Complexity of Trypanosomatid Endocytosis Pathways Revealed by Rab4 and Rab5 Isoforms in Trypanosoma brucei
1998; Elsevier BV; Volume: 273; Issue: 48 Linguagem: Inglês
10.1074/jbc.273.48.32102
ISSN1083-351X
AutoresHelen I. Field, Mariam Farjah, Arun Pal, Keith Gull, Mark C. Field,
Tópico(s)Calcium signaling and nucleotide metabolism
ResumoSmall G proteins of the Rab family are responsible for vesicle fusion and control flux during intracellular transport. Rab5 is important in endosome maturation and Rab4 in recycling of endocytic material. Three Rab5 isoforms identified so far in mammals and three in the yeast genome suggest that conservation of multiple Rab5 isoforms is required for sophisticated regulation of endocytosis. Trypanosoma brucei homologues of Rab5 and Rab4 (TbRab5A and TbRab4) have been identified. Here we report cloning of a second Rab5 homologue, TbRab5Bp. The TbRAB5Aand -5B genes are not linked in the genome, and phylogenetic reconstruction indicates that multiple Rab5 isoforms in yeast, mammals, and trypanosomes evolved independently. Northern blots demonstrate that TbRab5A, -5B, and TbRab4 messages are expressed in bloodstream form (BSF) and procyclic forms of the parasite even though endocytosis is not very active in the latter form. mRNA levels of TbRab5A and -4 are constitutive. Multiple-sized TbRab5B messages at very low abundance are detected, with greater expression in BSF. Also, the TbRab5B mRNA has a large 3′-untranslated region suggestive of potentially complex regulation, and therefore TbRab5Bp may be an important regulator of differential endocytosis levels between BSF and procyclic stage parasites. Affinity purified antibodies raised to C-terminal peptide sequences of all three TbRab proteins recognized small vesicular cytoplasmic structures, which for TbRab5Ap and -5Bp are predominantly near the flagellar pocket. TbRab5Bp colocalizes with invariant surface glycoprotein 100 (ISG100), a protein entering the endocytotic pathway in BSF parasites, whereas in procyclic cells populations of vesicles stained with both TbRab5Ap and -5Bp substantially overlap; TbRab5 proteins are therefore components of the endocytotic pathway. TbRab4p localizes to vesicular structures throughout the cytoplasm, with some overlap with TbRab5Bp, but the majority occupying a different compartment to the TbRab5s. Therefore the trypanosome endosomal system has been functionally dissected for the first time; these reagents provide a unique opportunity for manipulation of the protozoan endosomal system to further our understanding of drug uptake mechanisms and virulence. Small G proteins of the Rab family are responsible for vesicle fusion and control flux during intracellular transport. Rab5 is important in endosome maturation and Rab4 in recycling of endocytic material. Three Rab5 isoforms identified so far in mammals and three in the yeast genome suggest that conservation of multiple Rab5 isoforms is required for sophisticated regulation of endocytosis. Trypanosoma brucei homologues of Rab5 and Rab4 (TbRab5A and TbRab4) have been identified. Here we report cloning of a second Rab5 homologue, TbRab5Bp. The TbRAB5Aand -5B genes are not linked in the genome, and phylogenetic reconstruction indicates that multiple Rab5 isoforms in yeast, mammals, and trypanosomes evolved independently. Northern blots demonstrate that TbRab5A, -5B, and TbRab4 messages are expressed in bloodstream form (BSF) and procyclic forms of the parasite even though endocytosis is not very active in the latter form. mRNA levels of TbRab5A and -4 are constitutive. Multiple-sized TbRab5B messages at very low abundance are detected, with greater expression in BSF. Also, the TbRab5B mRNA has a large 3′-untranslated region