Identification of Calcium Binding Sites in the Trypanosome Flagellar

Molecular and Biochemical Parasitology 101 (1999) 61–70

Identiﬁcation of calcium binding sites in the trypanosome ﬂagellar calcium-acyl switch protein

Rosa A. Maldonado a, Salida Mirzoeva b, Lisa M. Godsel c, Thomas J. Lukas b, Samuel Goldenberg a, D. Martin Watterson b, David M. Engman c,*

a Department of Biochemistry and Molecular Biology, Fundac¸a˜o Oswaldo Cruz, Rio de Janeiro, Brazil b Drug Disco6ery Program and Department of Molecular Pharmacology and Biological Chemistry, Northwestern Uni6ersity Medical School, 303 E. Chicago A6e., Chicago IL, 60611 USA c Departments of Pathology and Microbiology-Immunology, Northwestern Uni6ersity Medical School, 303 E. Chicago A6e., Chicago IL, 60611 USA

Received 3 December 1998; accepted 24 February 1999

Abstract

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPD12) lacking the amino terminal pair of sites. FCaBPD12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPD34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction. © 1999 Elsevier Science B.V. All rights reserved.

Keywords: Trypanosoma; Calcium; Flagellum; Myristoylation; Recoverin

Abbre6iations: CaBP, calcium-binding protein; FCaBP, 24-kDa ﬂagellar calcium-binding protein. * Corresponding author. Tel.: +1-312-503-1288; fax: +1-312-503-1265. E-mail address: [email protected] (D.M. Engman)

1. Introduction sensor in the retinal rod cells of vertebrates that functions in cellular recovery from photoexcita- The central role of calcium as a regulator of tion [22]. Recoverin has four calcium binding cellular activity is well established [1,2]. Cell di- sequence motifs, but binds only two calcium vision, motility, gene expression and secretion ions [23]. In the 3-D structure of recoverin de- are regulated by calcium ions and the intracellu- termined by X-ray crystallography [24], a single lar calcium concentration must be precisely con- calcium is bound in the third calcium binding trolled during these processes. In the protozoan motif from the amino terminus. A more thor- Trypanosoma cruzi, parasite proliferation and ough NMR analysis showed that both motifs differentiation [3] and host cell invasion [4,5] are two and three bind calcium [25]. Recoverin as- regulated by calcium, and calcium-modulated sociates in a calcium-dependent manner via its cell signaling pathways similar to those of mam- amino terminal myristoyl group with the plasma malian cells are present [6]. Furthermore, T. membrane, where it binds to and inhibits the cruzi possesses a highly specialized calcium-con- activity of rhodopsin kinase [26]. When the in- taining organelle, the acidocalcisome [7], which tracellular calcium levels drop upon retinal cell is believed to permit the amastigote stage of the photoexcitation, the calcium dissociation from parasite to survive in the low calcium environ- recoverin results in a conformational change and ment of the mammalian cell cytoplasm in which sequestration of the myristoyl group in a hydro- it replicates [8]. Calcium is clearly an important phobic cleft [27]. Unable to associate with the modulator of parasite metabolism. membrane, recoverin moves into the cytoplasm, leaving rhodopsin kinase to phosphorylate and Intracellular calcium signals are transduced inactivate rhodopsin, the first step in the cellular into biological responses through their interac- recovery phase. When the intracellular calcium tion with calcium binding proteins (CaBPs) or increases, recoverin assumes its calcium-bound calcium binding domains in multidomain form and returns to the membrane, where it proteins. T. cruzi has a variety of proteins in again inactivates rhodopsin kinase, completing this class of signal transducing molecules. For the cycle. Both recoverin and FCaBP: (i) possess example, calmodulin [9] and its associated four calcium binding motifs but only bind two calmodulin-binding proteins [10] and calmodulin molecules of calcium; (ii) are myristoylated at regulated enzymes [11,12] have been reported. A the amino terminus; and (iii) display calcium- flagellar calcium binding protein (FCaBP) with a modulated membrane association (calcium-acyl computed mass of 24 kDa, is found in the flag- switch). The present study was undertaken to ellum of T. cruzi [13] and other trypanosomatids determine which of the four putative sites in [14–16], and is highly immunogenic [17–19]. FCaBP bind calcium. This is a critical first step FCaBP specifically localizes to the flagellar in our understanding of the protein’s calcium- plasma membrane via amino terminal myristoyl acyl switch mechanism and how it compares and palmitoyl modifications [20]. These acyl with that of recoverin. modifications are necessary, but not sufficient, for localization. Calcium binding is also required. For example, when calcium is chelated with EGTA in detergent-permeabilized try- 2. Materials and methods panosomes, FCaBP loses its flagellar membrane association. Thus, FCaBP is a calcium-acyl 2.1. Parasites switch protein: a lipoprotein whose membrane association is modulated by its calcium binding Epimastigotes of the Silvio X-10/4 clone of T. state [21]. cruzi were used for all studies and maintained at The best studied of the calcium-acyl switch 26°C in supplemented liver digest neutralized proteins is recoverin, a myristoylated calcium tryptose medium (LDNT+) as described [28]. R.A. Maldonado et al. / Molecular and Biochemical Parasitology 101 (1999) 61–70 63

