1 Zentrum für Molekulare Biologie der Universität Heidelberg, Im
Neuenheimer Feld 282, D-69120 Heidelberg, Germany
2 Centro de Biología Molecular `SO', Lab CX-203, Universidad
Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain
* Author for correspondence (e-mail: cclayton{at}zmbh.uni-heidelberg.de )
Accepted 1 April 2002
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Summary |
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Key words: Trypanosoma, Kinetoplastida, Glycosome, Peroxisome, biogenesis, Glycolysis
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Introduction |
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Glycosomes are peroxisome-related microbodies found in all kinetoplastids;
they contain the first 7-9 enzymes of glucose metabolism. The sleeping
sickness trypanosome, Trypanosoma brucei, is entirely dependent on
glycolysis for ATP generation while it multiplies in the mammal. Expression of
either phosphoglycerate kinase (PGK) or triosephosphate isomerase in the
cytosol inhibits parasite growth (Blattner
et al., 1998; Helfert et al.,
2001
), suggesting that correct localisation of glycolytic enzymes
is important. Various results, including metabolic modeling, suggest that in
bloodstream T. brucei, compartmentation plays a vital role in the
regulation of glycolysis (Bakker et al.,
2000
; Michels et al.,
2000
).
T. brucei is transmitted between mammals by tsetse flies. The
`procyclic' form that multiplies in the tsetse depends for ATP generation
mainly on the mitochondrial metabolism of amino acids
(Michels et al., 2000).
Several enzymes from pathways other than glycolysis can be associated with
trypanosomatid glycosomes, their activities varying considerably between
parasite species and life-cycle stages
(Michels et al., 2000
).
Microbody biogenesis depends on peroxin (PEX) proteins, which are needed
for microbody membrane formation and the import of matrix proteins from the
cytosol (Baerends et al., 2000;
Subramani et al., 2000
;
Titorenko and Rachubinski,
2001
). Mutations of the PEX2 protein lead to defective import of
matrix proteins into the peroxisomes of mammalian cells and yeasts
(Eitzen et al., 1996
;
Faust and Hatten, 1997
;
Waterham et al., 1996
); a
number of other defects could be secondary to metabolic disturbance
(Baerends et al., 2000
). The
peroxisomes of pex2 mutants (those lacking PEX2) are reduced to empty
ghosts (Hettema et al., 2000
).
PEX2 has two essential membrane-spanning regions and the N- and C-termini are
oriented towards the cytosol (Harano et
al., 1999
). The C-terminus contains a RING finger motif of
uncertain function (Shimozawa et al.,
2000
). Evidence from Yarrowia lipolytica indicates that
PEX2 traffics through the endoplasmatic reticulum and is involved in the early
stages of peroxisome formation (Titorenko
et al., 2000
; Titorenko and
Rachubinski, 2001
).
The PEX2 gene of the kinetoplastid Leishmania donovani
was identified by complementation of a mutant with a partial defect in
glycosomal matrix protein import
(Flaspohler et al., 1997). The
defect in the mutant was caused by premature termination in one copy of the
PEX2 gene. This encoded a protein lacking one transmembrane domain
and the RING finger (Flaspohler et al.,
1997
; Mannion-Henderson et
al., 2000
), which had a dominant-negative effect. It was not
possible to delete both copies of the LdPEX2 gene
(Flaspohler et al., 1999
).
Mammalian tissue culture cells survive indefinitely in the absence of PEX2
function; pex mutations are lethal in animals because of the
accumulation of metabolites or substrates and consequent defects. Yeasts
lacking peroxisomal membranes grow normally on glucose, but die when they are
restricted to a carbon source that is metabolised by peroxisomal enzymes, such
as oleate in Saccharomyces cerevisiae
(Erdmann et al., 1989) or
methanol in Hansenula polymorpha
(Waterham et al., 1994
). The
situation in pex mutants of bloodstream trypanosomes grown on glucose
should be analagous to that of oleate- or methanol-grown yeasts. One would
therefore predict that the glycosomal membrane should be essential in
bloodstream forms of T. brucei, but dispensable in the procyclic
forms, which do not need glycolysis to generate ATP and may thus cope without
glycosomes.
