Download RNA Interference of Signal Peptide-binding Protein SRP54 Elicits

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 277, No. 49, Issue of December 6, pp. 47348 –47357, 2002
Printed in U.S.A.
RNA Interference of Signal Peptide-binding Protein SRP54 Elicits
Deleterious Effects and Protein Sorting Defects in Trypanosomes*
Received for publication, July 31, 2002, and in revised form, September 19, 2002
Published, JBC Papers in Press, September 19, 2002, DOI 10.1074/jbc.M207736200
Li Liu, Xue-hai Liang, Shai Uliel, Ron Unger, Elisabetta Ullu‡, and Shulamit Michaeli§
From the Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel and the ‡Department of Medicine
and Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8022
Trypanosomes are protozoan parasites that have a
major impact on health. This family diverged very early
from the eukaryotic lineage and possesses unique RNA
processing mechanisms such as trans-splicing and RNA
editing. The trypanosome signal recognition particle
(SRP) has a unique composition compared with all
known SRP complexes, because it contains two RNA
molecules, the 7SL RNA and a tRNA-like molecule. RNA
interference was utilized to elucidate the essentiality of
the SRP pathway and its role in protein translocation in
Trypanosoma brucei. The production of double stranded
RNA specific for the signal peptide-binding protein
SRP54 induced the degradation of the mRNA and a loss
of the SRP54 protein. SRP54 depletion elicited inhibition in growth and cytokinesis, suggesting that the SRP
pathway is essential. The translocation of four signal
peptide-containing proteins was examined. Surprisingly, the proteins were translocated to the endoplasmic
reticulum and properly processed. However, the surface
EP procyclin, the lysosomal protein p67, and the flagellar pocket protein CRAM were mislocalized and accumulated in megavesicles, most likely because of a secondary effect on protein sorting. The translocation of
these proteins to the endoplasmic reticulum under
SRP54 depletion suggests that an alternative pathway
for protein translocation exists in trypanosomes.
The signal recognition particle (SRP)1 is an evolutionarily
conserved ribonucleoprotein (RNP) that exists in all three kingdoms (1–3). It couples the synthesis of membrane and secretory
proteins to the translocation across the eukaryotic endoplasmic
reticulum (ER) membrane, as well as the bacterial plasma
membrane and chloroplast thylakoid membranes (4).
In vitro studies have shown that SRP binds to the hydrophobic signal peptide as the N-terminal end of the nascent preprotein emerges from the ribosome, which results in a slowing or
pause of translation, a phenomenon termed “elongation arrest”
* This work was supported in part by a grant from the Israel Science
Foundation. The costs of publication of this article were defrayed in part
by the payment of page charges. This article must therefore be hereby
marked “advertisement” in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
§ Howard Hughes Medical Institute International Research Scholar.
To whom correspondence should be addressed: Faculty of Life Sciences,
Bar-Ilan University, Ramat-Gan 52900, Israel. Tel.: 972-3-5318068;
Fax: 972-3-5351824; E-mail: [email protected].
1
The abbreviations used are: SRP, signal recognition particle; RNAi,
RNA interference; RNP, ribonucleoprotein; Ffh, fifty-four homologue;
nt, nucleotide(s); PBS, phosphate-buffered saline; BiP, immunoglobulin
heavy chain-binding protein; PI, propidium iodide; GPI, glycosylphosphatidylinositol; hnRNP, heterogeneous nuclear ribonucleoprotein; N
domain, N-terminal domain; G domain, a Ras-like GTPase domain; M
domain, methionine-rich domain.
(5). SRP then targets the nascent chain-ribosome complex to
the ER membrane by interacting with a membrane-bound SRP
receptor. After this docking event that is GTP-dependent, the
translocation of the polypeptide takes place co-translationally
through the protein pore, the translocon (6, 7). The well defined
canine SRP complex is composed of a single RNA, the 7SL
RNA, and six proteins. The most studied protein of the SRP is
SRP54, which binds to the signal peptide. SRP54 also binds to
the SRP RNA and is a GTPase (8). SRP54 is a highly conserved
protein in nature. Much of what we know about this protein
comes from structural studies performed on the thermopile
bacteria Thermus aquaticus (9) and the Escherichia coli 54homologue (Ffh) protein (10). The Ffh is composed of three
domains: N-terminal domain (N domain), a Ras-like GTPase
domain (G domain), and a methionine-rich domain (M domain).
The N domain is a four-helix bundle that is closely associated
with the G domain. The G domain is a GTPase that contains a
motif unique to the SRP family of GTPases (11, 12). The N and
G domains of Ffh mediate the interaction of the SRP with
homologous N and G domains of FtsY, the SRP receptor.
The in vivo role of SRP in protein translocation was elucidated in bacteria and yeast. Disruption of the SRP pathway in
E. coli is lethal (13), most probably because of the defects in the
translocation of inner membrane proteins (14 –16). However,
the secretion of signal peptide-containing proteins such as
periplasmic or outer membrane proteins was only slightly affected (13, 14, 16, 17). Surprisingly, in yeast, different phenotypes were observed when the SRP pathway was disrupted. In
Saccharomyces cerevisiae SRP is not essential for cell growth,
although cells in which SRP was genetically disrupted grew
poorly (18). Moreover, the translocation of numerous secretory
and membrane proteins into the lumen of the ER is impaired.