suggestive of potentially complex regulation, and therefore TbRab5Bp may be an important regulator of differential endocytosis levels between BSF and procyclic stage parasites. Affinity purified antibodies raised to C-terminal peptide sequences of all three TbRab proteins recognized small vesicular cytoplasmic structures, which for TbRab5Ap and -5Bp are predominantly near the flagellar pocket. TbRab5Bp colocalizes with invariant surface glycoprotein 100 (ISG100), a protein entering the endocytotic pathway in BSF parasites, whereas in procyclic cells populations of vesicles stained with both TbRab5Ap and -5Bp substantially overlap; TbRab5 proteins are therefore components of the endocytotic pathway. TbRab4p localizes to vesicular structures throughout the cytoplasm, with some overlap with TbRab5Bp, but the majority occupying a different compartment to the TbRab5s. Therefore the trypanosome endosomal system has been functionally dissected for the first time; these reagents provide a unique opportunity for manipulation of the protozoan endosomal system to further our understanding of drug uptake mechanisms and virulence. bloodstream form expressed sequence tag flagellar pocket glutathione S-transferase immunofluorescence analysis invariant surface glycoprotein kilobase(s) nucleotides open reading frame polyacrylamide gel electrophoresis trypanosome lytic factor polymerase chain reaction 4-morpholinepropanesulfonic acid. Rabs are a family of small GTPases essential for membrane vesicle trafficking in eukaryotes (1Novick P. Brennwald P. Cell. 1993; 75: 597-601Abstract Full Text PDF PubMed Scopus (316) Google Scholar). Each Rab localizes to a distinct subset of organelles; the C terminus is responsible for intracellular localization (2Chavrier P. Gorvel J.P. Stelzer E. Simons K. Gruenberg J. Zerial M. Nature. 1991; 353: 769-772Crossref PubMed Scopus (318) Google Scholar), while the N terminus also recognizes the target organelle and is required for vesicle fusion (3Steele-Mortimer O. Clague M.J. Huber L.A. Chavrier P. Gruenberg J. Gorvel J.-P. EMBO J. 1994; 13: 34-41Crossref PubMed Scopus (33) Google Scholar). The Rab system controls organelle-specific flux by regulating the ability of the t- and v-SNARE partners to interact, promoting docking and fusion (4Pfeffer S.R. Annu. Rev. Cell Dev. Biol. 1996; 12: 441-461Crossref PubMed Scopus (180) Google Scholar, 5Lupashin V.V. Waters M.G. Science. 1997; 276: 1255-1258Crossref PubMed Scopus (183) Google Scholar). The amount of time that Rab5 remains GTP-bound governs the lifetime of the fusion-competent state of the docking vesicle (6Rybin V. Ullrich O. Rubino M. Alexandrov K. Simon I. Seabra M.C. Goody R. Zerial M. Nature. 1996; 383: 266-269Crossref PubMed Scopus (267) Google Scholar). Humans and yeast each have three identified Rab5 isoforms in the endosome system which colocalize, suggesting complex regulation or redundancy (7Bucci C. Lutcke A. Steele-Mortimer O. Olkkonen V.M. Dupree P. Chiariello M. Bruni C.B. Simons K. Zerial M. FEBS Lett. 1995; 366: 65-71Crossref PubMed Scopus (137) Google Scholar, 8Singer-Kruger B. Stenmark H. Dusterhoft A. Philippsen P. Yoo J.S. Gallwitz D. Zerial M. J. Cell Biol. 1994; 125: 283-298Crossref PubMed Scopus (184) Google Scholar, 9Singer-Kruger B. Stenmark H. Zerial M. J. Cell Sci. 1995; 108: 3509-3521Crossref PubMed Google Scholar, 10Lazar T. Götte M. Gallwitz D. Trends Biochem. Sci. 1997; 22: 468-472Abstract Full Text PDF PubMed Scopus (187) Google Scholar). Dominant negative Rab5 decreases endosome size, whereas constitutively active Rab5 leads to endosomal swelling and fusion (11Stenmark H. Parton R.G. Steele-Mortimer O. Lutcke A. Gruenberg J. Zerial M. EMBO J. 1994; 13: 1287-1296Crossref PubMed Scopus (770) Google Scholar). Phospholipase A2 and phosphatidylinositol 3-kinase are implicated in Rab5 function (12Barbieri M.A. Li G. Mayorga L.S. Stahl P.D. Arch. Biochem. Biophys. 