Báo cáo khoa học: Characterization of Trypanosoma brucei PEX14 and its role in the import of glycosomal matrix proteins pptx

Characterization of Trypanosoma brucei PEX14 and its rolein the import of glycosomal matrix proteins Juliette Moyersoen1,*, Jungwoo Choe2,*, Abhinav Kumar3,†, Frank G.. Michels1 1 Resear

Characterization of Trypanosoma brucei PEX14 and its role

in the import of glycosomal matrix proteins

Juliette Moyersoen1,*, Jungwoo Choe2,*, Abhinav Kumar3,†, Frank G J Voncken4, Wim G J Hol2,3 and Paul A M Michels1

1

Research Unit for Tropical Diseases, Christian de Duve Institute of Cellular Pathology and Laboratory of Biochemistry,

Universite´ Catholique de Louvain, Brussels, Belgium;2Department of Biochemistry, University of Washington, Seattle, USA;

3

Biomolecular Structure Center, Howard Hughes Medical Institute, University of Washington, Seattle, USA;

4

Zentrum fu¨r Molekulare Biologie der Universita¨t Heidelberg, Germany

It has been shown previously in various organisms that the

peroxin PEX14 is a component of a docking complex at the

peroxisomal membrane,where it is involved in the import of

matrix proteins into the organelle after their synthesis in the

cytosol and recognition by a receptor Here we present a

characterization of the Trypanosoma brucei homologue of

PEX14 It is shown that the protein is associated with

gly-cosomes,the peroxisome-like organelles of trypanosomatids

in which most glycolytic enzymes are compartmentalized

The N-terminal part of the protein binds specifically to

TbPEX5,the cytosolic receptor for glycosomal matrix

proteins with a peroxisome-targeting signal type 1 (PTS-1)

TbPEX14 mRNA depletion by RNA interference results,in

both bloodstream-form and procyclic,insect-stage T brucei,

in mislocalization of glycosomal proteins to the cytosol The mislocalization was observed for different classes of matrix proteins: proteins with a C-terminal PTS-1,a N-terminal PTS-2 and a polypeptide internal I-PTS The RNA inter-ference experiments also showed that TbPEX14 is essential for the survival of bloodstream-form and procyclic trypano-somes These data indicate the protein’s great potential as

a target for selective trypanocidal drugs

Keywords: trypanosome; glycosome biogenesis; PEX14; protein–protein interactions; RNA interference

All protists grouped in the order Kinetoplastida have a

unique form of metabolic compartmentation: the majority of

the glycolytic enzymes is present in organelles called

glyco-somes [1,2] This compartmentation has been studied in

detail in the kinetoplastid Trypanosoma brucei,the causative

agent of African sleeping sickness When present in the

mammalian bloodstream,this parasite is totally dependent

on glycolysis for its ATP supply as it lacks a functional

Krebs’ cycle and the mitochondrial system for oxidative

phosphorylation In contrast,the procyclic trypomastigote

living in the midgut of the tsetse fly relies more on

mitochondrial activity Because of its importance for the

mammalian infective stage,glycolysis has been indicated as a

validated target for the design of drugs against these

parasites The unique manner in which the pathway is organized in these organisms,and the observation that the structure and kinetic properties of most of their glycolytic enzymes display many special features,may allow the development of trypanocidal drugs that act through blocking specifically glycolysis in the parasites without interfering with the metabolism of the host [3]

The glycosome is an organelle related to peroxisomes [2,4,5] Peroxisomes are compartments of eukaryotic cells that may harbour different enzymatic systems in organisms belonging to distinct taxonomic groups,but all peroxisomes have in common a number of metabolic functions such as

an involvement in the metabolism of peroxides,fatty acids and ether lipids [6] These functions are also shared by kinetoplastid glycosomes,which however,have glycolysis as their most distinctive feature Considerable evidence is available to indicate that the presence of intact glycosomes and the proper targeting of glycolytic enzymes to the organelles is essential for kinetoplastid parasites [7–10] Therefore glycosome biogenesis may also be a good target for drug interference

At least 23 proteins,called peroxins (PEX),have been identified in yeasts and mammalian cells as factors involved

in the biogenesis of peroxisomes (reviewed in [11–14]) Peroxisomal matrix proteins are synthesized on free ribo-somes in the cytosol,and are translocated across the peroxisomal membrane usually without any form of pro-cessing The proteins to be imported are recognized by a cytosolic receptor,either PEX5 or PEX7,depending on their peroxisome-targeting signal,PTS-1 or PTS-2,respectively PTS-1 is a C-terminal tripeptide -SKL or a variation thereof

Correspondence to P A M Michels,ICP-TROP 74.39,

Avenue Hippocrate 74,B-1200 Brussels,Belgium.