2.2. Engineering of recombinant FCaBP and 2.3. Expression and puriﬁcation of recombinant FCaBP deletion mutants proteins

The FCaBP coding region was produced by pET23b constructs were introduced into the DNA amplification from a previously described Escherichia coli strain BL21 (DE3) (Novagen) for cDNA clone [29] using a sense primer contain- expression. Bacterial cultures were grown in LB m ing an NdeI restriction site, 5%-CATATGGGT- medium containing 100 g/ml ampicillin at 37°C in GCTTGTGGGTCG-3%, and an antisense primer a shaking incubator overnight. Cultures were containing an XhoI restriction site, 5%-GTC- diluted 1:5 in LB+ampicillin and grown at 30°C a b GAGCGCGCTCTCCGGCACG-3%. The PCR to an OD600 of 0.6, at which time -1,4- -D-iso- propylthiogalactopyrranoside was added to a final product was cloned into the pT7 Blue T vector concentration of 0.6 mM. After an additional 3 h (Novagen, Madison, WI) and directionally sub- of growth at 30°C, the cultures were placed on ice cloned into NdeI+XhoI-digested pET23b (No- for 5 min and bacterial cells were harvested by vagen). The termination codon was eliminated centrifugation at 5000×g for 5 min at 4°C. The during amplification, allowing expression of an cells were suspended in 0.2 pellet vol. of binding FCaBP fusion protein containing a carboxyl ter- buffer (5 mM imidazole, 500 mM NaCl, 20 mM minal hexahistidine tag. Two FCaBP deletion Tris–HCl, pH 7.9, 0.1 mM phenylmethyl- mutants were engineered in a similar manner. sulfonylfluoride) and sonicated in 5 s pulses, on ice, D The 12 mutant was constructed from two PCR for 10 min using an ultrasonic processor. Another products, one containing the 5% end of the cod- 0.2 vol. of binding buffer was added to the sonicate ing sequence up to the beginning of EF-1 (sense and the solution was clarified by centrifugation at primer containing an NdeI site as described 18 000×g for 20 min at 4°C and passed through above and antisense primer containing an a 0.22 mm Millipore filter. Recombinant proteins EcoRI site, 5%-GAATTCCTGTTTTGCCTCG- were isolated by affinity chromatography on GCGGT-3%) and another extending from the be- His-Bind™ resin (Novagen) according to the ginning of EF-3 through the end of the coding manufacturer’s instructions, and dialyzed for :20 region (sense primer containing an EcoRI site, h against 2×1 l changes of 20 mM Tris–HCl, pH 5%-GAATTCGAGCTGACGGTGATGTTCG-3%), 7.5, 1 mM DTT. Proteins were analysed by and XhoI-containing antisense primer described denaturing SDS-PAGE [31] and immunoblotting above. The D34 mutant was constructed from [32] using standard methods. two PCR products, one containing the 5% end of the coding sequence up to the end of EF-2 2.4. Calcium binding assays (NdeI-containing sense primer described above and antisense primer containing an SalI site, 5%- Protein samples were prepared and analysed by 45Ca2+ flow dialysis essentially as described [33]. GTCGACGATGTAGCACAGCATCAGACG- Briefly, the amino terminal sequences of purified, 3%) and another beginning directly downstream decalcified recombinant proteins were determined of EF-3 and extending through the end of the to assure polypeptide chain integrity and coding region (sense primer containing a SalI concentrations were assessed by amino acid site, 5%-GTCGACAAACTGGACGCCGACGG- composition analysis. This is the most accurate % C-3 , and XhoI-containing antisense primer de- method of protein determination available and is scribed above). All PCR products were cloned the key to accurate determination of stoichiometry into the pT7 Blue T vector and directionally in such studies. The sample in the upper chamber subcloned into NdeI+XhoI-digested pET23b. of the apparatus had a total volume of 400 ml, and DNA constructs were sequenced on both contained 20–50 mM protein in Buffer A (50 mM strands using the dideoxy chain termination HEPES, pH 7.5, 5 mM MgCl2, 150 mM KCl, 15 method [30]. mM NaCl). At the start of each experiment, 2–3 64 R.A. Maldonado et al. / Molecular and Biochemical Parasitology 101 (1999) 61–70 mCi of radioactive calcium was added (ICN Phar- 2.6. Immunofluorescence microscopy maceuticals, Costa Mesa, CA; specific activity 0.62

