cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding

Suspension-cultured cells of carrot (Daucus carota L.) synthesize and secrete a glycoprotein that is normally found only in dermal tissues (epidermis, endodermis and periderm). This protein, previously called GP57, is now referred to as EDGP (E xtracellular D ermal G lyco P rotein). We purified sufficient quantities of EDGP to obtain amino-acid sequences on two internal tryptic peptides and screened a cDNA library of young carrot roots with antiserum to EDGP and with oligonucleotides corresponding to the peptides. Here we report the derived amino-acid sequence of EDGP. Sequence comparisons show that it has 40% amino-acid sequence identity with 7S basic globulin, a protein that is released when soybean seeds are soaked in hot water for a few hours. We suggest that these two proteins belong to a new family of dermal proteins. As far as we know, this is the first reported derived amino-acid sequence for protein that is specific to the epidermis and other dermal tissues. The level of EDGP mRNA is low in dry seeds, but increases rapidly in growing seedlings as they develop dermal tissues. The level of mRNA is low in storage roots, but increases rapidly in response to wounding. The presence of EDGP in dermal tissues and its up-regulation in response to wounding indicate a role in the response of plants to biotic and-or abiotic stresses. An unusual feature of the amino-acid sequence of EDGP is that it contains a short motif, which is present at the active site of aspartyl proteases such as pepsin and chymosin.Abbreviations cDNA copy DNA - 2,4-D 2,4-dichlorophen-oxyacetic acid - EDGP extracellular dermal glycoprotein - 7SBG 7S basic globulin Supported by a contract from the United States Department of Energy (Energy Biosciences) (to M.J.C.) and a Grant-in-Aid for Special Research on Priority Areas (01660002, Cellular and Molecular Basis for Reproductive Processes in Plants) from the Ministry of Education, Science and Culture, and by the Fund from Basic Research Core System of Science and Technology Agency, Japan (to S.S.).     

Relationship of indole-3-acetic acid and tryptophan concentrations in normal and 5-methyltryptophan-resistant cell lines of wild carrots

A 5-methyltryptophan(5-MT)-resistant cell line of wild carrot (Daucus carota L.), W001, that exhibited auxin-independent callus growth, was found to accumulate indole-3-acetic acid (IAA) and tryptophan (trp). Anthranilate-synthetase activity in W001 cell extract was less sensitive to feedback inhibition by trp than in the original 5-MT-sensitive cell lines. It is hypothesized that the resistant enzyme allowed more trp synthesis and accumulation which, in turn, affected the IAA concentration in the cell. Since carrot cultures cannot regenerate in the presence of exogenous auxin, the elevated IAA concentration in W001 may be responsible for its drastically reduced capacity to regenerate. The relationship between trp and IAA levels was further investigated by examining the effect of 2,4-dichlorophenoxy acetic acid (2,4-D) on the endogenous concentration of trp and IAA. In general, the IAA level was reduced but the trp concentration was elevated when 2,4-D was present in the culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - 5-MT^r 5-MT-resistant - 5-MT^s 5-MT-sensitive - trp tryptophan     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

Quantitative studies of embryogenesis in normal and 5-methyltryptophan-resistant cell lines of wild carrot

The frequency of embryo formation was determined in normal and 5-methyltryptophan-resistant (5-MT^r) cell lines of wild carrot (Daucus carota L.) grown in the presence or absence of 2-isopentenyladenine (2-ip) and 2,4-dichlorophenoxyacetic acid (2,4-D). 2-ip stimulated the intitation of embryo formation and also accelerated embryo development. 2.4-D inhibited embryo differentiation at several stages: at 0.1 mg/l, it stopped regeneration at the earliest stage, resulting in callus growth instead of embryo formation; at 0.04 mg/l 2,4-D, some globular embryos were produced, but they did not develop into more advanced embryos. Variant cell lines with higher levels of auxin (indole-3-acetic acid, IAA) were used to study the effect of an elevated endogenous concentration of auxin on embryogenesis. IAA at these concentrations suppressed regeneration in the same manner as the exogenous auxin, 2,4-D, did. This result confirms the hypothesis that high levels of IAA are responsible for the suppression of regeneration in the 5-MT^r cell lines.     

Elicitor induction of the synthesis of a novel lectin-like arabinosylated hydroxyproline-rich glycoprotein in suspension cultures of Phaseolus vulgaris L.