However, different proteins show translocation defects of varying severity (18 –20). In Schizosaccharomyces pombe, the SRP
pathway is essential, because Srp54p depletion is lethal (21). In
another yeast strain, Yarrowia lipolytica, disruption of the two
7SL RNA-encoding genes is lethal (22), but SRP54 is important
but not essential for growth. The viability of SRP54-depleted
cells suggests that the SRP-independent pathway functions
well enough for survival (23).
Very little is known about the trypanosomatid SRP pathway.
Trypanosomes diverged very early in the eukaryotic lineage
(24); this provides an interesting system to study the evolution
of RNA-mediated processes, because many exotic and unique
processes were first described in trypanosomes including transsplicing (25) and RNA editing (26). The analysis of trypanosomatid 7SL RNA revealed that the RNA can be folded into the
canonical structure of eukaryotic 7SL RNA, except the Alu
domain, which is truncated, missing one of the loops, and most
importantly lacks the potential to form a pseudoknot (27). Most
intriguing is the finding that the trypanosomatid SRP complex
is composed of two RNA molecules: the 7SL RNA and a tRNA-
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This paper is available on line at http://www.jbc.org
RNAi of the Signal Peptide-binding Protein SRP54
like molecule, sRNA-76 in Trypanosoma brucei and sRNA-85 in
Leptomonas collosoma (28).2 Although we do not yet know the
function of this unique tRNA-like molecule, we hypothesize
that because it is a tRNA-like molecule, it may assist the
trypanosomatid 7SL RNA truncated Alu domain in the translational arrest function of the SRP. Another unusual feature
that was discovered for the L. collosoma 7SL RNA is that the
7SL RNA is found in two stable conformations: one enriched in
the ribosome fraction (7SL I) and the other, found in free SRP
(7SL II). The conversion of 7SL II to 7SL I is associated with a
novel RNA editing event of C to U in domain III of the RNA
(29).
Recently, RNA interference (RNAi) has become a very efficient means for deciphering the function of trypanosome gene.
RNAi is based on the degradation of mRNA by the production
of specific dsRNA (30). The dsRNA is cleaved to small interfering RNAs, which are 21–23 nt long that elicit the degradation
of the specific mRNA (30). dsRNA is produced in vivo either
from opposing T7 promoters, or in the form of a stem-loop
structure that is transcribed from the EP promoter (31, 32). In
both cases, the production of the dsRNA is regulated by the
tetracycline repressor. RNAi was first reported in trypanosomes for the silencing of tubulin, which resulted in the “fat
cells” phenotype (31, 33). Subsequently, this methodology was
used to silence different genes (32). Detailed studies were performed on the T. brucei exosome, mitochondrial topoisomerase
II, flagellum ontogeny, and RNA editing (34 –38)
Here, we used RNAi to examine the role of the SRP pathway
in protein sorting in trypanosomes. SRP depletion by RNAi
caused lethality, suggesting that this pathway is essential in
trypanosomes. Surprisingly, the signal peptide-containing proteins examined were translocated to the ER, and properly
processed, suggesting that an alternative translocation pathway must exist in trypanosomes. The secondary effect on protein sorting observed for procyclin EP, lysosomal protein p67,
and the flagellar pocket protein CRAM may stem from defects
in membrane integrity.
MATERIALS AND METHODS
Cell Growth and Transfection—Procyclic T. brucei strain 29-13 (obtained from Paul England’s laboratory, a gift from George Cross laboratory), which carries integrated genes for T7 polymerase and the
tetracycline repressor (39), were grown in SDM-79 medium supplemented with 10% fetal calf serum in the presence of 50 ␮g/ml hygromycin and 15 ␮g/ml G418. Cell growth was monitored by hemocytometer. To establish the cell lines with the dsRNA-expressing construct,
transformants were selected with 5 ␮g/ml phleomycin. For cloning,
individual cells were spread onto agarose plates as described in Ref. 40,
and the plates were incubated at 27 °C in a 5% CO2 atmosphere.
Individual colonies became visible after 5–7 days and were transferred
to medium for propagation in microtiter dishes and expanded in liquid
medium. Cells from cultures that showed the best characteristic phenotype upon tetracycline induction were grown and frozen. Every 2
weeks a new culture was started from the original frozen stock.