1996; 326: 64-72Crossref PubMed Scopus (52) Google Scholar, 13Li G. D'Souza-Schorey C. Barbieri M.A. Roberts R.L. Klippel A. Williams L.T. Stahl P.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10207-10211Crossref PubMed Scopus (246) Google Scholar), suggesting close integration with signal transduction pathways. Rab4 is involved in recycling of membrane material back to the plasma membrane from endosomal compartments (14Van Der Sluijs P. Hull M. Zahraoui A. Tavitian A. Goud B. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6313-6317Crossref PubMed Scopus (221) Google Scholar). Human Rab4a is subject to cell-cycle-dependent phosphorylation and is involved in insulin-mediated translocation of glucose transporters (15Bailly E. McCaffrey M. Touchot N. Zahraoui A. Goud B. Bornens M. Nature. 1991; 350: 715-718Crossref PubMed Scopus (130) Google Scholar, 16Shibata H. Omata W. Kojima I. J. Biol. Chem. 1997; 272: 14542-14546Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A second isoform, Rab4b, is not phosphorylated (17Bucci C. Zerial M. Zerial M. Huber L.A. Guidebook to the Small GTPases. Oxford University Press, Oxford, UK1995: 324-326Google Scholar). Trypanosoma brucei sp., the causative agent of Ngana in ungulates and sleeping sickness in humans, is a parasitic protozoan pathogen seriously affecting public health and the economy in endemic regions. Suramin, a drug currently used in management of African trypanosomiasis, is believed to enter the parasite via endocytosis (18Wang C.C. Annu. Rev. Pharmacol. Toxicol. 1995; 35: 93-127Crossref PubMed Google Scholar). Resistance to lysis by human serum is mediated by inducible, selective control of endocytosis (19Hager K.M. Hajduk S.L. Nature. 1997; 385: 823-826Crossref PubMed Scopus (47) Google Scholar). We can genetically engineer the Rab system to manipulate endocytosis in T. brucei and study drug uptake. 1H. Field and M. C. Field, unpublished data. 1H. Field and M. C. Field, unpublished data. T. brucei additionally provides a convenient model system for elucidation of cell biological processes in a divergent eukaryote with a strongly directional endocytotic/recycling system, where a very small portion of the total membrane (2–3%, Ref. 20Webster P. Russell D.G. Parasitol. Today. 1993; 9: 201-206Abstract Full Text PDF PubMed Scopus (110) Google Scholar) is the site of all endo- and exocytosis. Recycling is very active in T. brucei, with more than 95% of endocytosed variant surface glycoprotein (VSG) recycled (21Duszenko M. Seyfang A. Tartakoff A.M. Plattner H. Advances in Cell and Molecular Biology of Membranes. JAI Press Inc., CT1993: 227-258Google Scholar). Here we report on the subcellular location and evolution of three TbRab proteins involved in endocytosis in T. brucei. Culture-adapted blood stream form (BSF)2 T. brucei, strain 427, were grown as described (22Field H. Field M.C. J. Biol. Chem. 1997; 272: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Cells were quantitated with a Z2 Coulter Counter (Coulter Electronics, UK). For boiling SDS lysates, freshly harvested trypanosomes were added to SDS-PAGE sample buffer (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) at 95 °C, heated for 10 min, and reduced with dithiothreitol. Treatment with fluorescent endocytotic markers was at 3–4 mg/ml added from 10× stock, in 100 μl of culture medium, with (for Texas red-dextran, Molecular Probes) or without (for Lucifer yellow, Sigma) serum. Nitrocellulose for Western and Southern blotting was from Schleicher and Schuell, and nylon for Northern blots