Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p
1998; Springer Nature; Volume: 17; Issue: 13 Linguagem: Inglês
10.1093/emboj/17.13.3542
ISSN1460-2075
Autores Tópico(s)Lysosomal Storage Disorders Research
ResumoArticle1 July 1998free access Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p Patrick Lorenz Patrick Lorenz Present address: B.R.A.I.N GmbH, Darmstädter Strasse 34, D-64673 Zwingenburg, Germany Search for more papers by this author Alexander G. Maier Alexander G. Maier Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany Search for more papers by this author Eveline Baumgart Eveline Baumgart Institut für Anatomie und Zellbiologie II, Universität Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany Search for more papers by this author Ralf Erdmann Ralf Erdmann Institut für Physiologische Chemie, Abt. Systembiochemie, Ruhr Universität Bochum, D-44780 Bochum, Germany Search for more papers by this author Christine Clayton Corresponding Author Christine Clayton Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany Search for more papers by this author Patrick Lorenz Patrick Lorenz Present address: B.R.A.I.N GmbH, Darmstädter Strasse 34, D-64673 Zwingenburg, Germany Search for more papers by this author Alexander G. Maier Alexander G. Maier Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany Search for more papers by this author Eveline Baumgart Eveline Baumgart Institut für Anatomie und Zellbiologie II, Universität Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany Search for more papers by this author Ralf Erdmann Ralf Erdmann Institut für Physiologische Chemie, Abt. Systembiochemie, Ruhr Universität Bochum, D-44780 Bochum, Germany Search for more papers by this author Christine Clayton Corresponding Author Christine Clayton Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany Search for more papers by this author Author Information Patrick Lorenz2, Alexander G. Maier1, Eveline Baumgart3, Ralf Erdmann4 and Christine Clayton 1 1Zentrum für Molekulare Biologie, Universität Heidelberg, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany 2Present address: B.R.A.I.N GmbH, Darmstädter Strasse 34, D-64673 Zwingenburg, Germany 3Institut für Anatomie und Zellbiologie II, Universität Heidelberg, Im Neuenheimer Feld 307, D-69120 Heidelberg, Germany 4Institut für Physiologische Chemie, Abt. Systembiochemie, Ruhr Universität Bochum, D-44780 Bochum, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:3542-3555https://doi.org/10.1093/emboj/17.13.3542 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Kinetoplastid protozoa confine large parts of glycolysis within glycosomes, which are microbodies related to peroxisomes. We cloned the gene encoding the second most abundant integral membrane protein of Trypanosoma brucei glycosomes. The 24 kDa protein is very basic and hydrophobic, with two predicted transmembrane domains. It is targeted to peroxisomes when expressed in mammalian cells and yeast. The protein is a functional homologue of Pex11p from Saccharomyces cerevisiae: pex11Δ mutants, which are defective in peroxisome proliferation, can be complemented by the trypanosome gene. Sequence conservation is significant in the N- and C-terminal domains of all putative Pex11p homologues known, from trypanosomes, yeasts and mammals. Several lines of evidence indicate that these domains are oriented towards the cytosol. TbPex11p can form homodimers, like its yeast counterpart. The TbPEX11 gene is essential in trypanosomes. Inducible overexpression of the protein in T.brucei bloodstream forms causes growth arrest, the globular glycosomes being transformed to clusters of long tubules filling significant proportions of the cytoplasm. Reduced expression results in trypanosomes with fewer, but larger, organelles. Introduction Trypanosoma brucei is an extracellular protozoan parasite that causes severe diseases of livestock and humans in tropical Africa. It is transmitted between mammals by tsetse flies. Like other members of the order Kinetoplastida, T.brucei has compartmentalized the first seven enzymes of the glycolytic pathway inside specific organelles, the glycosomes (Clayton and Michels, 