Oligonucleotides and Plasmids—9/40, 5⬘-CGTCTAGAACGTGAAGGAGTTTGTAAAT-3⬘, sense, from position 80 to 99 of the SRP54 coding
region, including a XbaI site (underlined), was used for PCR amplification of the 535 bp for cloning into the pJM326 and pLew100 vectors,
respectively, to generate the stem-loop construct (32). 10/40, 5⬘CCCAAGCTTTCTCTTCGAACAGTGCCGAT-3⬘, antisense, complementary to positions from 594 to 613 of the SRP54 coding region,
including a HindIII site, was used for PCR amplification of the 535 bp
for cloning into the pJM326 vector to generate the stem-loop construct
(32). 3/59, 5⬘-CGACGCGTTCTCTTCGAACAGTGCCGAT-3⬘, antisense,
complementary to positions from 594 to 613 of the SRP54 coding region,
including a MluI, was used for PCR amplification of 535 bp for cloning
into the pLew100 vector to generate the stem-loop construct (32, 39).
34/251, 5⬘-ATTCTGGAGGTGCACAACCT-3⬘, sense, positions from
2
L. Liu, H. Ben-Shlomo, Y-X. Xu, Y. Zhang, I. Goncharov, and S.
Michaeli, submitted for publication.
47349
1228 to 1247 of the SRP54 coding region, was used for cloning and
sequencing. 35/251, 5⬘-TTCCCCATTTGTTACTTTTC-3⬘, antisense,
complementary to positions from 164 to 183 relative to the stop codon
was used for cloning and sequencing.
Northern Analysis—Total RNA was prepared with Trizol reagent
and 20 ␮g/lane were fractionated on a 1.2% agarose, 2.2 M formaldehyde
gel. The RNA was visualized with ethidium bromide. The SRP54 mRNA
and tubulin mRNA were detected with [␣-32P]-random labeled probe
(Random Primer DNA Labeling Mix, Biological Industries, Co.). For
analyzing small RNAs, total RNA was fractionated on a 10% polyacrylamide gel containing 7 M urea. The RNA was transferred to a nylon
membrane (Hybond; Amersham Biosciences) and probed with [␥-32P]end-labeled oligonucleotide, as was previously described (41).
Assembly of T. brucei SRP54 Gene using Bioinformatics—SRP54
proteins from other organisms were obtained from SwissProt. The T.
brucei data base (Sanger, GenBankTM, and TIGR) was searched for
SRP54 using TBLASNN (42). Ten overlapping fragments were assembled with the GELMERGE program of the Genetics Computer Group
(GCG). To confirm the sequence, portions of the gene were amplified by
PCR and sequenced.
In Vivo Labeling and Immunoprecipitation—Procyclic cells were
grown at 27 °C in SDM-79 medium. The cells were washed twice with
PBS and resuspended to 108 cells/ml in pre-warmed (27 °C) Met/Cys
minus Dulbecco’s modified Eagle’s medium (Invitrogen). The cells were
labeled with 200 ␮Ci/ml L-[35S]Met/Cys (Hybond; Amersham Biosciences) for 5 min, and then chased for 5 or 15 min by a 10-fold dilution
in prewarmed complete SDM-79 growth medium. Labeled cells (107, 1
ml) were washed once with PBS, and solubilized in 1 ml of wash buffer
(50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40,
0.5% deoxycholate, 0.1% SDS) containing protease inhibitor mixture
(Sigma). The extract was precleared by centrifugation. Antibodies (BiP,
5 ␮l/107 cells; p67, 3–5 ␮l/107 cells) were bound to the Protein ASepharose beads (Santa Cruz Biotech) by rotating for 1 h at room
temperature, washed once with wash buffer, and resuspended to the
original volume in wash buffer. 50 ␮l of antibody-protein A-Sepharose
suspension was mixed with 0.5 ml of cell lysate at 4 °C for 1 h. After the
supernatant was removed, the pellet was washed four times with wash
buffer and once with TEN buffer 50 mM Tris-HCl (pH 7.5), 150 mM
NaCl, 5 mM EDTA. The bound proteins were separated on a 10%
SDS-polyacrylamide gel.
Immunofluoresence and Confocal Microscopy—The cells were
washed with PBS, mounted on poly-L-lysine-coated slides, and fixed in
4% formaldehyde, 0.04% glutaraldehyde in PBS at room temperature
for 30 min. Cells were blotted in 10% fetal calf serum in PBS at room
temperature for 30 min and then incubated with the primary antibodies
with 0.1% Nonidet P-40 for 1 h. For the EP surface staining, the cells
were fixed with 4% formaldehyde but not permeabilized with Nonidet
P-40. The BiP antibodies (anti-rabbit) (43) and monoclonal p67 antibodies (44) were diluted 1:200 and 1:1000, respectively. The CRAM (45)
antibodies and the anti-EP monoclonal antibodies (Cedarline) were
diluted 1:300 and 1:500, respectively. Antibodies from a rabbit chronically infected with trypanosomes (CI serum, kindly provided by R. G.
Nelson) were diluted 1:400. After the cells were washed with PBS, they
were reacted with fluorescein isothiocyanate (Jackson ImmunoResearch). To stain the nucleus and kinetoplast, the cells were incubated
with propidium iodide (PI) (10 ␮g/ml) for 5 min. The cells were imaged
by a Bio-Rad MRC 1024 upright confocal microscope with a kryptonargon ion laser. The confocal microscope image analysis was performed
using the Laser Sharp 3.0 application.