was from Amersham Pharmacia Biotech. Radioisotopes were from Amersham International, ICN, and NEN Life Science Products. Vector pGEX2tk and glutathione-Sepharose 4B were from Amersham Pharmacia Biotech, and the pQE30 vector and nickel-Sepharose were from Qiagen Inc. Plasmids were grown in Escherichia coli XL1-Blue (Stratagene) following transformation by electroporation with a BTX 600 ECM electroporator. PCR products and gel-embedded DNA were purified using PCR cleanup kits (Promega), while plasmid and λ DNA were purified using Qiagen kits following the manufacturer's instructions. Small scale plasmid preparations were performed using the Promega Wizard system. Oligonucleotide primers were obtained from Genosys. PCR was performed in 50-μl reactions with 25 pmol of each primer in ammonium buffer (Bioline), 2.5 mm MgCl2, 1 unit of Taq polymerase (Bioline) in a 480 thermal cycler (Perkin Elmer) typically as follows: 98 °C, 5 min (1 cycle); 95 °C, 5 min (1 cycle); 94 °C, 1 min; 56 °C, 1 min; 72 °C, 3.5 min (25 cycles). Primers for subcloning and assembling ORFs for expression were: 5BH3 (5′-TCCAAGCTTTCAACCACAGCAACCGGA), 5BKON (5′-CGGGGTACCTCTGTGAAGACCGTTGCCG), 5A5′ (5′-CGCGAATTCATATGTCGTGTCAGCGACACC), and 5A3′ (5′-GAGAGCGGTAGCGCTCCTGCCC). 0.5-μg aliquots of total trypanosome DNA (24Medina-Acosta E. Cross G.A.M. Mol. Biochem. Parasitol. 1993; 59: 327-330Crossref PubMed Scopus (234) Google Scholar) were digested with restriction enzyme, and the fragments resolved on 1% Tris-acetate-EDTA agarose gels. Molecular weights were estimated from comigration of a 1-kb DNA ladder (Life Technologies, Inc.). Filters were prepared by high salt transfer Southern blotting (25Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley Interscience, New York1994Google Scholar) and probed at low (2× SSC, 0.1% SDS, room temperature) or high stringency (0.1× SSC, 0.1% SDS, 65 °C). Poly(A) enriched RNA was isolated (Hybaid Plc), and Northern blot filters were prepared using 20 μg of RNA or RNA from 108trypanosomes/lane, separated on a formamide-MOPS agarose gel in the presence of ethidium bromide followed by transfer in 20× SSC. Molecular weights were estimated from comigration of RNA markers (Life Technologies, Inc.) and trypanosome rRNA species at 1600, 1900, and 2300 bases. Probes were prepared as follows; full-length ORFs from 2 μg of TbRAB5A, -5B, or -4, and a fragment of tubulin (NotI/XbaI fragment from pXS2, Ref. 26Bangs J.D. Brouch E.M. Ransom D.M. Roggy J.L. J. Biol. Chem. 1996; 271: 18387-18393Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar) were excised and gel-purified. DNA was labeled by random hexamer priming (Boehringer-Mannheim) using 50 μCi [α-32P]dCTP and purified by size exclusion (Centrisep). Autoradiography was with BioMax film (Kodak) at −85 °C with an intensifying screen or by PhosphorImager (Molecular Dynamics). Images were quantitated with NIH Image 1.59 (National Institutes of Health). Sanger sequencing of the TbRAB5BORF and noncoding region was performed using a deaza-GTP kit (Amersham Pharmacia Biotech) and primer walking in both directions. All other sequence analysis was by dye terminator cycle sequencing on a 377 sequencer (Perkin Elmer) from polyethylene glycol-precipitated DNA (6.5% PEG8000, 0.4 m NaCl on ice 20 min, centrifuged at 13,000 × g, 4 °C, 30 min, washed with 70% EtOH). cDNA encoding TbRab5Bp was isolated from a T. brucei BSF cDNA library in λZAP (gift of Dr. John Mansfield, University of Wisconsin) using Rtb4 as a probe (27Field M.C. Boothroyd J.C. Exp. Parasitol. 1995; 81: 313-320Crossref PubMed Scopus (24) Google Scholar). Screening and recovery of the phagemid was as described (22Field H. Field M.C. J. Biol. Chem. 1997; 272: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Field M.C. Field H. Boothroyd J.C. Mol. Biochem. Parasitol. 