A novel lectin-like glycoprotein which accumulates in response to fungal elicitor action has been characterised in endomembranes from suspension cultures of French bean (Phaseolus vulgaris L.). The lectin, which has specificity towards N-acetylglucosamine oligomers, consists of a polypeptide of apparent molecular weight (M_r) 31 000 which is rich in glycine and contains 6.7% hydroxyproline O-linked to arabinose-containing oligosaccharides to give a glycoprotein of M_r 42500. A dual-labelling technique has been used to identify changes in the synthesis of the glycoprotein in cells exposed to fungal elicitor molecules. Thus, incorporation of [¹⁴C]proline into membranes in vivo and of [1^-3H]arabinose from uridine 5-diphosphate [1^-3H]arabinose in vitro and analysis by isoelectric focussing-polyacrylamide gel electrophoresis gave absolute correspondence of the labelled isoform of the glycoprotein. Having established the absence of contaminating polypeptides, subsequent analysis of microsomal fractions bysodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the peak of sythesis of the M_r-42500 glycoprotein occurred 4 h after the addition of fungal elicitor. The changes in the level of incorporation into the glycoprotein monomers were concomitant with increases in the activity of prolyl hydroxylase (EC 1.14.11.2)Incorporation of [¹⁴C]proline and its subsequent post-translational modification to hydroxyproline in microsomal polypeptides was followed by rapid transfer into the wall with an average t _1/2 of about 7 min. The M_r-42500 glycoprotein was rapidly transferred out of the endomembrane fraction with a t _1/2 of 2 min and could be detected in wall fractions where it became progressively less extractable. The glycoprotein, which clearly differs from bean extensin, accounts for up to 40% of the hydroxyproline newly exported in response to elicitor action. The lectin, which resembles those found in the Solanaceae and which is coinduced with enzymes of phytoalexin synthesis, may play some role in disease resistance.Abbreviations HRGP hydroxyproline-rich glycoprotein - IEF isoelectric focussing - M_r apparent molecular weight - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate     

Suppression of somatic embryogenesis in Citrus cell cultures by extracellular proteins

Nucellar-derived cell cultures of sour orange (Citrus aurantium L.) proliferate as proembryogenic masses. By a change in the carbon source of the medium from sucrose to glycerol they are induced to undergo synchronous embryogenesis forming embryo initials that develop into globular embryos. The proembryogenic masses released glycoproteins to the medium. Exogenous addition of the glycoproteins to cells in glycerol-containing medium modified the course of embryo development in a dose-dependent manner. Addition of 20 g · ml^–1 of glycoproteins blocked embryogenesis and resulted in an accumulation of embryo initials. When glycoproteins were added to cultures containing advanced globularstage embryos further development was suppressed. The inhibitory component of the glycoproteins was found to be a family of polypeptides with apparent molecular masses of 53–57 kDa. While these proteins normally accumulated only in cultures of proembryogenic masses, they could be induced to accumulate in glycerol-containing medium by the addition of the glycoproteins. Thus, their accumulation was not a direct consequence of the type of growth medium used or the developmental state of the cultures. The results indicate that the 53-to 57 kDa glycoproteins could play a regulatory role in in-vitro embryogenesis in sour orange. The normal progression of embryo development appears to depend, in an obligatory manner, on the absence of these glycosylated extracellular proteins from the medium.Abbreviations kDa kilodalton - PEM proembryogenic masses - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - 2D-PAGE Two-dimensional polyacrylamide gel electrophoresis We thank Dr. S. Satoh (Institute of Biological Sciences, Tsukuba, Japan) for sending protein samples of the purified 57-kDa glycoprotein. This research was supported by a grant from the Charles H. Revson Foundation for Basic Research in the Life Sciences of the Israel Academy of Sciences. R.F. is a recipient of the Jack and Florence Goodman Career Development Chair.     