We have previously shown that downregulation of the trypanosome
PEX11 gene reduces glycosome numbers
(Lorenz et al., 1998), and
that depletion of the GIM5 glycosomal membrane protein is deleterious to
bloodstream forms (Maier et al.,
2001
). However, neither of these mutations had any affect on
glycosomal matrix protein import. To find out the role of glycosomal
compartmentation in trypanosomes, we needed to relocate complete pathways to
the cytosol. We achieved this by downregulating PEX2 expression.
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Materials and Methods |
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Plasmid constructs
Clones from the bacteriophage P1 library of T. brucei TREU927/4
(Sarah Melville, University of Cambridge) were selected by screening with a
genomic PCR product. Sequencing was done by the ZMBH sequencing facility and
by TopLab (Martinsried, Germany). The complete open reading frame, and a
version with a termination codon replacing the codon for glutamine 239,
pex2r, were amplified using suitable primers and cloned into the vectors
pHD676 or pHD677 (Biebinger et al.,
1997
) for expression from the Tet-inducible EP1 promoter,
PEP1Ti. pHD789
(Irmer and Clayton, 2001
) was
used for inducible expression from the T7 promoter. In the resulting
construct, the hygromycin resistance cassette could be transcribed only from
the inducible T7 promoter, but the background transcription in the absence of
Tet was sufficient to allow selection of hygromycin-resistant cells. The pSP72
`stuffer' fragment (Shi et al.,
2000
) was a gift from Elisabetta Ullu (Yale University, New Haven,
CT). A 468 bp fragment from the 5' end of PEX2 open reading
frame,
pex2, was cloned upstream of the stuffer in the sense
orientation and downstream of the stuffer in the antisense (as)
orientation. The
pex2-stuffer-
pex2as was cloned
into the polylinker of the inducible trypanosomatid expression vector pHD677
[(Biebinger et al., 1997
)
http://www.zmbh.uni-heidelberg.de
)]. Reconstructed plasmid sequences are available from the authors on
request.
Northern blotting
Poly(A)+ RNA was isolated from procyclic cells using oligo(dT) cellulose
(Pharmacia). Resolution of 4 µg of mRNA on agarose/formaldehyde gels,
transfer to neutral nylon membranes, prehybridisation and hybridisation with
radioactive probes were done using standard procedures.
Immunofluorescence and western blotting
Cells were fixed and stained as described
(Maier et al., 2001) using
mouse polyclonal anti-T. brucei glyceraldehyde phosphate
dehydrogenase (GAPDH) or rabbit polyclonal anti-T. brucei PEX11,
aldolase (ALD), hexokinase (HXK) and phosphofructokinase (PFK) antisera.
Detection was with Cy2- or Cy3-linked secondary antibodies (Amersham). For
Fig. 2, the anti-ALD and
anti-PEX11 antibodies were purified on Protein A beads, coupled with
AlexaFluorTM 488 (Molecular Probes) following the manufacturer's
instructions, and applied after the PEX11 or the HXK stain. The software used
was OpenLabTM (Improvision) and Adobe® PhotoShop.
|
For western blotting, cells were fractionated with digitonin
(Helfert et al., 2001;
Lorenz et al., 1998
).
Incubations were with rabbit polyclonal anti-T. brucei PEX11, PFK,
GAPDH and ALD; and mouse monoclonal anti-T. brucei surface protein
EP1 (Cedarlane, Ontario). A rabbit polyclonal antibody raised against a
peptide from a T. brucei RNase II-like protein (accession number
AJ309002) recognises an unidentified cytosolic 100 kDa protein
(TbCSM1), which is not the original target and was used as a
cytosolic marker.