1996). We have no idea how this compartmentalization arose, and the metabolic advantages, if any, are also unclear. However, there is strong evidence that glycosomes are evolutionarily and functionally related to the peroxisomes of other eukaryotes (Michels and Hannaert, 1994). Beyond a similar ultrastructure, buoyant density and some common enzymatic constituents, glycosomes share with peroxisomes the two protein import pathways involving targeting signals, PTS1 and PTS2, that are responsible for post-translational import of peroxisomal matrix proteins (Opperdoes, 1987; Blattner et al., 1992, 1995; Sommer and Wang, 1994). All evidence so far indicates that the mechanism of microbody biogenesis has been conserved throughout eukaryotic evolution. In yeast and higher eukaryotes, a growing number of peroxin (peroxisomal biogenesis, PEX) genes have been defined by genetic complementation screens. Several of them encode membrane proteins (Erdmann et al., 1997; Waterham and Cregg, 1997). It has been reported previously that membranes of T.brucei glycosomes contain two predominant integral membrane proteins, estimated at 24 and 26 kDa (Aman and Wang, 1987). Here we report the cloning of the gene encoding the 24 kDa (p24) protein. p24 is structurally and functionally related to the 27 kDa peroxin Pex11p in yeast. Pex11p is required for peroxisome division in Saccharomyces cerevisiae (Erdmann and Blobel, 1995; Marshall et al., 1995) and Candida bodinii (Sakai et al., 1995). Previous reports have indicated that dimerization may be important in Pex11p function and that both N- and C-termini are oriented towards the peroxisomal matrix, thereby being inaccessible to cytosolic components (Marshall et al., 1996). Results Cloning of the gene encoding the 24 kDa membrane protein The major constituents of purified glycosomes have been identified as enzymes involved in glycolysis and glycerol metabolism (Misset et al., 1986). In addition, two proteins with an estimated molecular mass of 24 (p24) and 26 (p26) kDa previously have been shown to be integral membrane proteins that are expressed in both bloodstream (BF) and procyclic (Pro) forms of the parasite (Aman and Wang, 1987). We purified the 24 kDa protein, generated tryptic peptides and used the sequences to clone the corresponding cDNA and gene (see Materials and methods). The open reading frame (ORF) of 654 nucleotides predicts a protein of 218 amino acids with a molecular mass of 23 999.6 Da (Figure 1A), and includes the sequences of the p24 tryptic peptides. The calculated isoelectric point of 9.96 for p24 agrees with published estimates (Parsons and Nielsen, 1990). Using a specific antiserum raised against a predicted N-terminal peptide of p24, we performed a Western blot analysis to confirm that p24 is expressed in both life cycle stages of the parasite (see Figure 2A for BF and Figures 5 and 6 for Pro) and is largely resistant to carbonate extraction (Figure 5A). Figure 1.Sequence similarity of p24, RnPmp26 and yeast Pex11ps. (A) CLUSTAL-based multiple sequence alignment of T.brucei p24 (accession No. AJ005114) with peroxisomal membrane proteins. ScPEX11p (accession No. X81465) and CbPEX11p (accession No. U36243) are peroxin 11 from S.cerevisiae and C.boidinii respectively. RnPMP26 is a peroxisomal membrane protein from Rattus norvegicus. Shaded in black are blocks of homology (minimum 75% compliance with consensus). The N-terminal sequence of p24 is largely identical to a published T.brucei expressed sequence tag (accession No. W00255). The symbols used for the consensus are: a, acidic (D, E); b, basic (K, R, H); p, polar (S, T, Y, G, N, Q, C); f, hydrophobic (L, I, V, F, W, M, P, A). Shown above the alignment are the peptide sequences obtained from p24. (B) Kyte–Doolittle hydrophilicity plots of T.brucei p24 and S.cerevisiae Pex11p. Blocks 1–5 represent the regions of homology marked in (A) with the percentage identity/similarity calculated by the GAP program (default gap weight 3). Two transmembrane segments are predicted for p24 by each of two algorithms. Shown in black is the transmembrane domain of p24 (residues 130–147) predicted by both PredictProtein and TopPred II