfate/polyacrylamide gel analysis of the labeled protein indicated no major disturbances of protein synthesis in general; however, the syntheses of specific proteins in both the soluble and particulate fractions were enhanced or depressed. The present study demonstrates that endotoxin promotes the release of a mediator from exudate cells that regulates key anabolic activities in adipose cells.     

Palmitoylation of the GluR6 kainate receptor.

The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.     

Analogs of palmitoyl-CoA that are substrates for myristoyl-CoA:protein N-myristoyltransferase.

Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p; EC 2.3.1.97) is an essential enzyme that is highly selective for myristoyl-CoA in vivo. It is unclear why myristate (C14:0), a rare cellular fatty acid, has been selected for this covalent protein modification over more abundant fatty acids such as palmitate (C16:0), nor is it obvious how the enzyme's acyl-CoA binding site is able to discriminate between these two fatty acids. Introduction of a cis double bond between C5 and C6 of palmitate [(Z)-5-hexadecenoic acid] or a triple bond between C4 and C5 or C6 and C7 (Y4- and Y6-hexadecenoic acids) yields compounds that, when converted to their CoA derivatives, approach the activity of myristoyl-CoA as Nmt1p substrates in vitro. Kinetic studies of 42 C12-C18 fatty acids containing triple bonds, para-phenylene, or a 2,5-furyl group, as well as cis and trans double bonds, suggest that the geometry of the enzyme's acyl-CoA binding site requires that the acyl chain of active substrates assume a bent conformation in the vicinity of C5. Moreover, the distance between C1 and the bend appears to be a critical determinant for optimal positioning of the acyl-CoA in this binding site so that peptide substrates can subsequently bind in the sequential ordered bi-bi reaction mechanism. Identification of active, conformationally restricted analogs of palmitate offers an opportunity to "convert" wild-type or mutant Nmts to palmitoyltransferases so that they can deliver these C16 fatty acids to critical N-myristoylproteins in vivo. nmt181p contains a Gly-451-->Asp mutation, which causes a marked reduction in the enzyme's affinity for myristoyl-CoA. Strains of S. cerevisiae containing nmt1-181 exhibit temperature-sensitive myristic acid auxotrophy: their complete growth arrest at 37 degrees C is relieved when the medium is supplemented with 500 microM C14:0 but not with C16:0. The CoA derivatives of (Z)-5-hexadecenoic and Y6-hexadecynoic acids are as active substrates for the mutant enzyme as myristoyl-CoA at 24 degrees C. However, unlike C16:0, they produce growth arrest of nmt181p-producing cells at this "permissive" temperature, suggesting that these C16 fatty acids do not allow expression of the biological functions of essential S. cerevisiae N-myristoylproteins.     

Hydrolysis of inositol phospholipids precedes cellular proliferation in asbestos-stimulated tracheobronchial epithelial cells.

Metabolism of inositol phospholipids and phosphatidylcholine was investigated in tracheobronchial epithelial cells exposed to mitogenic concentrations of crocidolite asbestos. Alterations in levels of diacylglycerol, the endogenous activator of protein kinase C, and inositol polyphosphates, presumed mobilizers of intracellular calcium, were examined. Cultures labeled with [3H]glycerol and exposed to proliferative concentrations of crocidolite asbestos demonstrated significant elevations in [3H]diacylglycerol. In contrast, crocidolite-exposed cells labeled with [3H]myristic acid or [3H]choline did not display elevated production of [3H]diacylglycerol or release of [3H]choline metabolites--i.e., evidence of phosphatidylcholine hydrolysis. The soluble tumor promoter phorbol 12-myristate 13-acetate catalyzed both of these changes. myo-[3H]Inositol-labeled cells exposed as briefly as 10 min to mitogenic concentrations of crocidolite demonstrated elevations in [3H]inositol mono-, tris-, and terakisphosphates, phenomena indicating turnover of inositol phospholipids. The detection of diacylglycerol and inositol phosphates in crocidolite asbestos-exposed cells suggests that this fibrous tumor promoter activates phospholipase C as it stimulates cellular proliferation.     

Identification of two integral membrane proteins of Plasmodium falciparum.

We describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage lambda gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a Mr 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a Mr 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with [3H]glucosamine and [3H]myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the Mr 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.