Ci/mmol). Unlabeled CaCl2 was added to the flow T. cruzi transfectants expressing the various dialysis chamber every 1.5 min (made up from FCaBP proteins were isolated by centrifugation, calibrated 100 mM CaCl2 solution, Radiometer, washed in PBS and resuspended in PBS at a Copenhagen, Denmark). The lower chamber was density of 1×106 cells/ml. Twenty microliters of perfused at a flow rate of 3 ml/min and 1 ml this suspension was added to each well of a printed fractions were collected. Radioactivity was deter- slide (Cel-Tek, Glenview, IL), air dried, fixed at mined, after addition of 10 ml of Ecoscint A −20°C in anhydrous methanol for 30 min, and (National Diagnostics, Atlanta, GA), using a incubated in blocking buffer (1% bovine serum Beckman 6500 liquid scintillation counter. No albumin, 2% normal goat serum in PBS) for 30 quenching corrections were applied. For each min. Slides were incubated with c-myc specific, given total calcium concentration, free and bound 9E10 monoclonal antibody hybridoma superna- calcium was estimated, and mol calcium bound per tant (ATCC c CRL1729, myc 1-9E10.2) mol protein was plotted versus free calcium con- overnight at 4°C. Cells were washed with PBS and centration. incubated for 3–4 h with a 1:200 dilution of fluorescein isothiocyanate (FITC)-conjugated 2.5. Generation of T. cruzi transfectants goat-antimouse Ig (G+M) (Caltag Laboratories, expressing myc-tagged FCaBP proteins San Francisco, CA). Slides were then mounted in medium containing 10% polyvinyl alcohol (Air The T. cruzi episomal expression vector pTEX- Products and Chemicals, Allentown, PA), 25% 9E10 [34] was used for all studies. DNA fragments glycerol, 0.1 M Tris–HCl (pH 8.5), and 2.5% encoding FCaBP and FCaBP deletion mutants 1,4-diazobicyclo[2.2.2]octane (Sigma, St Louis, were directionally cloned into pTEX-9E10 for MO) and viewed by fluorescence microscopy. expression in transfected parasites as fusion proteins containing carboxyl terminal c-myc epitope tags. T. cruzi epimastigotes were washed once 3. Results with 132 mM NaCl, 8 mM KCl, 8 mM Na2HPO, 6 1.5 mM KH2PO4, 0.5 mM Mg(C2H3O2)2·4H2O, 3.1. Determination of the calcium binding acti ity 0.1 mM CaCl2, pH 7.0, and resuspended in this of wild type and mutant FCaBPs buffer at a density of 1×108/ml. A 400 ml volume of the cell suspension was placed into a 0.2 cm A previous calcium binding study using a rela- electrode gap cuvette (BioRad, Hercules, CA) with tively crude 45Ca2+overlay method indicated that 15–25 mg of supercoiled plasmid DNA and pulsed FCaBP binds 2 mol of calcium per mol protein 4 times at 0.3 kV and 500 mF using a BioRad Gene [13]. To predict which of the four putative binding Pulser. Electroporated cells were placed in 5 ml sites binds calcium with comparative selectivity LDNT+and G418 (0.5 mg/ml) was added 48 h and high affinity, we analyzed the sequence of later. Drug-resistant lines developed subsequently FCaBP using the recently updated EF-hand se- and were available for study after approximately 6 quence motif in the PROSITE database [35] (Fig. weeks. Proteins were fractionated by electrophore- 1). EF-1 and EF-4 conform to the binding consen- sis on reducing 12% SDS polyacrylamide gels [31] sus. EF-2 is missing the expected calcium ligating and analysed by Coomassie blue staining and site chain at position 3 and has an asparagine western blotting [32] using FCaBP-specific anti- instead of the highly conserved aspartic acid at serum [17]. Blots were developed using alkaline position 1, and EF-3 possesses the consensus cal- phosphatase-conjugated goat antimouse Ig (G+ cium ligating side chains, but contains a methion- M) (Caltag Laboratories, San Francisco, CA) and ine residue at position 6 [36,37]. To test the 5-bromo-4-chloro-3-indolyl phosphate (United hypothesis that FCaBP has only one calcium bind- States Biochemical, Cleveland, OH). ing site in each half of the molecule, we R.A. Maldonado et al. / Molecular and Biochemical Parasitology 101 (1999) 61–70 65 produced recombinant FCaBP and two FCaBP with a slightly larger mass on an SDS gel, as deletion mutants and analysed them using a direct observed previously for native FCaBP [17]. calcium binding method. Pairs of sites were FCaBP has two sites which bind calcium with deleted because the prevailing helix-loop-helix high affinity and selectivity in the presence of motifs generally demonstrate high affinity and physiological concentrations of magnesium (Fig. selective calcium binding activity only when they 3A). Surprisingly, the binding curves for the wild occur in pairs [38,39]. The unique amino and type and D12 mutant FCaBP proteins are very carboxyl terminal regions were preserved in both similar, indicating that FCaBP binds calcium deletion mutants (Fig. 2A) and carboxyl terminal through EF-3 and EF-4. Both proteins bind two hexahistidine tags were added to facilitate purifi- mols of calcium per mol of protein, while no cation. Production of the full-length protein (WT) binding activity was detected for the D34 mutant and each of the deletion mutants (D34 and D12) in under these conditions. Therefore, the experimen- E. coli and purification by nickel affinity chro- tal results do not bear out the predictions of the matography yielded protein of 85–95% purity most current EF-hand motif and emphasize the that reacted with FCaBP-specific antiserum (Fig. need to test experimentally the predictions based 2B). The molecular masses of the proteins were solely on motif analyses of open reading frames. predicted to be 25, 18 and 16 kDa, for the WT, In addition, since recoverin binds calcium through D34 and D12 proteins, respectively; each migrates sites EF-2 and EF-3, the results suggest that