programs. The first hatched domain (96–113) is predicted by PredictProtein and the second hatched domain (193–213) is predicted by TopPred II. Shaded in grey are three helical domains of ScPex11p (PredictProtein) that might interact with the membrane (residues 92–110, 133–145, 217–229). Download figure Download PowerPoint Figure 2.Overexpression of p24 leads to growth arrest in T.brucei. (A) Wild-type (BF 449) trypanosomes and cell lines harbouring tet-inducible transgenes encoding p24, p24Nmyc or p24Cmyc were cultured for 0, 10 or 20 h with or without tetracycline before harvesting and processing for Western blot analysis. The blots (1.5×106 cells per lane) were probed with antisera against aldolase (ALD), phosphoglycerate kinase (PGK), p24 and p26. (B) The same cell lines as in (A) were grown in the presence (▪) or absence (□) of tetracycline and counted at the intervals indicated. Fresh tetracycline was added after diluting cultures to 105 cells/ml every 24 h (arrows). Download figure Download PowerPoint Figure 3.p24 is an integral membrane protein with both the N- and C-termini facing the cytoplasm. (A) Carbonate extraction. Purified bloodstream form glycosomes (25 μg of protein) were extracted successively with low salt buffer, high salt buffer and sodium carbonate. After centrifugation, the resulting supernatants (LS, HS and CS) and the carbonate pellet were processed for Western blotting and probed with antisera against aldolase (ALD) and p24. (B) Protease protection assay. Procyclic trypanosomes were permeablized with digitonin. Increasing amounts of proteinase K (PK) were added to permeabilized cells on ice for either 0 or 30 min with or without Triton X-100. Separated proteins were detected with antisera to aldolase and p24. The top two panels show cells expressing p24Nmyc, the lower panels cells expressing p24Cmyc. (C) Antibodies have access to both N- and C-termini of p24 in a crude post-nuclear pellet fraction. Crude extracts of cells expressing either p24Nmyc or p24Cmyc received mouse antibodies to GAPDH (lanes 2 and 4) or myc (lanes 3 and 5) either pre- (lanes 2 and 3) or post-solubilization (lanes 4 and 5) followed by immunoprecipitation with protein A beads. After SDS–PAGE and Western blotting, detection was with rabbit anti-GAPDH and anti-p24 antisera. PNP, untreated post-nuclear pellet fraction; lanes 1, control precipitation using beads but no antibodies. Download figure Download PowerPoint Figure 4.p24 can homodimerize, yet covalent disulfide bonds are largely isolation artefacts. (A) High molecular weight forms of p24 are reduction sensitive. Total membranes of 107 procyclic trypanosomes were extracted with carbonate either in the presence or absence of DTT, separated by non-reducing SDS–PAGE and subjected to Western blot analysis (anti-p24 antiserum). Arrows indicate 24 (p24) and 27 kDa (p24myc) monomers and the high molecular weight dimers. (B) Co-immunoprecipitation of p24 and p24myc from 2×107 detergent-solubilized procyclic cells using antibodies against the myc epitope. The blot was probed with anti-p24 (upper panel) or anti-p26 antisera (lower panel). Lanes 1, total protein; lanes 2, pre-clear (protein A beads only); lanes 3, immunoprecipitate (beads plus anti-myc antibodies). (C) Most p24 high molecular weight forms are lost when free thiol groups are blocked. Western blot analysis of samples treated as in (A), but with 10 mM NEM included in the extraction buffers. Download figure Download PowerPoint Southern blot analysis of genomic DNA from T.brucei strain 427 revealed a pattern of fragments compatible with a single p24 gene per haploid genome (data not shown). Northern blot analysis showed transcripts of 1.35 kb hybridizing with p24 probes in total RNA from both Pro and BF form trypanosomes (data not shown), confirming the Western blot data. p24 shows homology to yeast Pex11p and a rat peroxisomal membrane protein A blastp database search revealed similarity between T.brucei p24 and the peroxisomal membrane protein Pex11p from S.cerevisiae (ScPex11p/PMP27) in two regions at the N- and C-termini. An alignment of p24, the two yeast peroxins ScPex11p and C.boidinii Pex11p (CbPex11p/PMP30), and a rat peroxisomal membrane protein is shown in Figure 1A. The overall identity (similarity) between T.brucei p24 and ScPex11p is 23% (47%). The strongest conservation (36% identity, 64% similarity) is in a 28 amino acid stretch at the N-terminus (block 1 in Figure 1A). Other blocks (2–5) with their respective similarity values are given in Figure 1B. Interestingly, the regions most strongly conserved between p24 and ScPex11p are also conserved in the sequence of a mammalian Pex11p homologue (PMP26) from Rattus norvegicus (Passreiter et al., 1998). Overexpression of p24 leads to growth arrest To study the properties of p24 in more detail, we generated trypanosomes expressing p24 and its myc epitope-tagged derivatives under control of a tetracycline- (tet) regulatable promotor (see Materials and methods). These lines possess the two endogenous p24 genes plus a tet-regulatable transgene. As shown in Figure 2A, induction of p24 transgene expression by addition of tetracycline leads to a marked increase in the p24 signal in Western blots of total cellular protein (∼18-fold after 20 h relative to the level in wild-type cells, as determined in a titration experiment; not shown). Induction of both the N-terminally tagged p24Nmyc and the C-terminally tagged p24Cmyc led to the appearance of an additional band at ∼27 kDa that is recognized by the p24 antiserum. The level of expression of tagged p24 relative to wild-type (449) levels of p24 was ∼2-fold for p24Nmyc and ∼35-fold for p24Cmyc. Interestingly, at late time points of p24 induction, the amount of p26, the other major membrane protein of T.brucei glycosomes (Aman and Wang, 1987; P.Lorenz, A.G.Maier and C.Clayton, in preparation), is strongly reduced. The levels of glycosomal matrix enzymes aldolase (targeting signal PTS2) and phosphoglycerate kinase (PTS1) were unchanged. After tetracycline addition to the cell lines, we observed a strong reduction in the growth rates of cells harbouring the p24 and p24Cmyc transgenes (Figure 2B). Induction of p24Nmyc only had a minor effect on cell growth. This might have been due to the smaller amount of protein produced. Effects of p24 overexpression on glycosome morphology Confocal laser scanning microscopy analysis confirmed the glycosomal location of p24 and its myc-tagged derivatives (Figure 3). Double immunofluorescence labelling of p24 and the glycosomal matrix enzymes GPDH or GAPDH revealed co-localization of both proteins on discrete globular structures in the cytoplasm (Figure 3A, B, D and E). This morphology was always seen in normal cells and was also dominant in the other cell lines when cultivated in the absence of tetracycline. In cells induced to overexpress either p24 or p24Cmyc, however, fluorescence was found in much larger, often elongated structures (Figure 3C and G). Ultrastructural analysis revealed that the overexpression of p24 and p24Cmyc in T.brucei resulted in an accumulation of intracellular tubules with an electron-dense matrix, bound by a single membrane and with a diameter about one-third that of normal glycosomes (Figure 4B and Table I). Clustered tubules often filled large parts of the cytoplasm. Immunogold labelling showed that the matrix of these tubules contained the glycolytic enzyme aldolase (Figure 4D–F), confirming that they were abnormal glycosomes. Their membranes were labelled specifically with an anti-p24 antiserum (Figure 4C) or, for p24myc, the anti-myc antiserum (Figure 4E and F). In addition to the clusters of tubules, some cells contained membrane whirls excluding detectable matrix and containing p24 (Figure 4G–I). Figure 5.Double immunofluorescence microscopy analysis of bloodstream form trypanosomes overexpressing p24. Trypanosomes were cultivated for 20 h with (A, C, E and G) or without (B, D, F and H) tetracycline before harvesting and processing for scanning laser confocal microscopy. (A and B) Wild-type (449) cells; (C and D) p24 transgenics; (E and F) p24Nmyc transgenics; (G and H) p24Cmyc transgenics. The first column in each row shows staining for p24 (Cy2, A–D) or p24myc (Cy5, E–H) using anti-p24 or anti-myc antibodies respectively. The second column shows staining