1995; 69: 131-134Crossref PubMed Scopus (20) Google Scholar). TheTbRAB5B ORF was PCR amplified from the cDNA, engineering a 5′ HindIII site and a 3′ KpnI site (primers 5BH3, 5BKON). The PCR product was ligated intoKpnI/HindIII-restricted pQE30 (Qiagen Inc.). The construct was verified by sequencing and expression in E. coli, producing a 23-kDa protein, the predicted molecular weight for a (His)6·TbRab5Bp fusion. TheTbRAB5A ORF (a gift from Dr. Nabib El-Sayed) was constructed from clone 22, encoding the N terminus of TbRab5Ap, 3N. El-Sayed, unpublished data. which was PCR amplified (primers 5A5′, 5A3′) to produce overlappingEcoRI/NdeI sites at the 5′ end and finishing at an existing Eco47III site; the PCR product was subcloned and then ligated into EcoRI- and Eco47III-cleaved clone T378, which encodes the majority of TbRab5Ap in pBluescript (29El-Sayed N.M.A. Alarcon C.M. Beck J.C. Sheffield V.C. Donelson J.E. Mol. Biochem. Parasitol. 1995; 73: 75-90Crossref PubMed Scopus (97) Google Scholar). The ORF was verified by restriction analysis and sequencing. The plasmid was cleaved with XhoI (3′ to the ORF) and blunt ended with Klenow, and the ORF was released with BamHI and ligated into BamHI/SmaI-cleaved pGEX-3X (Amersham Pharmacia Biotech) to create an in-frame fusion with glutathioneS-transferase (GST; a synthetic 4-amino acid linker is present). The construct was verified by expression in E. coli, producing a 56-kDa protein, the predicted molecular mass for a GST·TbRab5Ap fusion. TheTbRAB4 ORF (a gift from Dr. Nabib El-Sayed) was constructed from clone 452–5c, encoding the N terminus of TbRab4p,3was PCR amplified, producing a 5′ EcoRI site and finishing at an existing BsaBI site, and subcloned via these restriction sites directly into clone T452, encoding the majority of TbRab4p in pBluescript (29El-Sayed N.M.A. Alarcon C.M. Beck J.C. Sheffield V.C. Donelson J.E. Mol. Biochem. Parasitol. 1995; 73: 75-90Crossref PubMed Scopus (97) Google Scholar). The ORF was verified by restriction analysis and sequencing. Expression plasmids pGEX-3X·TbRAB5A and pQE30·TbRAB5B in E. coli were grown in L-broth and induced with 0.2–1.0 mm isopropyl-1-thio-β-d-galactopyranoside. GST-TbRab5Ap fusion protein was affinity purified on glutathione-Sepharose 4B as described previously (22Field H. Field M.C. J. Biol. Chem. 1997; 272: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 30Ridley A.J. Hall A. Cell. 1992; 70: 389-399Abstract Full Text PDF PubMed Scopus (3824) Google Scholar); cleavage was with Factor Xa (New England Biolabs) at room temperature for 2 h, adding more enzyme after 1 h. His6·TbRab5Bp protein was purified on nickel-Sepharose. Protein was estimated by SDS-PAGE and Coomassie staining. Typical yields were 0.4–4 mg of recombinant TbRab protein/liter E. coli. Antibodies to TbRab4p, -5Ap, and -5Bp were raised against synthetic peptides designed from the C termini and coupled to bovine serum albumin. Additional residues were included in TbRab4 and -5A peptides, to facilitate detection and derivatization. Peptides WGENGHAETLYDGPKRFSC (-4), WYGAQRLEPPTRQQKKEGGC (-5A), and ACKGVLGGQPNSTRRSSGC (-5B) were synthesized (Dr. R. Leatherbarrow, Department of Chemistry, Imperial College) and verified for purity by high performance liquid chromatography and for sequence by fast atom bombardment-mass spectrometry, following standard methods. A portion of the peptide was iodinated with IodoBeads (Pierce) or Bolton-Hunter reagent (-5B) and used as a tracer. Peptides were coupled to bovine serum albumin with an efficiency of ∼40%, usingm-maleimidobenzoyl-N-hydroxysuccinamide ester (Pierce) following standard procedures (24Medina-Acosta E. Cross G.A.M. Mol. Biochem. Parasitol. 