Fig. 1. Putative calcium-binding sites in FCaBP and recoverin. The amino acid sequence of FCaBP is shown with its four EF-hand sequence motifs (shaded) aligned with the four EF-hand motifs of recoverin below. Consensus amino acids for selected positions in the EF-hand motif are: D (position 1); D, N, or S (position 3); D, E, N, S, T, or G (position 5); any amino acid except G or P (position 7); D, E, N, Q, S, T, A, G, or C (position 9); E or D (position 12); I, L, V, M, F, Y, or W (position 13). Calcium ligating residues in the calcium binding loop are indicated. Amino acid substitutions (black highlights) at any of these critical positions may eliminate calcium binding. The D12 protein contains FCaBP amino acids M1-F57, EF (derived from an EcoRI cloning site), E141-A211, and hexahistidine tag. The D34 sequence contains FCaBP amino acids M1-F140, VE (derived from a SalI cloning site), K197-A211, and hexahistidine tag. 66 R.A. Maldonado et al. / Molecular and Biochemical Parasitology 101 (1999) 61–70

possible that the structural alteration in FCaBP resulting from deletion of the amino terminal sequence motifs might alter myristoyl/palmitoyl sequestration either positively (i.e. prevent membrane localization) or negatively (i.e. lead to constitutive membrane localization). Staining with