Fax: + 32 27626853,Tel.: + 32 27647473,

E-mail: michels@trop.ucl.ac.be

Abbreviations: ALD,aldolase; GAPDH,glyceraldehyde-3-phosphate

dehydrogenase; PEX,peroxin; PTS,peroxisome-targeting signal;

PYK,pyruvate kinase; RNAi,RNA interference; Tet,tetracycline;

TIM,triosephosphate isomerase.

*Note: These authors contributed equally to this study.

Present address: Plexxikon,Berkeley,California,USA.

Note: The novel nucleotide sequence data published here have been

deposited in the EMBL-EBI/GenBank and DDBJ databases and are

available under accession number AJ512212.

(Received 20 December 2002,revised 7 March 2003,

accepted 18 March 2003)

PTS-2 is the motif -R/K-L/V/I-X5-H/Q-A/L- close to the

N-terminus The receptor with its cargo docks at the

peroxisomal membrane to a protein complex comprising

PEX14,PEX13 and (so far only identified in yeast) PEX17

Several other peroxins are involved in the subsequent steps of

the import The import of matrix proteins seems to involve a

cascade of interactions between the cargo-loaded receptors

and different components of the import machinery: docking,

translocation,cargo release and receptor recycling In

addition,some proteins are directed to the peroxisomes by

a polypeptide-internal sequence (I-PTS),but so far,no

specific,common motif could be identified for such I-PTS

PEX14,a protein associated with the peroxisomal

mem-brane,has been identified in various yeasts,mammalian cells

and plants as a component of the docking complex and a

point of convergence of the PEX5- and PEX7-dependent

import pathways [15–17] The N-terminal part of PEX14

interacts with the repeated motifs WXXXF/Y of PEX5 [18]

and with the SH3 domain of PEX13 through a proline-rich

motif [15,17] Interaction of PEX14 with PEX7 has also been

shown [15] PEX14 behaves as an integral peroxisomal

mem-brane protein in Hansenula polymorpha [19] and humans [17]

and as a tightly bound peripheral peroxisomal membrane

protein exposed to the cytosol in Saccharomyces cerevisiae

[15] However,no membrane-spanning domains in PEX14

have been unambiguously identified and it was observed in

mammalian cells and S cerevisiae that both the N-terminal

and the C-terminal domain are at the cytosolic face of the

peroxisomal membrane [15,16,20] The prediction of a

membrane-anchoring subdomain is therefore speculative

Several peroxins involved in glycosome biogenesis have

been identified in the kinetoplastids Leishmania and

Trypanosoma[8,21–23] Previously, we have characterized

the T brucei PTS-1 receptor, TbPEX5,and its interaction

with glycosomal matrix proteins [22] We have now cloned

also the gene encoding PEX14 of T brucei Very recently,

Furuya et al [24],too,reported the identification of

trypanosome homologue of PEX14 They showed that

decreasing the level of this peroxin in procyclic T brucei,by

RNA interference,compromises import of a glycosomal

matrix protein and renders glucose toxic to the cells In this

paper,we describe a more detailed analysis of the role of

PEX14 in glycosome biogenesis in both bloodstream-form

and procyclic trypanosomes Moreover,we present data on

the interaction of TbPEX14 with TbPEX5

Experimental procedures

Organisms and growth conditions

Bloodstream and procyclic form of T brucei 427 (cell line

449 [25]) were cultured and transfected as described before

[25] The lines were not cloned These phleomycin-resistant

parasites express the tet repressor from the chromosomally

integrated plasmid pHD449 and are metabolically

indistin-guishable from the wild type [25]

Construction of expression clones; production

of recombinant proteins PEX14 and PEX5

A short potential PEX14 sequence was recognized in the

database of the T brucei (strain TREU927/4) genome

project The corresponding fragment was amplified on

T bruceistrain 427 genomic DNA and used as a hybridi-zation probe to screen a genomic library prepared of this strain in phage kGEM11 (Promega) [26] Each part of the gene was sequenced at least once in both directions,using a Beckman CEQ 2000 sequencer (Beckman Instruments, Inc.)

