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Characterization of the Gene for the Microbody (Glycosomal) Triosephosphate Isomerase of Trypanosoma Brucei

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Characterization of the Gene for the Microbody (Glycosomal) Triosephosphate Isomerase of Trypanosoma Brucei The EMBO Journal vol.5 no.6 pp. 1291 -1298, 1986 Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei Bart W.Swinkels1, Wendy C.Gibson1, Klaas A.Osinga13, isomerase, EC 5.3.1.1) is particularly suitable for such com- Roel Kramer1, Gerrit H.Veeneman2, parative studies. The enzyme is well characterized (see Straus Jacques H.van Boom2 and Piet Borst1 et al., 1985); the amino acid sequence of TIMs from both 'Division of Molecular Biology, The Netherlands Cancer Institute, eukaryotic (Kolb et al., 1974; Corran and Waley, 1975; Alber Plesmanlaan 121, 1066 CX Amsterdam, and 20rganic Chemistry and Kawasaki, 1982; Maquat et al., 1985; Straus and Gilbert, Laboratory, State University Leiden, Gorlaeus Laboratory, PO Box 9502, 1985a) and prokaryotic (Artavanis-Tsakonas and Harris, 1980; 2300 RA Leiden, The Netherlands Pichersky et al., 1984) sources has been determined and high 3Present address: Research and Development, Gist-brocades NV, Postbus 1, resolution structures for the chicken (Banner et al., 1975) and 2600 MA Delft, The Netherlands yeast (Alber et al., 1981) proteins are available. This makes TIM Communicated by P.Borst suitable for deducing long-range evolutionary relationships. To determine how microbody enzymes enter microbodies, we TIM has previously been purified from Trypanosoma brucei are studying the genes for glycosomal (microbody) enzymes (Misset and Opperdoes, 1984) and crystals for X-ray diffraction in Trypanosoma brucei. Here we present our results for triose- have been obtained (Weirenga et al., 1984), allowing the elucida- phosphate isomerase (TIM), which is found exclusively in the tion of the 3-D structure of the enzyme. Here we present the glycosome. We found a single TIM gene without introns, hav- sequence of the gene and an analysis of its transcription. ing one major polyadenylated transcript of 1500 nucleotides Results with a long untranslated tail of - 600 nucleotides. By a novel method, suitable for low abundance transcripts, we Isolation and characterization of the TIM gene of T. brucei demonstrate that TIM mRNA contains the 35-nucleotide We used a genomic DNA probe from the yeast TIM gene to iden- leader sequence (mini-exon) also found on several other tify the corresponding gene(s) in trypanosome nuclear DNA and trypanosome mRNAs. The TIM gene and a DNA segment in cDNA and genomic DNA recombinant clone banks. A single of at least 6 kbp upstream of the gene are transcribed at an trypanosome gene was found with homology to the yeast probe equal rate in isolated nuclei, suggesting that the gene is part (see also Gibson et al., 1985). The physical map of this gene of a much larger transcription unit. The predicted protein is presented in Figure 1 together with the genomic clones and is of the same size as TIMs from other organisms and shares subclones used to characterize the gene. -50% amino acid homology with other eukaryote TIMs, Figure 2 shows a Southern blot of trypanosome nuclear DNA somewhat less with prokaryote TIMs. Trypanosome TIM is hybridized with a trypanosome TIM cDNA probe. A single gene the most basic of all TIMs sequenced thus far. This is, in part, is present in strain 427 and in 20 other strains analysed; addi- due to the presence of two clusters of positively charged tional sub-stoichiometric bands seen previously and attributed to residues in the molecule which may act as a signal for entry the presence of related genes (Gibson et al., 1985) were in fact into glycosomes. due to a probe contaminated with an upstream fragment. Key words: glycosome/topogenesis/peroxisome biogenesis/micro- Sequence of the trypanosome TIM gene body evolution/mini-exon The nucleotide sequence of the TIM gene was determined by the chain termination method, using a serial sequencing approach as detailed in Figure 1. The sequence is presented in Figure 3; Introduction a comparison of the amino acid sequence homology of the The parasitic protozoa grouped in the family Trypanosomatidae trypanosome TIM with TIMs of other organisms is shown in are characterized by an unusual microbody, called the glycosome Figure 4 and Table I. Clearly, the trypanosome gene, isolated (Opperdoes and Borst, 1977; Opperdoes et al., 1984), which con- via its homology with the yeast TIM gene, is a typical TIM gene. tains enzymes of the glycolytic pathway in addition to (parts of) It specifies a protein of the same size as other TIMs, and shares several other metabolic pathways [see Opperdoes (1985) and -50% sequence homology, at both the nucleotide and amino references quoted therein]. The transfer of glycolysis