1993; 59: 327-330Crossref PubMed Scopus (234) Google Scholar). The conjugate was used to immunize rabbits and rats using the MPL® + TDM + CWS Adjuvant System (Sigma). Animals were exsanguinated, and serum was stored at 4 °C with azide. For affinity purification, peptide was coupled to either thiol-Sepharose (-5B) or CNBr-Sepharose following the manufacturer's instructions. Antibodies were affinity purified from serum after 50% ammonium sulfate precipitation (31Harlow, E., and Lane, D. (eds) (1988) Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar). Proteins were separated by reducing SDS-PAGE. For Western blots, 5 × 106−2 × 107 cells per lane were electrophoresed on 15% SDS-polyacrylamide minigels and blotted (Amersham Pharmacia Biotech) onto 0.45 μm nitrocellulose. Filters were processed at room temperature. After blocking for 1 h in buffer A (20 mm potassium-Hepes, 2 mmMgCl2, 0.1–1% Tween 20, 110 mm potassium acetate, pH 7.4, containing 2% non-fat milk (Marvel)), filters were washed three times briefly, incubated with primary antibody for 1 h, washed, incubated with horseradish peroxidase-conjugated anti-rabbit antisera at 1:20,000 (Sigma) for 1 h, washed in Buffer A, and then in Buffer A without milk. Visualization was with ECL™ (Amersham Pharmacia Biotech) and x-ray film (Kodak). GTP overlay assay was performed on 107 trypanosomes/lane electrophoresed on 15% SDS-polyacrylamide gels (32Wilson A.L. Sheridan K.M. Erdman R.A. Maltese W.A. Biochem. J. 1996; 318: 1007-1014Crossref PubMed Scopus (13) Google Scholar). IFA was as described previously (33Sherwin T. Read M. Methods Mol. Biol. 1993; 21: 407-414PubMed Google Scholar). BSF and procyclic trypanosomes were washed in phosphate-buffered saline (Sigma), allowed to settle onto poly-lysine coated slides (Sigma) for 3 min (on ice for BSF), fixed in 4% paraformaldehyde (Sigma) in phosphate-buffered saline for 20 min, and then treated with methanol at −20 °C for 5 min. Purified anti-TbRab peptide antibodies were used at 1:50–100 dilution. Secondary antibodies were conjugated with fluorescein isothiocyanate (Sigma), Texas Red, Oregon 488 (Molecular Probes), or Cy3 (Jackson). Cells were examined on a Leica DMRXA epifluorescence microscope fitted with a Photometrics CH250 Slow Scan CCD camera, and digital images were captured using IP Lab Spectrum 3.1. Alternatively, photography was as described (22Field H. Field M.C. J. Biol. Chem. 1997; 272: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar), with images digitized by scanning photographs (Scanmaker II, Microtek). Resulting images were false-colored, merged, and assembled into figures (Adobe® Photoshop 3.0.5, Adobe® Systems, Inc.) on a MacOS computer (Apple Inc.). Computer analysis was as described (22Field H. Field M.C. J. Biol. Chem. 1997; 272: 10498-10505Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Fig. 1 was generated using SEQVU (Garvan Institute of Medical Research, Sidney, Australia). AT. brucei homologue of mammalian Rab5 was previously isolated (T.b. Rab5, Ref. 29El-Sayed N.M.A. Alarcon C.M. Beck J.C. Sheffield V.C. Donelson J.E. Mol. Biochem. Parasitol. 1995; 73: 75-90Crossref PubMed Scopus (97) Google Scholar); we designate it TbRab5Ap. A second Rab5 homologue was identified in T. brucei by reverse transcriptase PCR using a degenerate primer to the WDTAGQE motif of Rab GTPases. An EST, Rtb4 (27Field M.C. Boothroyd J.C. Exp. Parasitol. 