Western Analysis—The whole cell extract (106 cell equivalent per
lane) was fractionated on a SDS-PAGE (10% gel), transferred to NitroBind (Micron Separations, Inc.), and probed with the antibodies.
Anti-EP monoclonal antibodies (Cedarline), and anti-CRAM antibodies
(kindly provided by Mary Lee) were diluted 1:2000 and 1:1500, respectively. The anti-hnRNP D0 monoclonal antibodies (kindly provided by
Gideon Dreyfuss) were diluted 1:1000. The bound antibodies were detected with goat anti-rabbit or goat anti-mouse IgG bound to horseradish peroxidase and visualized by ECL (Amersham Biosciences).
RESULTS
Identification and Characterization of Trypanosomatid
SRP54 —We searched the T. brucei genome project for the
SRP54 homologue using the protein sequence data from human, E. coli, and S. cerevisiae. The gene sequence was derived
from four different pieces that were clustered and the sequence
was confirmed by PCR and sequencing. The alignment of the T.
brucei protein with its homologues is presented in Fig. 1. The
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RNAi of the Signal Peptide-binding Protein SRP54
FIG. 1. Sequence alignment of T. brucei SRP54 with its homologues. The alignment was performed using the ClustalW multiple sequence
alignment program and is illustrated using the program SeqVu 1.0.1. Identity is represented by the gray background and the structural domains
are indicated. Secondary structure elements are shown above the aligned sequences. The deviated residues are circled. PGB, the putative guanine
nucleotide dissociation stimulator-binding site, and PRBM, the putative RNA binding motif, are indicated. The sequences were obtained from the
following sources: T. b., T. brucei, GenBankTM accession number AY1 35212; L. m., L. major, GenBankTM accession number AY064402; H. sap.,
Homo sapiens, GenBankTM accession number U51920; S. cer., S. cerevisiae, GenBankTM accession number 20424; E. coli, GenBankTM accession
number P07019.
BLAST search revealed a close identity to mammalian, S.
cerevisiae, and E. coli proteins, 51.3, 44.8, and 30.8%, respectively. The protein sequence was also compared with the
SRP54 from Leishmania major that we have recently cloned
and sequenced (GenBankTM accession number AY064402). As
expected, the protein is mostly related to the L. major protein
(identity of 77.5% and similarity of 83.3%). The protein carries
the conserved functional N, G, and M domains. Most striking
are the three amino acid changes (circled in Fig. 1). Interestingly, the changes are trypanosomatid-specific and are not
found in any other SRP54 protein homologues that exist in the
data base. Moreover, these changes are found in the most
conserved positions of the protein (position 82 in the N domain,
position 222 in the G domain, and position 335 in the M domain). Surprisingly, the C terminus of the L. major protein is
longer by 32 amino acids compared with the T. brucei protein.
In addition, the L. major C-terminal domain carries a high
glycine and methionine content compared with other homologous domains, for instance, 15 glycines and 15 methionines in
L. major, compared with 9 glycines and 11 methionines in
Drosophila. The proximity of the C-terminal extension to the
hydrophobic groove of the M domain, and the high content of
methionine in this portion of the protein suggest that this
domain perhaps also participates in signal sequence binding.
In fact, this hypothesis is supported by data demonstrating
that the deletion of this extension from the mammalian SRP
abolishes cross-linking to the signal sequence in vitro, suggesting the involvement of the C-terminal sequence in signal peptide recognition (46). It is reasonable to speculate that the
longer Leishmania C terminus, highly enriched in methionines
expands the hydrophobic surface area, which functions in recognizing the trypanosomatid signal peptides. The distinct
amino acid changes in the three conserved positions found in
the two trypanosomatid species as well as the changes in the C
terminus of the protein may explain the failure of the mammalian translocation machinery to recognize and properly translocate trypanosomatid signal peptide-containing proteins
(47). However, the shorter C terminus in the T. brucei protein, compared with that of L. major, is intriguing and may
affect the ability of these related proteins to function
interchangeably.
Silencing of SRP54 Expression by RNAi—As a first step
toward understanding the essentiality of the SRP pathway for
trypanosomes and developing criteria for translocation defects
because of depletion of the SRP pathway, we alleviated this
pathway by silencing SRP54 using the RNAi methodology.
We used a construct that expresses the dsRNA in the form of
a stem-loop structure (32). After linearizing the construct, the
RNAi of the Signal Peptide-binding Protein SRP54
FIG. 2. Northern and Western analyses of SRP54 mRNA and
protein upon SRP54 silencing. A, Northern analysis of SRP54
mRNA. RNA was prepared from the parental strain (PS), and cells
carrying the RNAi construct, uninduced (⫺Tet), and after 1– 8 days of
induction (⫹Tet). Total RNA (20 ␮g) was subjected to Northern analysis
with a random-labeled SRP54 and tubulin probes. The transcripts of
SRP54 mRNA, dsRNA, and tubulin are indicated by arrows. B, Western
analysis of SRP54 protein. Whole cell extract was fractionated on a 10%
SDS-polyacrylamide gel and subjected to Western analysis with the
anti-yeast Srp54p antibodies (18), and antibodies specific for mammalian cytoplasmic RNP protein hnRNP D0 (48). SRP54 and hnRNP D0
proteins are indicated by arrows. PS, parental stain, ⫺Tet and ⫹Tet,
uninduced and induced cells carrying the RNAi construct, respectively.