from its acid levels, with other eukaryotic TIMs. No major insertions are usual location in the cytosol to a microbody represents a required to align the amino acid sequences of the trypanosome remarkable step in eukaryotic evolution. We are interested in the TIM and those of other organisms, indicating that the trypano- mechanism of this translocation and in the changes required in some gene lacks introns. cytosolic glycolytic enzymes to allow their delivery into micro- Although we have no direct amino acid sequence data to link bodies. We hope that such changes may eventually be exploited the sequenced gene to the glycosomal TIM, the indirect evidence in chemotherapy (see Wierenga et al., 1984), and expect that for this link is strong. Only one TIM has been detected in they will provide information about the mechanism of entry of T. brucei (Misset and Opperdoes, 1984) and we find one gene. proteins into microbodies (peroxisomes, glyoxysomes) in general The subunit mass for T. brucei TIM, as calculated from the and, possibly, about the evolutionary origin of these organelles. nucleotide sequence, is 26 718 (not counting the initiator The glycolytic enzyme triosephosphate isomerase (Meyerhof methionine), which is in good agreement with the mass of 27 000 and Lohmann, 1934) (TIM; D-glyceraldehyde-3-phosphate ketol 4 500 deduced from SDS gel electrophoretic analysis of the © IRL Press Limited, Oxford, England 1291 B.W.Swinkels et al. TIM H H K R P C pK A H P pKp A K R C 5. IP,I I '1 I I I I I II 1 3. * mRNA 1500 nts I- I pTcTIM9 K H P PP RC P K R C P P, H pTgTIM12 1 I I I _ I I I R p C pK Hti A H PHif. R pTgTIM8 iK 1kb I I Hr-. c * p P Bc P K Ev .. R I.-,_ I .i.-- 200bp 5. 3. M13 subclones & , sequence strategy Fig. 1. Physical map of the TIM gene of T brucei 427. The genomic environment of the TIM gene (black box) is shown and below, maps of the inserts of two genomic clones, pTgTIM8 (in pAT153) and pTgTIM12 (in pBR322) are shown. The positions of the mRNA and cDNA clone pTcTIM9 are indicated below the gene. The broken line represents a polymorphic restriction site differing between the two TIM alleles in T. brucei 427. HincIl sites are only shown in pTgTIM8, where they were mapped. Part of the map of pTgTIM8 is enlarged to show the sequence strategy: bars indicate two PstI fragments subcloned into M13, the larger of which contains plasmid sequences (indicated by open box); vector arrows indicate length and orientation of sequence obtained with each primer (dot) or the Maxam and Gilbert method (cross). The 5' and 3' ends of the SI nuclease-mapped transcript are arrowed below the enlarged map. Restriction sites: A = AvaII, C = ClaI, Ev = EcoRV, H = HindIll, Hc = HincII, K = KpnI, P = PstI, Pv = PvuII and R = EcoI. purified glycosomal protein (O.Misset and F.R.Opperdoes, per- EcoRI Pst Avail Cla I sonal communication). Also the high net positive charge, calculated from the predicted amino acid sequence (see below), is in agreement with an isoelectric point >pH 9.0, found for kb the glycosomal TIM (O.Misset and F.R.Opperdoes, personal 8.0- W communication). Finally, X-ray diffraction studies of T. brucei TIM crystals show the enzyme to closely resemble other TIMs 5.3- _ and show divergence in electron density at positions predicted by divergence of the T. brucei TIM amino acid sequence from other sequences (Wierenga et al., 1984; R.K.Wierenga and W.G.J.Hol, personal communication). Hence, we conclude that our TIM sequence corresponds to the glycosomal TIM and that 2.6- wp we have not sequenced a gene for a second minor TIM or a non- functional gene. Our comparison of the amino acid sequences of the cytosolic and glycosomal phosphoglycerate kinases (PGK) of T. brucei (Osinga et al., 1985) revealed two types of difference: the 1.3- glycosomal enzyme has a C-terminal extension of 20 residues and a much higher positive charge than the cytosolic isoenzymes. For TIM there is no cytosolic equivalent of the glycosomal en- zyme, but the comparison of its amino acid sequence with those of TIMs from other organisms shows that the trypanosomal TIM lacks a C-terminal extension (Figure 4). This has been confirm- 0.5- ed by X-ray diffraction of TIM crystals (R.K.Wierenga and W.G.J.Hol, personal communication). The charge of different probe: TIM c DNA TIMs, as calculated from their amino acid sequences, assuming neutral pH, is presented in Figure 5. We do not know how this relates to the pH in situ. However, a large difference in pH bet- Fig. 2. The TIM gene is present in a single copy in T. brucei. Nuclear DNA from T. brucei was digested with the restriction enzymes shown, size ween the cytosol and the glycosomal matrix seems unlikely in fractionated through a 0.7% agarose gel and hybridized with the TIM view of the pH optima of the cytosolic and glycosomal PGK, cDNA clone pTcTIM9.
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