Tryptophan and indole-3-acetic acid accumulation in Acer cell cultures and its relationship with cell autolysis

The accumulation and decline of free indole-3-acetic acid (IAA) and tryptophan has been monitored in cells of Acer pseudoplatanus L. grown in batch suspension cultures. The period of maximal IAA accumulation per cell or per unit dry weight of tissue was found to precede the peak of tryptophan accumulation by several days. A study of cell viability throughout a growth passage indicated the presence of a basal level of non-viable cells of 5–7%, with only minor increases occurring during the first week of the three-week growth passage. The results suggest that IAA biosynthesis is not regulated by substrate availability arising from proteolysis in dead cells.Abbreviation GC-MS Gas chromatography-mass spectrometry - IAA indole-3-acetic acid - 5-MT 5-methyltryptophan - TLC thin-layer chromatography     

Quantitation of gibberellins and the metabolism of [3H]gibberellin A1 during somatic embryogenesis in carrot and anise cell cultures

In a carrot (Daucus carota L.) cell line lacking the ability to undergo somatic embryogenasis, and in carrot and anise (Pimpinella anisum L.) cell lines in which embryogenesis could be regulated by presence or absence of 2,4-dichlorophen-oxyacetic acid (2,4-D), in the medium (+2,4-D=no embryogenesis,-2,4-D=embryo differentiation and development), the levels of endogenous gibberellin(s) (GA) were determined by the dwarfrice bioassay, and the metabolism of [³H]GA₁ was followed. Embryos harvested after 14 d of subculture in-2,4-D had low levels (0.2–0.3 g g^-1 dry weight) of polar GA (e.g. GA₁-like), but much (3–22 times) higher levels of less-polar GA (GA_4/7-like); GA₁, GA₄ and GA₇ are native to these cultures. Conversely, the undifferentiated cells in a non-embryogenic strain, and proembryos of an embryogenic strain (+2,4-D) showed very high levels of polar GA (2.9–4.4 g g^-1), and somewhat reduced levels of less-polar GA. Cultures of anise undergoing somatic embryo development (-2,4-D) metabolized [³H]GA₁ very quickly, whereas proembryo cultures of anise (+2,4-D) metabolized [³H]GA₁ slowly. The major metabolites of [³H]GA₁ in anise were tentatively identified as GA₈-glucoside (24%), GA₈ (15%), GA₁-glucoside (8%) and the ¹⁽¹⁰⁾GA₁-counterpart (2%). Thus, high levels of a GA₁-like substance and a reduced ability to metabolize GA₁ are correlated with the absence of embryo development, while lowered levels of GA₁-like substance and a rapid metabolism of GA₁ into GA₈ and GA-conjugates are correlated with continued embryo development. Exogenous application of GA₃ is known to reduce somatic embryogenesis in carrot cultures; GA₄ was found to have the same effect in anise cultures. Thus, a role (albeit negative) in somatic embryogenesis for a polar, biologically active GA is implied.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellin(s) or gibberellin-like substances - GC-RC gas chromatography-radiochromatogram counting - HPLC high-presare liquid chromatography - Rt retention time - TLC thinlaver chromatography     

Immunocytochemical localization of patatin,the major glycoprotein in potato (Solanum tuberosum L.) tubers

Patatin is a family of glycoproteins with an apparent molecular weight of 40 kDa. The protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization of the patatin protein. Since antibodies raised against patatin showed crossreactivity with glycans of other glycoproteins, antibodies specific for the protein portion of the glycoprotein were purified. Using these antibodies for electron-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanum tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers.Abbreviations PHA phytohemagglutinin - TFMS trifluoromethanesulfonic acid     

Partial amino-acid sequence of ECP31, a carrot embryogenic-cell protein,and enhancement of its accumulation by abscisic acid in somatic embryos

ECP31, an embryogenic-cell protein from carrot (Daucus carota L.), was purified by sequential column-chromatographic steps and digested by V8 protease on a nitrocellulose membrane. The resultant peptides were separated by reverse-phased column chromatography and sequenced. The sequences obtained were 70–80% homologous to those of a late-embryogenesis-abundant protein (D34) from cotton (Baker et al, 1988, Plant Mol. Biol. 11, 227–291). The level of ECP31 in somatic embryos of carrot was increased by treatment of the embryos with 3.7 · 10^–6 M abscisic acid (ABA) for 48 h, and there was no change in this enhanced level for up to 192 h in the presence of ABA. No similar enhancing effect of ABA was observed on the level of ECP31 in embryogenic callus or segments of carrot hypocotyls. In an immunohistochemical analysis, ECP31 was found in epidermal tissue and in the vascular system of ABA-treated somatic embryos.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - LEA protein late-embryogenesis-abundant protein To whom correspondence should be addressedThis work was supported in part by a grant-in-aid for Special Research in Priority Areas (Project No. 02242102) from the Ministry of Education, Science and Culture, Japan, and by Special Coordination Funds of the Science and Technology Agency of the Japanese Government.     