1– 8 are the dates after induction.
DNA was used to transfect T. brucei 29-13 cells that express
the tetracycline repressor as well as the T7 polymerase (39).
The stem-loop RNA (dsRNA) was designed to contain 1620 nt
composed of 535 nt sense, a 550-nt loop derived from the
Pex11␤ gene, and 535-nt antisense RNA. The EP promoter,
which is under the control of the tetracycline repressor, is used
to transcribe this RNA. In addition, the construct also carries
the phleomycin resistance gene, which is under the control of
the T7 promoter and the rRNA spacer regions, which enable its
integration to the nontranscribed rDNA spacer (32). Because
the population of transformants is heterologous due to integration into different rDNA loci, the cells were cloned by selection
on agar plates. Different colonies were selected and the growth
was monitored after the addition of tetracycline. For further
analysis, we chose the clones whose growth was severely affected upon the addition of tetracycline.
The correlation between the growth defects and the level of
SRP54 mRNA was examined. Briefly, RNA was extracted from
the parental 29-13 cells and from the strain carrying the
SRP54 RNAi construct, before and after induction with tetracycline, and was subjected to Northern analysis with a probe
derived from the coding region of the SRP54. The results are
presented in Fig. 2A and indicate that 1–2 days after induction,
the mRNA was reduced by ⬃90% (determined by densitometric
analysis). The production of dsRNA was also observed in the
absence of tetracycline, and its production was slightly reduced
during the course of silencing. However, reduction in the level
of dsRNA did not change the reduction in the level of SRP54
mRNA. The degradation of SRP54 was specific because no
reduction was observed for tubulin mRNA (Fig. 2A).
To examine whether the reduction at the level of SRP54
mRNA was accompanied by a reduction at the level of the
protein, extracts from parental strain and cells carrying the
RNAi construct, induced and uninduced, were subjected to
Western analysis with antibodies raised against the yeast
SRP54 homologue. The results (Fig. 2B) indicate that there was
almost a 95% reduction at the level of the protein from the
second day of silencing. The level of SRP54 did not change
during the course of silencing (8 days). Antibodies to the mammalian cytoplasmic hnRNP D0 protein (48) that cross-reacts
with the T. brucei protein were used as a control to demonstrate the specific reduction at the level of SRP54.
47351
The Effects of SRP54 Silencing on Cell Growth, Cytokinesis,
and the Kinetoplast Content—The growth of cells carrying the
RNAi construct upon tetracycline induction was compared with
those cells carrying the RNAi construct (uninduced) and with
the parental strain. The results, presented in Fig. 3A, a, indicate that the growth of the induced cells was severely affected.
Three days after tetracycline induction the growth of the silenced cells ceased, whereas the uninduced cells grew at much
a higher rate than the induced cells but were slightly inhibited
compared with the parental strain. The growth inhibition of
the uninduced cells originates from “leaky” dsRNA production.
In other experiments (data not shown) massive death was
observed after 10 days. Interestingly, the inhibition of growth
was reversible; if induction was for 3 days and tetracycline was
removed after that time, the cells continued to grow (Fig. 3A,
b). However, if silencing was for 6 days, the cells never grew
again after tetracycline removal and eventually died. These
results indicate that SRP54 is essential for parasite growth.
The most remarkable phenotype of the SRP54-silenced cells
was visualized by confocal microscopy. Three parameters were
examined: nuclear content, number of kinetoplasts, and the
shape of the cells. The cells were subjected to immunofluorescence with CI antibodies described under “Materials and Methods.” The nucleus and kinetoplast were stained with PI. The
results are presented in Fig. 3, B and C. Major changes in the
shape of the cells were evident already 1 to 3 days upon the
addition of tetracycline. The different phenotypes observed
were: 1) cells lost their normal shape; 2) cells possessing detached flagella; and 3) multiple conjoined cells, two or more
cells that were physically connected and moving in different
directions. After 4 days of SRP54 silencing, most of the cells
contained more than a single nucleus. These phenotypes suggest that the silencing disrupted normal cytokinesis. At the
later stages of silencing (10 days or more), the cells were
rounded and contained numerous nuclei. Anucleate cells were
also observed and many of the cells lost their flagellum. At this
point these “monstrous” cells stopped moving and died. Interestingly, during later stages of induction (day 8 and onward),
the staining with CI serum and PI was yellow because of the
merging between the red PI staining and the green fluorescence of the antibodies (Fig. 3B). The yellow staining suggests
that either the nuclear envelope became more permeable to the
antibody or that during silencing, epitopes, which are normally
internal, were exposed, like in apoptotic cells. To examine
whether the cells undergo apoptosis, we analyzed the DNA
from induced and uninduced cells, but no signs for apoptotic
DNA degradation were observed in the silenced cells.