Enzyme assays
2x108 cells were washed in cold 25 mM Tris-HCl pH 7.8,
containing 1 mM EDTA and 10% sucrose, resuspended in 0.5 ml of the same buffer
with protease inhibitors (Mini-complete, Roche) and 50 mg of glass beads
(212-300 µm, Sigma) and broken at 4°C by three (bloodstream) or six
(procyclic) vibration pulses (5 seconds each, mini-bead beater). A
post-nuclear supernatant was microfuged for 15 minutes at 20,000 g to
give a cytosolic fraction and a pellet containing glycosomes and other
organelles. HXK activity was measured by following the reduction of
NADP+ at 340 nm in 10 mM MgCl2, 10 mM D-glucose, 660
µM ATP, 5.635 mM NADP+, 4.75 mM NaHCO3 and 5 µg/ml
of glucose-6-phosphate dehydrogenase (G6PDH) in 0.1 M TEA buffer pH 7.3 at
25°C. Phosphoglucoisomerase (PGI) activity was measured in the same way,
but the reaction mix contained 7 mM MgCl2, 1.3 mM fructose-6-P, 400
µM NADP+ and 5 µg/ml of G6PDH in 0.1 M TEA buffer.
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Results |
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The PEX2 mRNA was detectable on northern blots as two bands of 1.3 kb and 1.7 kb (Fig. 1A, Tet), and at similar abundance in poly(A)+ RNA from bloodstream and procyclic trypanosomes. These bands were extremely faint or undetectable when total RNA was used. We do not know the reason for the presence of the two bands. Attempts to map polyadenylation sites and splice sites by reverse transcription and PCR failed to yield specific products. By restriction digestion and Southern blot hybridisation, we found just two apparently identical allelic copies of PEX2 in genomic DNA (data not shown; C. Guerra-Giraldez, PhD Thesis, University of Heidelberg, 2001), so the different mRNAs could come from alternative splicing or polyadenylation.
|
For detection of PEX2 protein, rabbits were immunized with two peptides, corresponding to amino acids 151-168 and 253-278. None of the four sera obtained recognised any protein of the expected size, either in total cell extracts or in purified glycosomes, although they all detected a TbPEX2-maltose-binding-protein fusion overexpressed in E. coli (C. Guerra-Giraldez, PhD Thesis, University of Heidelberg, 2001). This suggests that PEX2 might be present at rather low abundance in trypanosomes.
To overexpress PEX2 in trypanosomes, the open reading frame was cloned downstream of the Tet-inducible RNA polymerase I promoter, PEP1Ti. The construct was transfected into transgenic bloodstream trypanosomes expressing the Tet repressor (449 cells) and clonal cell lines selected. When Tet was added, the expression of two PEX2 mRNAs, slightly bigger than the endogenous ones, was strongly induced, but no protein was detected with the anti-TbPEX2 antibodies. Finally, a myc tag was added to the inducible copy of PEX2; upon addition of Tet no myc-tagged protein was detectable, either in total lysates or in the organellar pellet made by digitonin or carbonate fractionation. We also failed to detect PEX2 inducibly expressed from a bacteriophage T7 promoter in trypanosomes expressing T7 polymerase. Attempts to detect the PEX2 protein after pulse-labeling with [35S]methionine were similarly unsuccessful. The addition of tetracycline to all these inducible cell lines had no effect at all on cell growth (in Fig. 1B, open squares show the growth of cells inducibly overexpressing PEX2; filled squares correspond to the same cells grown without Tet) and morphology, suggesting that overproduction of PEX2, if it occurs, has no deleterious consequences.
These results suggest that PEX2 is expressed at low abundance in trypanosomes, and expression of high levels is not possible. The most likely explanation is that PEX2 can occupy a limited number of sites on the glycosomal membrane and any superfluous PEX2 is degraded.