1995; 81: 313-320Crossref PubMed Scopus (24) Google Scholar), was used to probe a T. brucei BSF cDNA library. A full-length cDNA of ∼2.0 kb was cloned and sequenced. The ORF was hypothetically translated and identified as a Rab5 homologue (Fig. 1 A) by BLAST search of GenBankTM and designated TbRab5Bp. Neither T. brucei Rab5 clearly corresponded to a particular Rab5 subclass. Comparison of TbRab5Bp with -5Ap and other Rab5 protein sequences (Fig. 1 A, and Table I) confirmed the conservation of canonical Rab protein features, i.e.GTP-binding regions, effector loops, and dicysteinyl C-terminal isoprenylation signal (34Seabra M.C. Goldstein J.L. Sudhof T.C. Brown M.S. J. Biol. Chem. 1992; 267: 14497-14503Abstract Full Text PDF PubMed Google Scholar), but two differences were seen between TbRab5Ap and -5Bp. The first is an insertion in TbRab5Ap (residues 61–77) with a high Ala and Gly content; this region corresponds to a loop between β-sheets 2 and 3 in the Ras structure and is predicted to be on the protein surface and highly mobile. The second is a deletion in -5Bp within the hypervariable region (Fig. 1 A); this is more common as similar but smaller indels are found in this region of mammalian Rab5 s and TbRab5Ap when compared with Ypt51p, -52p, and -53p (not shown).Table IHomology between TbRab5Ap, −5Bp, and closest homologues in the data baseThe hypothetical translations of the TbRAB5A and −5B ORFs were used to search GenBank.™ The top scoring match is shown in each case. Values above the diagonal are percent similarity and below percent identity. Open table in a new tab The hypothetical translations of the TbRAB5A and −5B ORFs were used to search GenBank.™ The top scoring match is shown in each case. Values above the diagonal are percent similarity and below percent identity. By BLAST search, the nearest homologue of TbRab5Ap was Ypt51p and of -5Bp wasCanis familiaris Rab5c (Table I). TbRab5Ap and -5Bp were as similar to each other as to their nearest homologues (Table I) so could not be assigned to specific Rab5 subgroups. A phylogeny for the Rab5 lineage was constructed, using Clustal (4.0) followed by PAUP, on sequences representative of the subclasses of Rab5 so far identified in mammals (Rab5a, -5b, and -5c) and the three Rab5 homologues fromSaccharomyces cerevisiae (Ypt51p, -52p, and -53p). Rab5 proteins from any one of the three lineages were monophyletic (Fig. 1 B). The simplest interpretation of this topology is that proliferation of the Rab5 subclasses occurred after speciation and that a single progenitor Rab5 gene was present in the eukaryote common ancestor. Because TbRab5Ap and -5Bp are closely related, we asked whether the genes encoding them (TbRAB5Aand -5B) were linked in the genome. Trypanosome genomic DNA, digested with a panel of restriction enzymes, was Southern blotted and probed with the complete ORFs of TbRAB5A and -5B(Fig. 2 A). No cross-hybridization between probes was detected using plasmids as targets at low stringency (not shown). There was no coincidence between the hybridization patterns obtained with TbRAB5A and -5B, reflected in the different maps obtained for the two genes (Fig. 2 B). The TbRAB5B probe revealed the presence of a restriction fragment-length polymorphism (Fig. 2 B, asterisk) giving a doublet in SacII digests although previous Southern blot analysis of the trypanosome genome with the Rtb4 probe (the 5′ third of the -5B ORF) suggested that TbRAB5Bwas single copy (27Field M.C. Boothroyd J.C. Exp. Parasitol. 1995; 81: 313-320Crossref PubMed Scopus (24) Google Scholar). In our original EST study, we isolated a second partial cDNA (Rtb9) closely related to Rtb4but with a small number of substitutions (27Field M.C. Boothroyd J.C. Exp. Parasitol. 