In asynchronous trypanosome culture, there are either cells
that carry one nucleus and one kinetoplast (1K1N), or cells
carrying 2N2K and therefore have completed mitosis and are
ready for cytokinesis. Whereas staining the cells with PI (Fig.
3C), we noticed that during silencing (starting from day 4)
multiple nuclei started to accumulate, which was not accompanied by kinetoplast multiplication, suggesting that uncoupling takes place between nuclear and kinetoplast duplication
and segregation. At later stages of induction we could observe
cells carrying four, even up to nine nuclei per cell with a single
kinetoplast. These results suggest that during silencing, both
cytokinesis and kinetoplast multiplication were severely
affected.
To correlate the deleterious effects of SRP54 silencing with
specific defects in protein translocation, we examined the processing of four different proteins harboring the signal peptide.
We chose for most of the analyses cells on the second day after
tetracycline induction, because this is the first day that the
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RNAi of the Signal Peptide-binding Protein SRP54
FIG. 3. A, growth curves of cells carrying the SRP54 RNAi construct. a, the growth of parental stain (PS) was compared with the
silenced cells. The number of cells of uninduced (⫺Tet) is designated
by filled triangles, of induced cells (⫹Tet) by filled squares, and the PS
by circles. The arrow indicates the time when tetracycline was added.
b, the reversibility of cell growth after the removal of tetracycline.
Growth was monitored as in A. The addition of tetracycline is indicated by an arrow. Cell growth in the presence of tetracycline is
designated by filled triangles. After 3 or 6 days the tetracycline was
removed (⫺Tet) (indicated in circles and squares, respectively) and
growth was monitored thereafter. B, changes in cell shape and kinetoplast during SRP54 silencing. Uninduced cells (⫺Tet) and induced
cells (⫹Tet) 1– 6, 7– 8, and 10 days after tetracycline induction were
fixed, permeabilized, and reacted with CI serum as described under
“Materials and Methods.” The nucleus and kinetoplast were stained
with PI. The cells were incubated with antisera obtained from a
rabbit that was chromically infected with T. brucei. The fluorescence
images were visualized in a confocal microscope. Detached flagellums
(DF) are indicated. C, the content of kinetoplast and nuclei upon
SRP54 silencing. Uninduced (⫺Tet) and induced cells (⫹Tet) 4 days
after tetracycline induction were stained with PI. The numbers of
kinetoplast (K) and nuclei (N) per cell are indicated. Scale bar, 5 ␮m.
level of SRP54 protein was minimal and no dramatic effect on
cell shape was yet observed.
Changes in the Distribution and Content of the Procyclin
Surface Protein EP during SRP54 Silencing—The first protein
examined was the glycosylphosphatidylinositol (GPI)-anchored
surface glycoprotein, the procyclin EP. EP contains up to 30
tandem repeats of glutamic acid and proline. The EP protein is
attached to the membrane by a complex glycosylated GPI anchor that is capped with sialic acid (49).
The EP gene encodes for a 15-kDa protein. The protein
undergoes different modifications including GPI anchoring and
glycosylation. The fully glycosylated protein migrates as a heterogeneous population of the 45–50-kDa protein on a 12%
polyacrylamide gel. The heterogeneity is derived from the addition of branched poly-N-acetyllactosamine to the GPI anchor.
Further contributing to heterogeneity is the sialylation of the
GPI branches by trans-sialidase when EP arrives at the cell
surface (50). A monoclonal antibody that recognizes the EP by
immunofluorescence and Western analysis was used to examine the effects of silencing on EP maturation and cellular localization. To this end, proteins prepared from uninduced and
induced cells were subjected to Western analysis (Fig. 4A). No
changes were observed between induced and uninduced cells.
The fully processed proteins formed during silencing can represent protein translocated by the residual level of SRP (less
than 10%) or most likely proteins that are translocated by an
alternative chaperone pathway as in yeast (18, 21). The putative precursor, 35-kDa polypeptide (designated PEP in Fig. 4A)
was also present in uninduced cells. Recent studies on EP
processing suggest that the EP precursor carrying the GPI
anchor is already modified with a short carbohydrate side
chain, suggesting that PEP is an ER-precursor (51).
To further explore the processing of EP under SRP54 silencing, we examined the cellular distribution of the protein.
Briefly, cells were fixed by paraformaldehyde and reacted with
anti-EP antibodies. This type of staining detects only the protein present on the surface of the parasite. The results, presented in Fig. 4B, indicate that while in the wild-type cells the
EP was uniformly distributed on the surface, forming a tight
and heavy coat, upon silencing, the coat became much thinner.
In the presence of detergent (Nonidet P-40) the antibodies can
enter the cell and stain the EP internally. Indeed, staining in
the presence of detergent revealed very strong punctated staining of the induced cells compared with uninduced cells that
showed only staining around the flagellar pocket (Fig. 4C),
suggesting that the protein is synthesized but cannot be properly translocated to the surface of the parasite. The punctated
staining observed during silencing is reminiscent of staining
observed during overproduction of the T. brucei TbRab2p protein that regulates intracellular membrane trafficking (52). If
the phenotype observed in SRP54 silenced cells is similar to
overexpression of TbRab2p, this suggests that the mislocalization observed for EP is because of a secondary defect that may
result from augmented production of ER transport vesicles.