In order to overexpress TbPEX14 in Escherichia coli, the gene was introduced in the expression vector pET28a (Novagen) that directs the production of the protein fused

to a N-terminal sequence including a histidine tag E coli strain BL21(DE3) which has the T7 RNA polymerase gene under the control of the lacUV5 promoter [27] Also a fragment containing the first 146 amino acids (PEX14-N) was expressed,without tag To this end,the corresponding 5¢ part of the gene and the complete T brucei PEX14 gene were amplified by PCR using two sets of oligonucleo-tides: sense 5¢-GACCATGGCTTTGCTGCTGTCGGG-3¢; 5¢-GTGGATCCTAATCCCTCCAGTCC-3¢ and antisense 5¢-GAGGATCCATGTCTTTGCTGCTG-3¢; 5¢-CGAA GCTTTCAAGCTGCCTCGC-3¢,respectively The sense primers contain a NcoI or BamHI site (underlined) adjacent

to a sequence corresponding to the 5¢ end of the PEX14 gene,and the antisense primers are complementary to a gene-internal region or the terminal coding region of the gene,followed by a BamHI or HindIII restriction site (underlined),respectively PCR was performed using Taq DNA polymerase (TaKaRa biomedicals) The amplified fragments were purified and ligated in the pGEM-T Easy vector (Promega) After checking their sequence,the amplified fragments were liberated from the recombinant plasmid by digestion with NcoI and BamHI or BamHI and HindIII,respectively,and ligated in the expression vector pET28a (Novagen) E coli BL21(DE3) was then trans-formed with the recombinant pET28a-TbPEX14-N or pET28a-TbPEX14 plasmid Only this latter construct directs the production of a fusion protein bearing an N-terminal extension of 20 residues including a His6-tag At

a later stage,a point mutation was introduced in the vector pET28a-TbPEX14-N in order to fuse the vector-encoded histidine tag also to the N-terminus of the TbPEX14-N sequence This plasmid,pET28a-TbPEX14-N,H, was also introduced in E coli BL21(DE3) and used for bacterial expression of recombinant protein TbPEX14-N,H

Cells harbouring a recombinant plasmid were grown at

37C in 50 mL of Luria–Bertani medium supplemented with 30 lgÆmL)1 kanamycin Isopropyl thio-b-D -galacto-side was added to a final concentration of 1 mMwhen the culture reached an D600nmof approximately 0.5 to induce the expression of the protein and growth was continued for

3 h Cells were collected by centrifugation (3000 g,10 min,

4C) and resuspended in cell lysis buffer containing 50 mM sodium phosphate,pH 8,100 mM NaCl and a protease inhibitor mixture (Roche) Cells were lysed by two passages through an SLM-Aminco French pressure cell (SLM Instruments Inc.) at 90 MPa The lysate was centrifuged (12 000 g,15 min,4C) and TbPEX14-N,H or TbPEX14 was purified from the soluble cell fraction Nucleic acids were removed by treatment with 250 units Benzonase (Merck) for 30 min at 37C and protamine sulfate (0.5 mgÆmL)1) followed by centrifugation for 10 min at

12 000 g The His-tagged proteins were further purified by

metal affinity chromatography (TALON resin,Clontech)

followed by anion-exchange chromatography (column

20HQ) and gel filtration (Superdex 200) to at least 90%

purity

Gel-retardation, gel filtration

Purified TbPEX5 [22] and TbPEX14-N,H were taken up

in 5 mM Tris/HCl,pH 7.0,100 mM NaCl and 1 mM

dithiothreitol and incubated for 30 min at room

tempera-ture The sample was loaded on a 4–20% polyacrylamide

gel (Novagen) and run under nonreducing and

nondena-turing conditions for 90 min at 140 V with a Tris-glycine

buffer of pH 8.5 The gel was stained with a gelcode blue

stain solution (Pierce)

Gel-filtration chromatography was performed on

Super-dex 200 HR 10/30 (Amersham Biosciences) with 25 mM

Tris/HCl,pH 7.0,100 mMNaCl and 1 mMdithiothreitol at

a flow rate of 0.4 mLÆmin)1 at 4C A total volume of

100 lL of sample was loaded on the column Six runs were

performed with TbPEX5 alone, TbPEX14-N,H alone and a

mixture of TbPEX5 and TbPEX14-N,H at various molar

ratios

Protein concentrations were determined by the Bradford

method using bovine serum albumin as standard [28] This

method was also used in experiments involving subcellular

fractionation (see below)

Construction of trypanosome expression vectors; RNAi

To induce RNA interference (RNAi) in trypanosomes

by the production of a double-stranded RNA,a 800-bp

fragment [PEX14-(2–269)] from the PEX14 open-reading

frame was cloned in the sense orientation upstream of the

pSP72 stuffer fragment [29] and in the antisense orientation

downstream of the stuffer The

PEX14-(2–269)-stuffer-PEX14-(2–269) was cloned into the polylinker downstream

of the tetracycline (Tet) inducible PARP promoter of the

trypanosomatid vector pHD677 (giving pRP14) [25]