1995; 81: 313-320Crossref PubMed Scopus (24) Google Scholar). These data suggest thatRtb4 and Rtb9 are derived from allelic copies of TbRAB5B. There was no additional hybridization with the TbRAB5A or-5B probes. As these two probes do not cross-hybridize at low stringency, there are no sequences more closely related toTbRAB5A than -5B and vice versa in the trypanosome genome, but more distantly related TbRAB5 genes may have been undetectable and therefore cannot be formally excluded. Southern analysis with the TbRAB4 ORF revealed a single copy, with no evidence for linkage between TbRAB4 and any other TbRAB gene so far studied (Fig. 2 A, Ref.27Field M.C. Boothroyd J.C. Exp. Parasitol. 1995; 81: 313-320Crossref PubMed Scopus (24) Google Scholar). Poly(A)-enriched RNA from 108 trypanosomes (3.4 μg from BSF and 10 μg from procyclics: the differing RNA yield is a known phenomenon 4J. Boothroyd, personal communication. ) was used in each lane of a Northern blot and probed with ORFs from TbRAB4, -5A, and -5B (Fig. 3). TbRAB4 detected a single major species migrating at ∼0.7 kb, with the ORF of 718 nt suggesting minimal untranslated sequence (UTR), and TbRAB5A detected a single species of ∼1.0 kb, with the ORF of 930 nt so there is again very little UTR. In contrast, TbRAB5B detected larger message species. As the 5′ UTR is 150 nt and the ORF 612 nt, the 3′ UTR is ∼1.2 kb and could potentially include regulatory sequences. The message levels were at a ratio of ∼10:20:1 for TbRab4:-5A:-5B, with -5B particularly difficult to detect because of low abundance and molecular weight heterogeneity, differing in BSF (3.8, 2.4, 2.1*, 1.8, 1.35 kb) and procyclics (3.8*, 2.3, 1.95* kb, where asterisks indicate the prominent message sizes). The 3.8-kb message probably represents an unprocessed nuclear transcript. The 2.0-kb cDNA clone containingTbRAB5B is consistent with a message size of 2.1–2.4 kb. For quantitation, the blots were reprobed with tubulin as a constitutively expressed standard; the signal intensity was at ∼1:4 for BSF:procyclics and ∼1:1 with the mass of RNA loaded. When normalized to tubulin, both TbRab4 and -5A messages were constitutive. The 3.8-kb TbRab5B message is also constitutive; conversely the ∼1.8–2.4-kb -5B transcripts normalized to tubulin at a ratio of 1:0.5, indicating more processed -5B message in BSF than in procyclics. In separate experiments loading equal masses of BSF and procyclic poly(A)-enriched RNA or poly(A)-selected RNA, hybridization signals forTbRAB5B were more intense in BSF, confirming that TbRab5Bp is developmentally regulated 5H. Field, A. Pal, and M. C. Field, unpublished data. (Ref. 27Field M.C. Boothroyd J.C. Exp. Parasitol. 1995; 81: 313-320Crossref PubMed Scopus (24) Google Scholar). GTPase activity was assessed in the two life stages by GTP overlay of lysates from 107 BSF or procyclic cells (Fig. 4). Total protein levels are approximately equal for both BSF and procyclic cells (Fig. 4).1 The pattern of GTP-bound bands was essentially identical, while significantly more GTP was bound by BSF lysate than procyclic. Rat (anti-TbRab4p and -5Ap) and rabbit antisera (-5Bp) were raised against synthetic peptides corresponding to the C termini of the TbRab sequences and affinity purified, and the specificity of anti-TbRab5Ap and -5Bp sera were checked by Western blotting against lysates of E. coli overexpressing recombinant TbRab (Fig. 5 A). Anti-TbRab5Ap recognized recombinant -5Ap but not -5Bp, and vice versa. No signal was observed using anti-TbRab4p or -5Ap antibodies in Western blotting on trypanosome lysates (Fig. 5 B, and data not shown). Extra residues added to the TbRab5Ap and -4p peptides may explain their relative lack of reactivity in Westerns, but inactivity in Western blotting is commonly observed for anti-Rab C-terminal peptide antibodies that work well in IFA (35Zerial M. Parton R. Chavrier P. Frank R. Methods Enzymol. 1992; 219: 398-407Crossref PubMed Scopus (33
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