The Translocation of BiP Takes Place during SRP54 Silencing—The BiP is an ER resident chaperone. SRP depletion in S.
cerevisiae but not in S. pombe affected the translocation of BiP
(18, 21). The distribution of BiP was examined upon silencing
by immunoprecipitation and confocal microscopy. The uninduced and SRP54-silenced cells on the second day after induction were subjected to pulse-chase labeling with a [35S]methionine/cysteine mixture. The extracts were subjected to
immunoprecipitation with anti-BiP antibodies. Even during a
short pulse-chase time, no preprotein was detected (Fig. 5A),
suggesting that the BiP is normally translocated during SRP54
silencing, like in S. pombe (21). To verify that BiP is normally
RNAi of the Signal Peptide-binding Protein SRP54
FIG. 4. The effect of SRP54 silencing on EP translocation. A,
Western analysis with anti-EP antibodies. Extracts (106 cells per lane)
were prepared from uninduced cells (⫺Tet) and induced cells 1, 3, and
6 days upon tetracycline induction (⫹Tet). After separating the proteins
in a 12% SDS-polyacrylamide gel, the blot was reacted with monoclonal
antibodies against EP. The size of the marker proteins is indicated in
kDa. The positions of the PEP (a putative ER precursor) and the mature
glycosylated protein (MEP) are indicated. B, immunofluorescence assay
(IFA) with anti-EP (surface staining). Cells were fixed with 4% formaldehyde for 20 min. The cells were visualized by indirect immunofluorescence after incubation with anti-EP antibodies. Uninduced cells
(⫺Tet) and induced cells (⫹Tet) on the second day after induction are
indicated. C, immunofluorescence assay with anti-EP (internal staining). Cells were stained as described in A, except that the cells were
permeabilized with 0.1% Nonidet P-40. Scale bar, 5 ␮m.
translocated during SRP54 depletion, the uninduced and silenced cells on the second day after induction were stained with
anti-BiP antibodies. The results, presented in Fig. 5B, demonstrate that in induced cells, BiP is still distributed within the
ER, suggesting that despite the SRP54 depletion, BiP is normally translocated to the ER. It should be noted that exactly
the same results were obtained when the cells were analyzed
on later days of induction, indicating that the ability to translocate the protein to the ER was maintained through the course
of silencing.
The Translocation of Lysosomal Protein p67 during SRP54
Silencing—To examine the defects of SRP 54 depletion on
47353
FIG. 5. Distribution of BiP in uninduced and induced cells. A,
immunoprecipitation of in vivo labeled BiP protein. Uninduced (⫺Tet)
and induced cells (⫹Tet) on the second day after tetracycline induction
were labeled with [35S]methionine/cysteine mixture (5-min pulse and 5or 15-min chase). Extracts were prepared and immunoprecipitation was
performed as described under “Materials and Methods.” The total cell
extracts and the immunoprecipitated products were analyzed on a 8%
SDS-polyacrylamide gel. The size of the marker proteins is indicated in
kDa. The specific immunoprecipitated product is indicated by an arrow.
B, immunofluorescence with anti-BiP antibodies. Uninduced cells
(⫺Tet) and induced cells (⫹Tet) on the second day after induction were
fixed and reacted with anti-BiP antibodies. The images were visualized
using the confocal microscope. Scale bar, 5 ␮m.
integral membrane protein, we examined the localization of
p67, which is a lysosomal protein that is highly glycosylated
(44). The protein is a type I transmembrane protein that harbors a luminal domain and short cytoplasmic domain. In bloodstream trypanosomes, p67 is synthesized as a 100-kDa glycoform (gp100) harboring an estimated 14 N-linked oligosaccharides. It is then transported to the Golgi where elongation of the N-glycans takes place. In procyclic form, p67 is
synthesized exclusively as the gp100 glycoform, which is transported directly to the lysosome (44).
To follow the translocation of p67 during silencing, we monitored the synthesis and processing of nascent p67. Cells on the
second day of silencing were subjected to pulse-chase with
[35S]methionine/cysteine. Extracts were prepared and subjected to immunoprecipitation. A major 100-kDa protein was
observed but the p67 precursor was hardly detected (Fig. 6A).
However, two additional immunoprecipitated products, 50- and
37-kDa polypeptides, were observed upon silencing. Interestingly, a mature glycosylated protein appeared in the induced
cells in amounts identical to the uninduced cells. The synthesis
47354
RNAi of the Signal Peptide-binding Protein SRP54
FIG. 6. Distribution of the lysosomal protein p67 upon SRP54 silencing. A, immunoprecipitation of p67. Uninduced cells (⫺Tet) and induced cells
(⫹Tet) on the second day after induction
were labeled with [35S]methionine/cysteine mixture (5-min pulse and 5-min
chase). The procedure is as in Fig. 5A. The
extracts and the immunoprecipitated
products were analyzed on a 10% SDSpolyacrylamide gel. The molecular mass
of marker proteins is indicated in kDa.