For induction of double-stranded RNA,cells were

cultured in the appropriate medium [25] containing

250 ngÆmL)1of Tet for bloodstream-form and 5 lgÆmL)1

of Tet for procyclic-form cells Cultures were diluted daily to

2· 105cellsÆmL)1for bloodstream-form trypanosomes or

1· 106cellsÆmL)1for procyclic trypanosomes Cell

densi-ties were determined using a cell counting grid (Merck) and

growth curves were plotted as the product of the cell density

and the total dilution vs time

Subcellular fractionation by treatment of cells

with digitonin

Trypanosomes (108) were washed twice in a buffer

contain-ing 25 mM Hepes,pH 7.4,250 mM sucrose and 1 mM

EDTA,and then resuspended in the same buffer Aliquots

of a suspension of intact cells were diluted to a protein

concentration of 1 mgÆmL)1in HBSS buffer (Invitrogen)

Subsequently,variable amounts of digitonin were added to

the different aliquots followed by an incubation of 4 min at

25C The suspensions were centrifuged at 12 000 g for

2 min Aliquots of supernatants were used for Western

blotting with rabbit polyclonal antisera raised against

T brucei pyruvate kinase (PYK,antiserum used at a dilution of 1 : 100 000),glyceraldehyde-3-phosphate dehy-drogenase (GAPDH,at 1 : 150 000),aldolase (ALD,at

1 : 150 000) and triosephosphate isomerase (TIM,at

1 : 10 000) Blots were processed for antigen detection using a peroxidase-conjugated affinity-purified rabbit anti-serum (Rockland) followed by a chemiluminescence-based detection system (ECL,Amersham Biosciences)

Immunofluorescence Untreated procyclic trypanosomes or treated for 3 days with Tet were fixed and stained as described [30] using rabbit polyclonal anti-T brucei ALD,GAPDH,PEX14 and PEX11 antisera A synthetic peptide of the N-terminal part

of TbPEX14 (DGKSKPEVEH),coupled to keyhole limpet hemocyanin,was used to raise a polyclonal antiserum in rabbit The specificity of the antiserum was checked using a Western blot prepared after SDS/PAGE with a lysate of

E coli cells expressing TbPEX14-N,H The single band detected has the molecular mass of the PEX14 fragment and was absent from noninduced bacteria The anti-ALD Ig were purified on Protein A beads,coupled with Alexa-FluorTM 488 (Molecular Probes) following the manufac-turer’s instructions Detection of GAPDH,PEX14 and PEX11 was performed with Cy3-linked secondary antibody (Amersham,Biosciences) The software used was OPEN-LABTM(Improvision) andADOBEPHOTOSHOP

Results

T brucei PEX14 interacts specifically with T brucei PEX5

We have cloned and sequenced the PEX14 homologue of

T brucei strain 427,as described in Experimental proce-dures The gene codes for a polypeptide (TbPEX14) of 365 amino acids,with a calculated molecular mass of 39 806 Da and a predicted isoelectric point of 6.07 The sequence is nearly identical to that reported recently by Furuya et al [24] for the PEX14 of another strain (EATRO 110) Only one substitution was observed: a valine at position 156 instead of the alanine reported in [24] In yeast,human,and Arabidopsis,it has been shown that the N-terminal part of PEX14 is responsible for the interaction with PEX5 [16,31,32] This N-terminal domain of the polypeptide of each of these organisms is followed by a stretch of hydrophobic amino acids that is presumably involved in the protein’s anchoring to the membrane Also in PEX14 of

T brucei a series of hydrophobic residues is found at the corresponding region of the polypeptide (amino acids 148– 166,LVIGAGAAVIGGFAAFKAF) Because this stretch may affect the solubility of the protein,we preferred to use only the soluble N-terminal segment of the molecule for

in vitrointeraction studies between TbPEX14 and TbPEX5

To this end,the first 146 amino acids of TbPEX14 were overexpressed in E coli as a fusion protein with a His-tag called TbPEX14-N,H and purified (see Experimental pro-cedures) TbPEX5 was overexpressed and purified as described by de Walque et al [22]