Fig. 2. (A) Schematic of recombinant histidine-tagged FCaBP (WT), D12 and D34 mutants, which lack the first pair and second pair of EF-hand motifs, respectively. (B) Expression and purification of recombinant proteins. Recombinant proteins were produced in E. coli and purified by affinity chromatography on nickel sepharose. Proteins were analysed by SDS-PAGE followed by Coomassie blue staining and immunoblotting with FCaBP-specific antiserum. Fig. 3. (A) Calcium binding properties of wild type FCaBP () FCaBP and recoverin differ fundamentally in and the D12 deletion mutant (). Calcium binding experi- their calcium-acyl switch mechanisms. ments were performed on decalcified proteins (20–50 mM) using 45Ca2+flow method as described [33], except that the dialysis buffer was 50 mM HEPES, pH 7.5, 5 mM MgCl , 150 Z 2 3.2. Localization of the 12 mutant FCaBP mM KCl, 15 mM NaCl. Prior to the experiments, protein protein samples were decalcified by three cycles of trichloroacetic acid precipitation or passage through a Chelex column. The protein To determine whether the calcium binding D12 concentration in decalcified samples was determined by amino mutant protein displays the same flagellum-spe- acid analysis after hydrolysis. Analysis of binding data reveals that WT and D12 proteins bind two calcium ions (mol/mol) cific localization in the parasite as does the native while D34 protein does not bind calcium under these condi- protein, T. cruzi epimastigotes were transfected tions (not shown). (B) Localization of WT-Myc and D12-Myc with constructs encoding myc-tagged full-length proteins in T. cruzi transfectants. T. cruzi epimastigotes were (WT-Myc) and mutant (D12-Myc) proteins and transfected with episomal constructs expressing FCaBP or D12 analysed by immunofluorescence microscopy (Fig. mutant proteins containing carboxyl terminal myc epitope tags. A vector-only control was included as well. Transfectants 3B). Although we have previously shown that the were analyzed by immunofluorescence microscopy using the amino terminal 24 amino acids are necessary and myc-specific 9E10 monoclonal antibody. Both proteins localize sufficient for flagellar localization [20], it remained to the flagellum, as does endogenous FCaBP [13,20]. R.A. Maldonado et al. / Molecular and Biochemical Parasitology 101 (1999) 61–70 67 the tag-specific monoclonal antibody showed that ner for FCaBP that is unique to the parasite can both proteins localize to the flagellum, indicating be found, then it or FCaBP might be novel drug that deletion of the amino terminal set of se- discovery targets. quence motifs does not result in constitutive acyl Considered together with our recent study [20], sequestration. Upon calcium chelation, the wild the results presented in this paper suggest a model type protein loses flagellar membrane localization for calcium-regulated flagellar membrane localiza- [20], the defining feature of a calcium switch. A tion of FCaBP (Fig. 4). FCaBP possesses at its similar analysis of the D12 mutant gave ambigu- amino terminus a consensus sequence for coupled ous results, with some cells showing flagellar myristoyl/palmitoyl modification, the former oc- staining upon calcium chelation and others show- curring cotranslationally and the latter at a mem- ing a lack of staining (not shown). More conclu- brane site where the palmitoyltransferase enzyme sive determination of the role for each sequence is believed to reside. Calcium binding to EF-3 and motif awaits analysis of FCaBP point mutants, EF-4 triggers a switch to an open conformation in especially those in motifs three and four. which the myristate is extruded from a hydrophobic cleft, thereby permitting transient membrane association. Palmitoylation then provides the ad- 4. Discussion ditional hydrophobic group that stabilizes membrane localization and, possibly, calcium binding Despite the fact that the first sequence motif of [41]. Thus, FCaBP can be regulated by two dis- FCaBP conforms to the binding consensus, cal- tinct, but complementary mechanisms—calcium- cium binding could not be detected to the D34 acyl switching and dynamic palmitoylation. In mutant that contains this site. Direct 45Ca2+ this regard it is unique among all acylated CaBPs binding data of full-length and D12 mutant described to date. It is possible that both fatty proteins demonstrated that only FCaBP proteins acids are sequestered in the calcium-free state, containing motifs three and four have calcium although this may be difficult to demonstrate binding activity. Based on precedents with since the lability of the thioester linkage of palmi- calmodulin, it is possible that the calcium binding tate hinders in vitro study. Regardless, if FCaBP activity of motif one in FCaBP is suppressed by functions in a manner similar to that of recoverin, defects in sequence motif two. Specifically, it was we would predict that it binds to a flagellar mem- shown that a point mutation in one calcium bind- brane protein in the ‘resting’, calcium-bound state ing site of calmodulin can suppress the calcium and dissociates from that protein under condi- binding activity of the neighboring site [37,40]. tions of low intracellular calcium. Where and how Regardless, it is clear from the data presented this may occur during the T. cruzi life cycle or cell here that the calcium sites used by FCaBP and cycle are not known. Although the host cell cyto- recoverin are distinct and only a subset of the plasm provides a calcium-poor environment for those identified by amino acid sequence motif the T. cruzi amastigote, the parasite’s acidocal- analysis. Even if both proteins evolved from a cisome maintains calcium homeostasis [7]. Indeed, common ancestor, the sites used for the calcium- FCaBP is expressed in the amastigote, where it is acyl switching differ. Like recoverin, FCaBP is localized to the small flagellar remnant [20]. It is likely to transduce calcium signals through differ- also possible that FCaBP regulation does not ential engagement of partner proteins. In the case occur in a life cycle or cell cycle stage-specific of recoverin, rhodopsin kinase has been identified manner, but that it occurs on a much smaller time as one potential target protein which is negatively frame, such as the flagellar beat cycle. regulated by interaction with the acyl switch One of the most interesting aspects about protein [26]. Such a partner has yet to be found FCaBP is its flagellar membrane localization and for FCaBP, but likely candidates are protein ki- apparent exclusion from the pellicular (cell body) nases or other proteins that need to be regulated membrane. This may occur because the chemical in a calcium regulated fashion. If a binding part- composition of the flagellar membrane is uniquely 68 R.A. Maldonado et al. / Molecular and Biochemical Parasitology 101 (1999) 61–70