Overexpression of truncated PEX2 is lethal in bloodstream forms
Flaspohler, Parsons and colleagues had previously shown that expression of
a version of PEX2 that lacked one transmembrane domain and the C-terminal RING
finger caused a partial defect in glycosomal protein localization in L.
major, even with a background of normal PEX2 expression
(Flaspohler et al., 1999;
Flaspohler et al., 1997
). These
studies were done when no inducible expression system was available for
Leishmania, and the cell lines showed no growth defects. We therefore
thought it might be possible to see rather more severe effects in trypanosomes
using an inducible gene expression system. To construct a gene encoding the
truncated trypanosome PEX2, we replaced the codon for glutamine 239 with a
termination codon, just as had been done for Leishmania. The
truncated gene pex2
r was then placed downstream of the
Tet-inducible RNA polymerase I promoter,
PEP1Ti. The construct was
transfected into 449 bloodstream trypanosomes and clonal cell lines selected.
When Tet was added, the expression of pex2
r mRNA was strongly
induced and a very marginal decrease in the growth rate of the parasites was
seen (Fig. 1B, open
diamonds).
We next placed the truncated gene downstream of a Tet-inducible T7
polymerase promoter, transfected the construct into bloodstream trypanosomes
expressing T7 polymerase, and selected clones in the absence of Tet. Upon
addition of Tet, the growth of the cells was severely impaired
(Fig. 1B, open circles),
although even here the truncated pex2r could not be detected.
Examination of the cells by immunofluorescence, using anti-PEX11 antibodies,
revealed only a low number of surviving trypanosomes with intact glycosomes
(punctate pattern), and cell fractionation studies with digitonin revealed no
mislocalised glycosomal enzymes after 24 hours
(Fig. 1C); samples of cells
grown with Tet for longer times were difficult to obtain.
Destruction of PEX2 mRNA causes cell-growth arrest in both
forms of the parasite
The experiments with dominant-negative PEX2 had not resulted in the
creation of living trypanosomes with glycolytic enzymes in the cytosol, as we
had expected from the results previously obtained with Leishmania. We
adopted an alternative approach, attempting to reduce the level of PEX2
expression. Attempts to knock out both genes by homologous recombination
failed, as had previously been reported with Leishmania. We therefore
expressed a PEX2 double-stranded RNA (dsRNA) under the control of a
Tet-inducible promoter. The expression of dsRNA in trypanosomes causes RNA
interference (RNAi): highly sequence-specific destruction of the homologous
mRNA (Ngô et al., 1998).
In normal cells, or in the transfected cells grown without Tet, two
PEX2 mRNAs were present at low abundance. When Tet was added, these
RNAs became undetectable within 13 hours in procyclic forms
(Fig. 1A); a control mRNA
(tubulin) was unaffected.
In bloodstream trypanosomes the destruction of PEX2 mRNA resulted in reduced growth within 24 hours (Fig. 1D). (We were unable to analyse the RNA in these cells because even after only 24 hours, it was not possible to isolate sufficient good-quality mRNA.) By the second day many cells looked abnormal and growth was strongly impaired. Procyclic trypanosomes grew relatively normally for about 2 days, but by the third day, cells of both stages were dying.
The computer model of trypanosome glycolysis predicts that bloodstream
trypanosomes lacking glycosomal membranes and grown in 5 mM glucose should be
able to maintain glycolysis, whereas parasites grown in 25 mM glucose (normal
bloodstream-form medium) will undergo lethal runaway hexose phosphorylation
(Bakker et al., 2000). We
tested the growth of the bloodstream forms over 3 days after Tet addition, in
a range of glucose concentrations (1, 2, 4, 5, 10, 25 mM). Growth inhibition
was similar at all concentrations (not shown). Control of glycolytic flux is
clearly not the only role of the glycosomal membrane in bloodstream
trypanosomes.
Depletion of PEX2 reduces the number of glycosomes in both
stages
We next examined the distributions of glycosomal matrix enzymes and a
glycosomal membrane marker, PEX11, by immunofluorescence. 24 hours after Tet
addition, most bloodstream-form cells were normal but some had fewer
glycosomes than usual (Fig. 2).