Gel retardation assays under nondenaturing and non-reducing conditions were performed to ascertain the

association of the proteins TbPEX5 and TbPEX14-N,H

(Fig 1) The proteins were mixed and incubated for 30 min

before subjected to electrophoresis In lanes 1 and 7

(Fig 1A), TbPEX5 and TbPEX14-N,H alone were,

respectively,loaded on the gel From the second lane to

the sixth,mixtures of TbPEX5 and TbPEX14-N,H are

present,with their ratio increasing from 1 : 0.6 to 1 : 6.8

These lanes show a shifted band supposedly corresponding

to the complex formed by the two proteins (band 3) As the

molar ratio increases,more unbound TbPEX14-N,H is seen

in the upper part of the gel (Fig 1A,band 5) To confirm

the composition of the various bands,the proteins were

extracted from the native gel and analysed by SDS/PAGE

(Fig 1B) Indeed both proteins form a complex and run

together as they are present in the same band 3 On the

native gel,band 4 (lane 6) seems to represent some

TbPEX5 which does not bind to TbPEX14-N,H even in

the presence of excess of the latter This could be explained

by release of some TbPEX5 from the interacting complex

during migration However,when proteins from band 4

were extracted from the native gel and subjected to SDS/

PAGE,not only TbPEX5,but also some TbPEX14-N,H

was located in the band This may be due to diffusion of

some complexed protein as the band migrates in the native

gel Band 5 contains the excess of TbPEX14-N,H

In a second experiment,the same two components were

also mixed in different molar ratios and analysed by

gel-filtration chromatography The elution profiles of the

protein mixture, TbPEX5 and TbPEX14-N,H, contained

an early eluting species,which was not detectable when each

protein was analysed individually This high molecular mass

complex consists of TbPEX5 and TbPEX14-N,H as

con-firmed by SDS/PAGE analysis SDS/PAGE analyses of

corresponding peak fractions confirmed the presence of both

proteins in the shifted peak whereas the unbound TbPEX5

or TbPEX14-N,H was found as the late peak in all elution

profiles (Fig 2) From the experiments presented in Figs 1 and 2,we conclude that the N-terminal part of TbPEX14 (TbPEX14-N,H) directly interacts with TbPEX5 in vitro

Destruction of PEX14 mRNA by RNA interference (RNAi)

is lethal for trypanosomes The effect of suppression of TbPEX14’s expression on the parasite’s growth rate and glycolytic enzyme localization was studied Double-stranded RNA corresponding to essential genes is deleterious for trypanosomes because it induces specifically the degradation of the mRNAs by a process called RNA interference (RNAi) [33] A construct that leads to the production of double-stranded TbPEX14 RNA in T brucei was prepared as described in Experimen-tal procedures The linearized plasmid DNA was inserted into a transcriptionally silent region of the genome by homologous recombination The production of the double-stranded RNA was induced by the addition of Tet and resulted,in bloodstream-form trypanosomes,in reduced growth within 24 h followed by death of parasites In contrast,the effect on procyclic-form trypanosome growth was less acute Indeed,a decrease in cell number could only

be seen after 4 days of the Tet system’s induction (Fig 3A) Whereas all cells of the procyclic culture were killed,this appeared not to be the case for the culture of bloodstream-form trypanosomes This will be discussed below

Western blotting using lysates of bloodstream-form trypanosomes and a polyclonal antiserum raised in rabbits against a synthetic peptide (see Experimental procedures) showed that PEX14 is a low abundance protein This observation is similar to that reported by Guerra-Giraldez

et al [34] for T brucei PEX2 Attempts to confirm the decrease of the PEX14 protein as a result of RNAi remained inconclusive On Northern blots made with RNA from procyclic trypanosomes,however,low levels of PEX14 mRNA could be detected,which decreased upon induction

of RNAi,whereas the concentration of control mRNA (for the glycolytic enzyme enolase) remained unchanged (Fig 3B) The effect of RNAi was more difficult to show for bloodstream-form T brucei because the level of PEX14 mRNA in these cells was significantly lower (not shown)

In order to determine the influence of the reduction of the expression of TbPEX14 on the import of glycosomal matrix proteins,the subcellular distribution of glycolytic enzymes was analysed This was carried out by treating the cells with increasing concentrations of digitonin This detergent forms insoluble complexes with sterols,thus permeabilizing the different cellular membranes in a selective manner The plasma membrane,having a higher sterol content than the glycosomal membrane,will have its integrity affected at

a lower concentration of digitonin than the glycosomal membrane [34–37] Therefore,enzymes sequestered in glycosomes will only be released from the cells at higher concentrations of the detergent than enzymes present in the cytosol The appearance of the enzymes outside the cells was assayed by Western blotting (Fig 4) In procyclic-form trypanosomes,0.05 mgÆ(mg protein))1 affects only the plasma membrane,as indicated by the release of the cytosolic marker PYK from the cells (into the supernatant fraction),whereas glycosomal enzymes (GAPDH,ALD and TIM) remain in the cells (pellet fraction,not shown) In