Fig. 4. FCaBP: model for the calcium myristoyl/palmitoyl switch mechanism of flagellar membrane localization. The FCaBP coding region contains an amino terminal acylation domain (white; G2, myristoylation site, C4, palmitoylation site) and four EF-hand calcium binding sites (black), of which only EF-3 and EF-4 bind calcium. FCaBP is cotranslationally myristoylated, but adopts a conformation in which the myristoyl group is sequestered within a hydrophobic cleft in the protein. The calcium myristoyl switch mechanism (Ca2+, white circles) leads to a conformational change in which the myristoyl moiety is extruded. This may permit transient membrane association, which is stabilized via subsequent palmitoyl addition by a putative membrane-associated palmitoyl transferase. The diacylated protein is stably associated with the flagellar plasma membrane as long as the protein is: (i) palmitoylated; and (ii) bound by calcium. In this model, three steps are reversible—the calcium switch, palmitoylation and flagellar membrane association. It is possible that the diacylated protein is the substrate for the switch; i.e. that the calcium switch and palmitoylation are reversed in this scheme. suitable for insertion of FCaBP’s acyl groups; i.e. Landfear, personal communication). Whether the the flagellar membrane constitutes a unique do- latter possibility is true remains to be determined, main or functional ‘raft’, as has been described in but the cumulative work on recoverin and other CaBPs strongly suggests that FCaBP functions as mammalian cells [42]. Alternatively, the palmi- a calcium sensor, regulating the function of its toyltransferase may be associated only with the target at its flagellar membrane or nonmembrane flagellar membrane. Finally, localization may be location. mediated by association with a flagellum-specific binding partner, which may be mediated by a hexapeptide identified common to a variety of Acknowledgements flagellar membrane proteins in trypanosomatids, including FCaBP (R/KS/TA/GS/TS/TS/T, We thank Cheryl Olson for technical assistance FCaBP amino acids 6–11; E.L. Snapp and S.M. and Kojo Mensa-Wilmot and James Ames for R.A. Maldonado et al. / Molecular and Biochemical Parasitology 101 (1999) 61–70 69 helpful suggestions. This work was supported by DM, Goldenberg S. Homologues of the 24-kDa flagellar 2+ research grants AI38022 and GM30861 from the Ca -binding protein gene of Trypanosoma cruzi are National Institute of Health. R.A.M. was sup- present in other members of the Trypanosomatidae fam- ily. Exp Parasitol 1997;86:200–5. ported by a research fellowship from CONICIT [15] Lee MG, Chen JF, Ho AW, D’Alesandro PA, Van der (Venezuela), D.M.E. is an Established Investiga- Ploeg LH. 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