The matrix enzymes ALD, HXK (Fig.
2), PFK and GAPDH (not shown) still showed punctuate fluorescence;
cytosolic staining for matrix enzymes was never observed. Both enzymes
colocalised with the glycosomal membrane marker PEX11. Attempts to repeat
these experiments at later time points yielded essentially similar results,
but were much more problematic because of the number of dead cells.
Transmission electron microscopy of PEX2-depleted bloodstream trypanosomes was
also difficult because hardly any of them survived the fixation. The few
intact cells looked normal and the rest were lysed (not shown). These results
suggested that in all the living bloodstream-form trypanosomes, the glycosomal
matrix enzymes remained predominantly compartmented within the organelle. A
short treatment (5 minutes) with digitonin allowed the glycosomal compartment
to be left intact before immunofluorescence. In this case, there was no
staining of glycosomal enzymes (not shown), confirming that these were not
cytosolic.
Examination of PEX2-depleted procyclic trypanosomes, in contrast, showed that the mutation had the effects that were expected from a `silenced' PEX2. Trypanosomes grown in the absence of Tet showed the punctate PEX11 and GAPDH staining pattern typical for cells with intact glycosomes (Fig. 3, top row). After 60 hours of RNAi induction, the number of PEX11-labeled structures was drastically reduced in most of the cells and the GAPDH signal was spread throughout the cell and excluded from the glycosomal remnants (Fig. 3, middle and bottom rows). This suggests that PEX2 depletion does indeed prevent proper targeting of glycosomal matrix enzymes in trypanosomes.
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Glycolytic enzymes are abnormally distributed in cells lacking
PEX2
To confirm the effects of PEX2 depletion on glycosomal matrix enzymes, we
subjected the mutants to cell fractionation. In no case was the total enzyme
level affected by PEX2 reduction. Fig.
4A shows the effects on the distribution of HXK and PGI
activities. HXK activity was always found mainly in the organellar pellet
fraction, consistent with the results from the immunofluorescence in
bloodstream forms (Fig. 2). In
contrast, PGI activity showed a clear shift to the cytosolic fractions of both
bloodstream and procyclic cells. Unfortunately we could not confirm this by
immunofluorescence because suitable antibodies are not available. The
mis-localisation of PGI could therefore be due either to intrinsic cytosolic
location, or leakage from weakened organelles (or remnants) during the
fractionation procedure.
|
Western blots of three other glycosomal enzymes from digitonin-fractionated procyclic cells are shown in Fig. 4B. The pex2 procyclic forms were defective in targeting of ALD, PFK and GAPDH. No redistribution of any of these enzymes to the cytosol was detected in the bloodstream mutants, as seen by immunoflourescence (ALD shown in Fig. 2).
In Y. lipolytica pex2 mutants, levels of a surface and a secreted
protein were reduced by 80% (Titorenko et
al., 1997). We checked the synthesis and processing of the variant
surface glycoprotein (VSG) in bloodstream cells by pulse chase (5 minutes
pulse, 60 minutes chase); no differences could be detected between mutants
grown in the absence or the presence of Tet (data not shown; C. Guerra
Giraldez, PhD Thesis, University of Heidelberg, 2001). For procyclics, we
examined the level of the major surface protein EP for up to 3 days after
addition of Tet; the pex2 mutation also had no effect
(Fig. 4C).
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Discussion |
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ALD, GAPDH and PFK have basic isoelectric points and associate in
high-molecular-weight complexes (`cores') which sediment during centrifugation
even after dissolution of the glycosomal membrane
(Opperdoes, 1987). Their
cytosolic location in pex2 procyclic trypanosomes clearly indicated
an import defect. PGI has a neutral pI
(Parsons and Nielsen, 1990
),
does not associate with cores and is readily released from glycosomes during
fractionation [(Opperdoes,
1987
) and see Fig.