Fig 1 Gel-retardation assay of TbPEX5 and TbPEX14 interaction,

under nondenaturing and nonreducing conditions (A) Purified His 6

-tagged TbPEX5 and a protein comprising the N-terminal part of

TbPEX14 (TbPEX14-N,H) were loaded on the gel separately (first and

last lane) or mixed together (lane 2–6) The molar ratios of TbPEX5/

TbPEX14-N,H run in the various lanes are indicated underneath the

gel (B) SDS/PAGE analysis of native gel bands Band 1 corresponds

to TbPEX5 (first lane),band 2 to TbPEX14-N,H (last lane), band 3 to

the complex formed by the two proteins (lane 3),and bands 4 and 5

correspond to noncomplexed TbPEX5 and TbPEX14-N,H,

respect-ively (lane 4 and 5).

Fig 2 Gel-filtration chromatography of TbPEX5 and TbPEX14 interaction (A) Purified His 6 -tagged TbPEX5 and TbPEX14-N,H were loaded separately (first and last profile) or mixed together in various molar ratios: 1 : 0.6,1 : 0.9,1 : 1.7,1 : 3.4 (from bottom to top) Absorbance,OD,of the column eluate was monitored at 280 nm The peaks at an elution volume of 12 mL and 15 mL correspond to TbPEX5 and TbPEX14-N,H, respectively (B) SDS/PAGE analysis of the different peak fractions The numbers at the top of the gel correspond to the different peaks of the elution profile.

Fig 3 Effect of PEX14-specific RNAi on the growth of bloodstream-form(BF) or procyclic-form(PF) T brucei (A) The growth profiles of cells transfected with the plasmid producing double-stranded RNA of TbPEX14 were determined by growing the cells in the absence (j) or in the presence of Tet (r) (250 ngÆmL)1for BF and 5 lgÆmL)1for PF) Shown are cumulative cell numbers (determined as described in Experimental procedures) (B) Northern analysis of total RNA from uninduced (–Tet) and induced for 24 h (+Tet) procyclic-form trypanosomes Each lane contains 10 lg total RNA.

contrast,the glycosomal enzymes were only released at a

higher concentration of digitonin,necessary to permeabilize

the glycosomal membrane In trypanosomes grown for

3 days with Tet,the subcellular distribution of enzymes was

clearly affected: part of the GAPDH (a PTS-1 protein),

ALD (a PTS-2 protein) and TIM (an I-PTS protein) is

already released from the cells at concentrations of digitonin

that leave the glycosomal membrane intact A similar,

partially cytosolic localization of these three enzymes was

observed when bloodstream-form cells were treated for 24 h

with Tet (data not shown) The partial mislocalization of all

three enzymes in these cells clearly shows the involvement of

PEX14 in the targeting of PTS-1,PTS-2 and I-PTS proteins

to the glycosome

To further analyse the effect of the destruction of PEX14

mRNA on the parasite,we performed immunofluorescence

experiments (Fig 5) First we confirmed the glycosomal

localization of TbPEX14 Indeed,trypanosomes labelled

with anti-TbPEX14 presented a punctuate pattern and a

colocalization of the protein with the glycosomal enzyme

ALD in wild-type cells We next examined the distribution

of glycosomal matrix enzymes and a glycosomal membrane

marker,PEX11 (Fig 5B,C) In cells that were not treated

with Tet,ALD and GAPDH,and ALD and PEX11,

respectively,colocalized and showed a punctuate

fluores-cence pattern corresponding to a glycosomal localization

However,many cells treated for 3 days with Tet were

already severely affected as concluded from their round

shape and disintegration of the nucleus (data not shown)

Cells with apparently normal morphology were analysed

In the first +Tet panel (Fig 5B),both ALD and GAPDH

show a clear cytoplasmic localization In the second panel, cells at an intermediate stage are shown,where ALD is still present in glycosomes while most of the GAPDH is present

in the cytoplasm In Fig 5C,the nontreated trypanosomes have the same punctuate pattern as the wild-type cell in Fig 5A Together,these data indicate that TbPEX14 depletion does prevent proper targeting of glycosomal matrix enzymes Surprisingly,the Tet-treated cells showed a partial cytoplasmic localization of PEX11 similar to that of ALD

Discussion

In peroxisomal matrix protein import,PEX14 seems to be involved in the first event occurring at the peroxisomal membrane The cytosolic receptors,PEX5 and PEX7,

Fig 4 Subcellular fractionation by treatment of cells with digitonin.