Uridine and dolichyl diphosphate activated oligosaccharides are intermediates in the biosynthesis of the S-layer glycoprotein of Methanothermus fervidus

The surface of the extremely thermophilic archaebacterium Methanothermus fervidus is covered by glycoprotein subunits. The carbohydrate moiety of the surface glycoprtein accounts for about 17 mol%. It is composed of mannose, 3-O-methylglucose, galactose, N-acetylglucosamine and N-acetylgalactosamine. From cell extracts the corresponding surgar-1-phosphates and nucleotide activated derivatives of Man, Gal, GlcNAc and GalNAc were isolated. Furthermore UDP-and dolichyl activated oligosaccharides were obtained. On the basis of the isolated precursors a pathway for the biosynthesis of the oligosaccharide chains is proposed.Abbreviations DNP-Glu N-2,4-dinitrophenyl-glutamic acid - Dol dolichol - Gal galactose - Gal-1-P galactose-1-phosphate - GalNAc N-acetylgalactosamine - GalNAc-1-P N-acetylgalactosamine-1-phosphate - Glc glucose - GlcNAc N-acetylglucosamine - GlcNAc-1-P N-acetylglucosamine-1-phosphate - Man mannose - Man-1-P mannose-1-phosphate - 3-O-MeGlc 3-O-methylglucose - P phosphate - TCA trichloroacetic acid - TLC thin-layer chromatography - Tris tris(hydroxymethyl)aminomethan     

A major stress-inducible Mr-42000 wall glycoprotein of French bean (Phaseolus vulgaris L.)

A major wall protein of suspension-cultured cells of French bean has been isolated and characterised. It can be prepared from walls or the culture filtrate and in composition it is particularly rich in proline, valine and glutamic acid/glutamine and contains appreciable amounts of hydroxyproline. The N-terminus shows some glycosylation, while following chemical deglycosylation the first 38 residues were found to be identical to those of proline-rich proteins from soybean. However, the composition of the highly purified M_r-42000 bean protein differs considerably from the soybean proteins and must contain its own specific domains. An antibody was raised and used to demonstrate the inducibility of the M_r-42000 bean protein in response to elicitor action. The protein was found to be mainly localised in the intercellular spaces of the cortical cells of bean hypocotyls and at the wall-plasmalemma interface of xylem vessels, another potentially accessible compartment for pathogens. Following wounding, the protein was found to be generally distributed in the wall of epidermal and cortical cells of the hypocotyls. The M_r-42000 protein is cross reactive with antibodies raised to glycoproteins of the Rhizobium infection thread and the chitin-binding hydroxyproline-rich glycoprotein, potato lectin. These common epitopes together with the previously demonstrated chitin-binding properties of the bean protein indicate a role in host-microbial interactions. Furthermore, the M_r-42000 protein itself bound to the growing hyphal tips of the bean pathogen, Colletotrichum lindemuthianum.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - PAL phenylalanine ammonia-lyase We thank Dr Nick Brewin for advice on interpretation of immunolocalisations and for the gift of MCA 265. We thank Dudley Fernandino for carrying out the confocal microscopy. GPB thanks the Science and Engineering Research Council for funding.     

Variation in endoplasmic-reticulum-associated glycoproteins of carrot cells cultured in vitro