for matrix markers GPDH (Cy5, A–D) or GAPDH (Cy2, E–H). The third and fourth column show interference contrast image and overlay. Scale bar 10 μm. Download figure Download PowerPoint Figure 6.Overexpression of p24 in T.brucei leads to the proliferation of glycosomal tubules. (A and B) The ultrastructure of p24Cmyc transgenic bloodstream form trypanosomes cultured for 20 h in the absence (A) or presence (B) of tetracycline. The tubules were also found in cells overexpressing p24 (C and D) and less frequently in cells expressing p24Nmyc (E), and could be decorated with antibodies against the glycosomal matrix marker aldolase (D) and p24 (C). (E and F) Co-localization of p24myc (6 nm gold) and aldolase (15 nm gold) on the same tubules in cells expressing p24Nmyc and p24Cmyc. Overexpression of p24 sometimes resulted in the formation of membrane whirls and club-shaped structures reflecting membrane proliferation (G and H, arrows). These whirls could also be labelled with anti-p24 gold complexes (I). A quantification of glycosome morphology on electron microscopical sections is shown in Table I. Gly: glycosomes, N: nucleus. Download figure Download PowerPoint Table 1. Quantification of glycosome morphology in ultrathin cell sections Cell linea Globular Globular + tubular Tubular (clusters) + − + − + − 449 52 50 44 46 4 4 p24 16 44 26 32 58 24 p24Nmyc 34 42 24 48 42 10 p24Cmyc 12 26 12 36 76 38 Using electron microscopy, 50 sections were evaluated for each incubation condition and grouped into three categories, depending on the morphology of their glycosomes. a Cells were incubated for 20 h ± 1μg/ml tetracycline. All results are given as percentages. The moderate overexpression of p24Nmyc did not change glycosome morphology as dramatically as seen with p24 and p24Cmyc, consistent with the lack of other phenotypic effects. Although there was a tetracycline-induced increase in the number of cell sections with clusters (Table I), the tubules mostly had a larger diameter compared with those seen with p24 and p24Cmyc (not shown). The N- and C-terminal ends of p24 are facing the cytosol The p24 sequence contains two hydrophobic amino acid stretches that are predicted to form transmembrane helices (Figure 1B). To confirm the previously described membrane association of p24 (Aman and Wang, 1987), we extracted purified glycosomes successively with low salt buffer, high salt buffer and finally carbonate (Figure 5A). Whereas aldolase, a soluble matrix enzyme, is readily released into the high salt and carbonate supernatants, most of p24 remains inextractable in the carbonate pellet, suggesting membrane integration. To establish the membrane topology of p24, we performed protease protection assays using digitonin-permeabilized cells expressing the native and myc-tagged versions of p24. Native p24 completely resisted proteinase K digestion in the absence of Triton X-100, suggesting that it might reside largely on the inner side of the glycosomal membrane (Figure 5B), as suggested previously for ScPex11p (Marshall et al., 1996). The N- and C-terminally tagged p24myc proteins, however, behaved differently. The addition of proteinase without Triton X-100 resulted in the disappearance of the 27 kDa p24myc species and the appearance of a smear intermediate in size between p24 and p24myc. Under these conditions, the glycosomal matrix enzyme aldolase is protease resistant. p24 is degraded more strongly and aldolase is degraded to a slightly smaller protease-resistant core (Clayton, 1987) when detergent is added to the reaction (Figure 5B). This suggests that both the N- and C-terminal ends of p24 are oriented towards the cytosol. We next assessed the accessibility of p24myc to antibodies. The exposure of the myc epitope on glycosomes in a post-nuclear pellet fraction, and hence the cytosolic orientations of both N- and C-termini, were confirmed by an antibody capture assay. Whereas anti-myc antibodies precipitated similar amounts of p24myc from both p24Nmyc- and p24Cmyc-expressing cell lines, independently of whether they were added to the assay before or after detergent solubilization, anti-GAPDH antibodies precipitated significant