Intact procyclic-form T brucei cells were incubated for 4 min at

con-centrations of digitonin as indicated The release of PYK,ALD,

GAPDH and TIM from the cells was assayed after centrifugation of

the treated cell suspensions and the preparation of a Western blot of

the resulting supernatants The subcellular distribution of PYK,ALD,

GAPDH and TIM is shown for procyclic-form cells grown without

(–Tet) or with Tet for 3 days (+Tet).

Fig 5 Effects of PEX14 depletion on the distribution of glycosomal proteins of T brucei, as determined by immunofluorescence (A) Sub-cellular localization of TbPEX14 in wild type (WT) procyclic trypan-osomes ALD is shown in green and PEX14 in red (B) Effects of TbPEX14 depletion on matrix protein import ALD is shown in green, and GAPDH in red (C) Effects of TbPEX14 depletion on glycosomal matrix and membrane proteins in procyclic trypanosomes ALD is shown in green,and PEX11 in red In panels B and C,cells were grown

in the presence (+Tet) or the absence (–Tet) of 5 lgÆmL)1Tet for

3 days Control experiments using only one antibody at a time and looking in two different channels have been performed to eliminate possible artefacts (not shown).

loaded with their PTS-1 or PTS-2 proteins,respectively,

dock at the peroxisomal membrane through PEX14 In this

respect,some studies showed that the N-terminal part of

PEX14 interacts with pentapeptide motifs,WXXXF/Y,

present in several copies in the N-terminal domain of all

PEX5s [22,32,38] In this paper, in vitro experiments showed

that also in T brucei the N-terminal part of TbPEX14

(amino acids 1–146) interacts specifically with TbPEX5

This result is consistent with a role of PEX14 in glycosomal

matrix protein import

While this manuscript was in preparation,Jardim et al

[39] reported the cloning and characterization of the

Leishmania donovani PEX14 homologue The leishmania

PEX14 was shown to be tightly associated with the

glycosomal membrane Moreover,similar to our work,

these authors showed by an in vitro analysis that the

LdPEX14 binds to the previously characterized LdPEX5

[39] with a binding constant Kdof 2.75 lM,and that the

amino-terminal domains of the two peroxins were

respon-sible for this interaction

The role of TbPEX14 in glycosome biogenesis received

further confirmation by analysing the subcellular

distribu-tion of the glycosomal enzymes GAPDH,ALD and TIM

upon reduction of the PEX14 mRNA level RNAi

treatment of T brucei resulted in the release of part of the

glycosomal proteins from the cell at lower concentrations of

digitonin than in untreated cells (Fig 4) We interpret this

observation as the retainment of newly synthesized proteins

in the cytosol due to the inability to import matrix proteins

by the fraction of growing and dividing glycosomes The

miscompartmentation of PTS-1 and PTS-2 proteins

(GAP-DH and ALD,respectively) observed in both the procyclic

and bloodstream-form trypanosomes when PEX14

expres-sion is affected,strongly suggests that also in T brucei,this

peroxin is the point of convergence of both matrix proteins

import pathways Highly interestingly,also TIM,which has

no PTS-1 or PTS-2,is mislocalized in these cells In

trypanosomes transfected with different parts of the TIM

gene fused to the gene of a reporter protein,the import

signal was located in a 22-residue peptide in the center of the

primary structure of TIM (S de Walque & P A M

Michels,unpublished data) Moreover,we have not been

able to show an interaction between T brucei TIM and

PEX5 in vitro under conditions that allowed PEX5 to

interact specifically with a PTS-1 protein [22] To our

knowledge,no evidence for the involvement of PEX14 in

I-PTS protein import has been described so far The

mechanism of I-PTS protein import in the peroxisomes is,at

present,not known We propose that I-PTS proteins such as

TbTIM enter glycosomes by piggy backing,involving the

formation of a heteromeric complex with a PTS-1 or PTS-2

protein (see also [40,41]) Indeed, our observation of an

altered TIM compartmentation in RNAi-treated cells is in

line with such a mechanism This hypothesis is currently

under investigation in our laboratory Surprisingly,also

PEX11,a glycosomal membrane protein [8],was partially

shifted to the cytosol At present,it is not clear yet if this

should be interpreted as an involvement of PEX14 in the

insertion of PEX11 into the membrane Alternatively,it

could be imagined that reduction of (functional) glycosomes

due to PEX14 depletion results in aberrant distribution of

PEX11 (and TIM) in the cell

The study of the importance of TbPEX14 for the parasite has shown that the suppression of peroxin expression by RNAi leads to a reduction in the growth rate of T brucei, both of the bloodstream and the procyclic form,followed by death of the cells Therefore, TbPEX14 appears to be an essential protein for T brucei However,the reduction of growth rate appears in the bloodstream-form cells after already 24 h whereas the effect on growth of the procyclic form is seen only after 4 days of treatment Indeed, bloodstream-form trypanosomes are entirely dependent