Glycoproteins extracted from microsomes of in-vitro-cultured cells of Daucus carota L. cv. US-Harumakigosun were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by peroxidase-conjugated concanavalin A. The appearance of a glycoprotein with M_r 31 000 (GP 31) was correlated with the ability of cells to form somatic embryos. GP 31 appeared in embryogenic cells cultured in 2,4-dichlorophenoxyacetic acid (2,4-D)-containing medium, but not in somatic embryos and non-embryogenic cells; it disappeared when the cultures were transferred to auxin-free medium. Another glycoprotein with M_r 32 000 (GP 32) was detected only in non-embryogenic cells, regardless of the presence or absence of 2,4-D. Both glycoproteins, GP 31 and GP 32, were associated with the rough endoplasmic reticulum and were extractable with 0.05% deoxycholate.Abbreviations Con A concanavalin A - 2,4-D 2,4-dichlorophenoxyacetic acid - ER endoplasmic reticulum - GP 31, GP 32 a glycoprotein with an apparent molecular mass of 31 or 32 kdalton - kDa kilodalton - MS Murashige and Skoog - M_r apparent molecular mass - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) secrete a number of acid hydrolases and other proteins that have both highmannose and complex asparagine-linked glycans. We used affinity chromatography with concanavalin A and an antiserum specific for complex glycans in conjunction with in vivo-labeling studies to show that all of the secreted proteins carry glycans. The presence of complex glycans on secretory proteins indicates that they are passing through the Golgi complex on the way to the extracellular compartment. The sodium ionophore, monensin, did not block the transport of proteins to the extracellular medium, even though monensin efficiently inhibited the Golgi-mediated processing of complex glycans. The inhibition of N-glycosylation by tunicamycin reduced by 76% to 84% the accumulation of newly synthesized (i.e. radioactively labeled) protein that was secreted by the sycamore cells, while cytoplasmic protein biosynthesis was not affected by this antibiotic. However, in the presence of glycoprotein-processing inhibitors, such as castanospermine and deoxymannojirimycin, the formation of complex glycans was prevented but glycoprotein secretion was unchanged. These results support the conclusion that N-linked glycan processing is not necessary for sorting, but glycosylation is required for accumulation of secreted proteins in the extracellular compartment.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Thioredoxin and NADP-thioredoxin reductase from cultured carrot cells

Dark-grown carrot (Daucus carota L.) tissue cultures were found to contain both protein components of the NADP/thioredoxin system—NADP—thioredoxin reductase and the thioredoxin characteristic of heterotrophic systems, thioredoxin h. Thioredoxin h was purified to apparent homogeneity and, like typical bacterial counterparts, was a 12-kdalton (kDa) acidic protein capable of activating chloroplast NADP-malate dehydrogenase (EC 1.1.1.82) more effectively than fructose-1,6-bisphosphatase (EC 3.1.3.11). NADP-thioredoxin reductase (EC 1.6.4.5) was partially purified and found to be an arsenite-sensitive enzyme composed of two 34-kDa subunits. Carrot NADP-thioredoxin reductase resembled more closely its counterpart from bacteria rather than animal cells in acceptor (thioredoxin) specificity. Upon greening of the cells, the content of NADP-thioredoxin-reductase activity, and, to a lesser extent, thioredoxin h decreased. The results confirm the presence of a heterotrophic-type thioredoxin system in plant cells and raise the question of its physiological function.Abbreviations DTNB dithiolbis(2-nitrobenzoic acid) - FBPase fructose-1,6-bisphosphatase - FTR terredoxin-thioredoxin, reductase - NADP-MDH NADP-malate dehydrogenase - NTR NADP-thioredoxin reductase - SDS sodium-dodecyl sulfate     

Purification of an autocatalytic protein-glycosylating enzyme from cell suspensions of Daucus carota L.

A glycosyltransferase was identified in the 174 000 · g membrane pellet and the supernatant from extracts of cell suspensions of Daucus carota L. The enzyme from the supernatant was enriched 475-fold, and sodium dodecyl sulfate-gel electrophoresis and fluorography of this purified sample showed that the only enriched protein band (40 000 Da) was simultaneously an enzyme and a glucose-acceptor. Gel filtration and electrophoresis under non-denaturing conditions proved that in vivo this protein provides the subunits for a very large molecule. Radio-gas-liquid chromatography demonstrated that only one glucosyl moiety was transferred from UDP-glucose to the protein.Abbreviations DEAE diethylaminoethyl - GT IsU glycosyltransferase I, soluble, substrate UDPglucose - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Trypanosoma cruzi glycoprotein 72: Immunological analysis and cellular localization

Two monoclonal antibodies were used to biochemically characterize glycoprotein 72 (GP72) from Trypanosoma cruzi and to localize the protein in live and fixed parasites by indirect immunofluorescence and in thin section of parasites by immunogold electron microscopy. GP72 was shown in immunoblots to be specific for the epimastigote stage; the protein could not be detected in trypomastigotes. Each antibody reacted with a different epitope on the glycoprotein and deglycosylation of GP72 ablated reactivity with one of the antibodies. Indirect immunofluorescence and electron microscopic evaluation of parasite associated gold particles showed the presence of GP72 in the cell surface membrane including the flagellar pocket and the cytostome. In addition, cytoplasmic membrane vesicles of the endosomal-lysosomal system stained intensely.