amounts of GAPDH only when added after solubilization, reflecting the matrix localization of this glycolytic enzyme (Figure 5C). p24 can form homodimers The dimerization of Pex11p in yeast has been reported previously (Marshall et al., 1996). Dimerization of glycosomal p24 is readily demonstrated in cells overexpressing p24, but is also detectable in wild-type cells. Figure 6A illustrates the behaviour of p24 when total membranes were isolated from various cell lines in either the presence or absence of reducing agents. In the absence of dithiothreitol (DTT), a 40 kDa band was recognized by the anti-p24 antiserum (Figure 6A, lanes 449− and p24−). The intensity of this band was strongly reduced, but not abolished, if DTT was included in the extraction buffers (Figure 6A, lanes 449+ and p24+). In the cell lines expressing p24Nmyc, the p24 antiserum recognized three reduction-sensitive p24 bands of ∼50, 45 and 40 kDa; in the p24Cmyc cell line, there were high molecular weight bands at 45, 42 and 40 kDa (Figure 6A). The 40 kDa band probably represents the p24 homodimer as it is present also in the wild-type (449) cells possessing only the endogenous p24 genes. In the lines overexpressing tagged versions of p24, one might expect two dimers larger than that, namely a p24myc homodimer and a p24–p24myc heterodimer. The top bands (running at 50 or 45 kDa) were indeed recognized by an anti-myc antibody in extracts of p24Nmyc- and p24Cmyc-expressing cell lines (data not shown). Unexpectedly, the intermediate bands (45 or 42 kDa) were not recognized; possibly the myc tag is conformationally masked in the heterodimer. A second approach to investigate p24 dimerization was to test for co-immunoprecipitation of p24 from cells expressing p24myc using an anti-myc antibody. As shown in Figure 6B, anti-myc antibodies precipitate endogenous p24 only in extracts from cells also expressing p24Nmyc or p24Cmyc but not from wild-type cells expressing only p24. The other major protein in the glycosomal membrane, p26 (containing 10 cysteines in its primary structure, P.Lorenz, A.G.Maier and C.Clayton, in preparation), does not co-precipitate with p24myc, suggesting that homodimerization of p24 is specific. To test the possibility that covalent disulfide bridge formation was an oxidation artefact occurring during isolation, we included in the extraction buffers alkylating agents known to block free sulfhydryl groups. The addition of 10 mM N-ethylmaleimide (NEM) led to a 90% reduction of p24 dimers in cells expressing wild-type levels of p24 (Figure 6C), suggesting that most sulfhydryl groups shown to be cross-linked in the earlier experiments were in a reduced state at the time of breaking the cells. Intriguingly, when using only 1 mM NEM, it appeared that the extra high molecular bands seen in cell lines expressing p24myc were more strongly affected than the wild-type p24 homodimer (not shown). Heterologous expression of p24 in mammalian cells and yeast To investigate whether glycosomal sorting of p24 was mediated by an evolutionarily conserved mechanism, we transiently expressed the protein in monkey CV-1 cells. As seen in Figure 7, there was a clear co-localization of the peroxisomal matrix marker acyl-CoA oxidase and p24Cmyc on discrete spots scattered over the entire cytoplasm. Similarly, when expressed in S.cerevisiae, p24 was sorted to peroxisomes as evidenced by the co-localization of p24Cmyc and thiolase on discrete spots inside the cells (Figure 8B). Figure 7.p24 is targeted to peroxisomes in monkey CV-1 cells. Double immunofluorescence labelling of transiently transfected CV-1 cells showing p24Cmyc co-localizing with acyl-CoA oxidase. (A) DAPI staining of nuclei, (B) Cy2-coupled anti-myc staining for p24Cmyc, (C) Cy3-coupled anti-acyl-CoA oxidase staining. Only the cell in the middle of the panels expresses p24Cmyc. Scale bar 12 μm. Download figure Download PowerPoint Figure 8.Expression of p24 functionally complements the pex11Δ mutation of S.cerevisiae. Yeast pex11Δ mutants were transformed with the low copy plasmid pRSterm or pRSterm containing the genes encoding ScPEX11 (pRS-PEX11), p24 (pRS-p24) or p24Cmyc (pRS-p24C) under the