on glycolysis and glycosomes for their ATP supply From

a computer simulation of the metabolism,it was concluded that an intact glycosomal membrane and proper enzyme compartmentation are of vital importance for these cells to compensate for a lack of activity regulation of hexokinase and phosphofructokinase by feedback loops [9] In contrast, procyclic cells have a highly branched metabolic network by which the harmful effects of the unregulated kinases may be alleviated In addition,the partially cytosolically located pentose-phosphate pathway in procyclics [42,43] may pro-vide an extra-glycosomal shunt for glucose catabolism that partially overcomes the problems caused by the miscom-partmentation of glycolytic enzymes Thus a mistargeting of glycosomal enzymes could result in a more severe pheno-type in bloodstream-form trypanosomes than in procyclics Similar differences between bloodstream-form and procyclic

T bruceihave been observed when PEX2 was targeted by RNAi [34]

Also surprising was the observation that the culture of bloodstream-form trypanosomes,in contrast to that of procyclic cells,was not entirely killed within 4 days after RNAi induction This could be attributed to the develop-ment of cells that became nonresponsive to RNAi In three experiments we observed that the number of cells increased again after 4 days (see Fig 3A) Indeed,the spontaneous development of such RNAi revertants has been observed by

us and others also in other cases and appears to occur more frequently in bloodstream-form trypanosomes than in procyclic cell lines [44] The effect may be due to either a rearrangement of the Tet repressor gene [45] or to deletion

of the target gene insert [44]

Furuya et al [24],too,reported recently the cloning and sequencing of the gene and confirmed the glycosomal localization of the encoded protein These authors demon-strated that disruption of PEX14 function by RNAi resulted

in death of trypanosomes of both bloodstream and procyclic-form cultures Interestingly,removal of simple sugars from the medium allowed the procyclic trypano-somes to survive We interpret these data as additional support for our hypothesis that proper compartmentation is particularly important during growth on sugars to prevent the unrestrained accumulation of sugar-phosphates that would otherwise result from the lack of regulation of sugar kinases,as mentioned above [9] During growth on other carbon sources such as amino acids,trypanosomes do not rely on glycosomes for catabolism and,consequently,lesser harm may occur to the cell

Glycolysis in bloodstream-form trypanosomes is consid-ered as a valid and highly appropriate target for the design

of trypanocidal drugs,because this process is of vital importance for the energy supply of the parasites and the sequestering of their glycolytic pathway into glycosomes is

different from that in the host where it is a cytosolic process.

This compartmentation endows the parasite enzymes with

unique structural and kinetic features [3] It has now also

been shown that the compartmentation itself is essential to

the trypanosomes [8,9,34] (this paper) This vital importance

of proper compartmentation and intact organelles together

with the very low level of conservation of peroxins provide

additional promising drug targets: specific compounds may

be designed that,in a selective manner,would interfere

effectively with glycosome biogenesis,without affecting

peroxisome biogenesis in the cells of the human host The

amino-acid sequence of T brucei PEX14 has only 31%

identity with that of its human homologue,and both we and

Furuya et al [24] showed that this peroxin is an essential

protein for both bloodstream and procyclic-form

trypano-somes Therefore,PEX14 is a new,potentially good target

for drugs that would act by disrupting the specific

inter-actions between TbPEX14 and other peroxins,e.g TbPEX5

or other T brucei peroxins still to be identified and

characterized (e.g PEX13,PEX7)

Acknowledgements

This research was supported by grants from the Belgian Fonds de la

Recherche Scientifique Me´dicale (FRSM) and the Interuniversity

Attraction Poles – Federal Office for Scientific,Technical and Cultural

Affairs Juliette Moyersoen is a recipient of a scholarship from the

Fonds a` la Recherche dans l’Industrie et l’Agriculture (Belgium) We

thank Dr Barbara Bakker (Amsterdam,the Netherlands) for

stimula-ting discussions and critical reading of the manuscript We also thank

Dr Julius Lukes (Ceske Budejovice,Czech Republic) for raising an

antiserum against PEX14.

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