4, Tet columns]. The partitioning of PGI into the soluble
fraction in pex2 cells could therefore reflect either mis-targeting
or organelle fragility. HXK was always associated with the pellet, even in the
mutants, and retained a punctate distribution by immunofluorescence. HXK has a
PTS-2 signal (de Walque et al.,
1999
); it is unlikely to be imported into the PEX2-defective
glycosomes via this signal, so the most likely explanation is that an
alternative signal is present. This could be a membrane-targeting signal, or
hexokinase might be imported attached to a membrane protein; HXK has a strong
tendency to aggregate and stick to membranes
(Opperdoes, 1987
) (P. Michels,
personal communication).
Procyclic trypanosomes depend mainly upon amino acids and the mitochondrion
for ATP generation. (Although there is some glucose in our standard procyclic
media, proline is supplied as the principal carbon source.) The fact that
depletion of PEX2 is nevertheless lethal implies that pathways other than
glycolysis are essential and require compartmentation. Enzymes of fatty acid
beta oxidation, ether lipid biosynthesis, purine salvage, sterol synthesis and
pyrimidine synthesis are found in kinetoplastid glycosomes
(Michels et al., 2000),
although some of these are probably absent in bloodstream trypanosomes. A
properly assembled, intact organelle could be essential for the functioning of
these pathways or enzymes. As the procyclic parasites survive for at least 24
hours with defective compartmentation, the growth impairment seems likely to
be caused by gradual depletion of critical products or accumulation of toxic
intermediates.
In bloodstream trypanosomes, the effects of PEX2 depletion were much more
severe than in procyclics. Some parasites with abnormally low numbers of
glycosomes were seen but neither ALD, PFK nor GAPDH was detected in the
cytosol. Very similar results were obtained when we overexpressed a
C-terminally truncated, dominant-negative version of PEX2 in bloodstream
trypanosomes, as had been done in Leishmania
(Flaspohler et al., 1999).
Cell growth rapidly ceased and the resulting population was a mixture of few
normal-looking and mostly dead cells; no matrix enzyme relocalisation was
detectable. The most likely explanation for these observations is that in
PEX2-depleted bloodstream trypanosomes, a decrease in the number of glycosomes
can be tolerated, but as soon as overall import of enzymes fails or
the level of cytosolic enzymes rises above a critical threshold the
parasites die. The most likely cause of death is a failure of ATP production.
It may be that glycolysis cannot be maintained at an adequate rate when the
enzymes are in the cytosol. It is possible that import into the glycosome is
required for the glycolytic enzymes to assemble into a multienzyme complex,
allowing metabolite channeling, but no evidence of such channeling has ever
been found. The results of metabolic modeling indicate that dilution of the
glycolytic enzymes into the whole cell volume would have no effect at all on
the glycolytic rate. However, the regulatory and kinetic properties of
trypanosome glycolytic enzymes are unusual (for example, no feed-back control
is exerted on HXK or PFK) and may be adapted so that adequate glycolytic flux
can be sustained only when the enzymes are in a specialised (glycosomal)
environment. It has, for example, been suggested that if hexokinase and
phosphofructokinase were exposed to the ATP/ADP ratio of the cytosol, runaway
phosphorylation would ensue (Bakker et al.,
2000
). An alternative explanation is afforded by our observation
that hexokinase appears to remain glycosome-associated in pex2 cells.
If the glycosomal membrane represents a permeability barrier for hexose
phophates, the retention of hexokinase in the glycosomes would result in a
break in the pathway after the formation of glucose-6-phosphate.
Even minor mis-localisation of glycosomal enzymes is lethal in bloodstream trypanosomes. Furthermore, our results imply that compartmentation of glycosomal enzymes is essential for survival of procyclic T. brucei. Given the low sequence conservation between the PEX proteins of kinetoplastids and mammals, it will be worthwhile to look for specific inhibitors of glycosomal biogenesis.
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Acknowledgments |
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References |
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