control of the yeast FOX3 promoter. Similar results were obtained for both UTL7A and W303A strains. (A) p24 restores growth of UTL7A pex11Δ cells on oleate. Wild-type, pex11Δ cells and the indicated transformants were plated on YNO at different densities. (B) p24 is targeted to yeast peroxisomes and restores normal peroxisome morphology of oleic acid-induced pex11Δ cells. Immunofluorescence localization of thiolase (in W303A wild-type and pex11Δ harbouring pRSterm, pRS-PEX11, pRS-p24) or thiolase plus myc-tagged p24 (in UTL7A pex11Δ harbouring pRS-p24C as indicated). Scale bar 5 μm. Download figure Download PowerPoint Prompted by the apparent sequence similarities between p24 and the yeast peroxin Pex11p, we attempted to complement functionally pex11Δ knockout mutants with the trypanosome gene. The S.cerevisiae pex11Δ mutants are characterized by a defect in peroxisome proliferation that leads to a growth defect on YNO plates containing oleate as the single carbon source and to the appearance of giant peroxisomes (Erdmann and Blobel, 1995; Marshall et al., 1995, 1996). As expected, introduction of ScPEX11 restores peroxisome morphology as well as growth on YNO plates of pex11Δ cells (Figure 8A and B). Growth on YNO was also restored when the mutant strain was transformed with plasmids encoding p24 or p24Cmyc under the control of the FOX3 promoter (Figure 8A). Furthermore, the heterologous expression of p24 and p24Cmyc led to the restoration of peroxisome morphology of pex11Δ mutant cells. Instead of the very few giant peroxisomes typical for oleate-grown pex11Δ mutant cells, transformants of pex11Δ that harbour the trypanosomal proteins showed normally sized and numbered organelles (Figure 8B). These observations suggest that trypanosomal p24 is able functionally to replace the ScPex11p in yeast peroxisome biogenesis. As p24Cmyc maintained the complementing activity, the tagging obviously did not interfere with p24 function in peroxisome biogenesis. Double immunofluorescence localization of p24Cmyc and peroxisomal thiolase (Fox3p) revealed a congruent fluorescence pattern (Figure 8B), indicating that p24Cmyc is targeted to peroxisomes in yeast. The heterologous complementation of the yeast mutant together with the peroxisomal localization of the protein in yeast and CV-1 cells strongly supports the notion that p24 is the T.brucei orthologue of Pex11p from yeast. Trypanosome p24 is involved in glycosome division To see if the absence of p24 in trypanosomes gives a similar phenotype to the deletion of PEX11 in yeast, we attempted to create cell lines lacking the p24 gene. Yeast pex11 mutants are viable on rich media, showing growth defects only when grown on media that require peroxisome function for catabolism. Bloodstream trypanosomes are obligatorily dependent on the glycolytic enzymes in the glycosome for survival. Procyclic (insect) forms, in contrast, metabolize amino acids and generate energy via mitochondrial pathways and so conceivably might be able to survive without glycosomal compartmentation. We therefore attempted to delete the p24 gene from wild-type (449) Pro trypanosomes, using constructs containing the neomycin phosphotransferase (NPT) or hygromycin phosphotransferase (HYG) genes as a selectable markers. Constructs contained untranslated regions from 5′ and 3′ of p24, and were designed to enable clean replacement of the p24 gene by homologous recombination (tenAsbroek et al., 1990). Upon transfection with the NPT construct, G418-resistant clones with only a single remaining copy of the p24 gene were obtained without difficulty. After a second transfection with the HYG construct, half of the cells were selected for hygromycin resistance alone, and half for resistance to both drugs. Of the 19 viable clones obtained using hygromycin selection alone, none were resistant to G418, suggesting that the HYG gene had replaced the NPT gene. Only five cell lines grew out of double drug selection, but all still expressed p24 (data not shown), indicating retention of the gene. These results suggest that p24 is essential in T.brucei. To assess the knockout phe
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