The specific immunoprecipitated products of the fully glycosylated glycoprotein
100, and two additional degradation products are marked by arrows. Lanes 1 and 2
are extracts from the uninduced and induced cells, respectively; lanes 3 and 4 are
immunoprecipitated products from uninduced and induced cells, respectively. B,
immunofluorescence with anti-p67 antibodies. Uninduced cells (⫺Tet) and induced cells (⫹Tet) on the second day after
tetracycline induction were fixed and reacted with anti-p67 antibodies (44). The
images were visualized using a confocal
microscope. Kinetoplast (K), nucleus (N),
and lysosome (L) are indicated. Scale bar,
5 ␮m.
of the fully glycosylated form under the silencing condition may
reflect either the residual activity of the SRP or the activity of
an alternative translocation pathway as already discussed. The
50- and 37-kDa polypeptides are strikingly similar to the
standard p67 degradation products that can be observed in
normal cells (44). However, these degradation products are
normally seen only after several hours of chase in procyclic
cells, whereas the duration of chase was only for few minutes in
the experiment presented in Fig. 6A. The degradation of p67 is
mediated by trypanopain, the major lysosomal thiol protease,
which also has to enter the secretory pathway. Perhaps trypanopain is also mislocalized to the same aberrant secretory compartment discussed above and cleavage takes place in the same
locale prior to its entry to the lysosome. Interestingly, however,
the majority of p67 is properly translocated to the ER and
glycosylated during SRP54 silencing, suggesting that also p67
can utilize an alternative pathway for translocation.
To investigate the effect of silencing on the processing of p67,
SRP54-silenced cells on the second day of induction were stained
with antibodies. The results indicate that in uninduced cells
staining was confined to the lysosome localized near the nucleus.
However, in induced cells, staining was not confined to a single
site but gave punctated staining, like in the case of EP (Fig. 4C).
The data therefore suggest that despite proper translocation to
the ER, the p67 protein is mislocalized and is also found in these
aberrant megavesicular structures. Also in the case of p67, the
same results were observed in the later days of silencing.
Processing and Localization of the Flagellar Pocket Protein
CRAM during SRP54 Silencing—The CRAM protein is the
trypanosome homologue to the human low density lipoprotein
receptor (45). CRAM is a type I membrane protein that is
concentrated in the flagellar pocket, an invagination of the cell
surface of trypanosomes where endocytosis and exocytosis take
place. CRAM has a predicted molecular mass of 130 kDa. The
glycosylated form of the protein migrates as a 200-kDa protein.
FIG. 7. Distribution of CRAM upon SRP54 silencing. A, Western
analysis was as described in the legend to Fig. 4A. The anti-hnRNPD0
was used as a control for equal loading. B, immunofluorescence of cells
with anti-CRAM antibodies. Uninduced cells (⫺Tet) and induced cells
(⫹Tet) on the second day after induction are indicated. The position of
the flagellar pocket is marked. Scale bar, 5 ␮m.
RNAi of the Signal Peptide-binding Protein SRP54
The protein harbors a transmembrane domain and a 41-amino
acid cytoplasmic extension. CRAM also possesses a large intracellular domain with a cysteine-rich repeat.
To investigate the effect of silencing on the processing of
CRAM, Western analysis was performed on whole cell lysates
from induced and uninduced cells. The results presented in Fig.
7A indicate that the protein was synthesized at the level identical to the level in the uninduced cells and the protein was
perfectly glycosylated. This result resembles the data obtained
for EP. We further examined the localization of CRAM by
immunofluorescence. The results, presented in Fig. 7B, reveal
major differences between the uninduced and induced cells.
Whereas in uninduced cells, the CRAM was confined to the
flagellar pocket, in the induced cells the staining was punctated and not concentrated in the flagellar pocket similar to the
staining observed for p67 and EP, indicating defects in localization but not in translocation and glycosylation.
DISCUSSION
The signal peptide-binding protein SRP54 was identified in
trypanosomatids and compared with its homologues from E.
coli to mammals. RNAi of the SRP54 gene was utilized to
elucidate the essentiality of the SRP pathway and its role in
protein translocation in T. brucei. SRP54 depletion elicits dramatic effects on cell shape, cytokinesis, and kinetoplast duplication. Surprisingly, all the signal peptide-containing proteins
examined were translocated to the ER during silencing. However, a secondary effect on protein sorting was observed for the
GPI-anchored surface protein (EP) and two type I integral
membrane proteins (p67 and CRAM). These proteins were
translocated to the ER and properly glycosylated but mislocalized. The translocation of EP, BiP, p67, and CRAM to the ER
upon depletion of SRP54 indicates that an alternative protein
translocation pathway exists in trypanosomes. However, the
mislocalization of these proteins suggests that the proper sorting of these proteins was not compromised by this alternative
pathway. The secondary effect on protein sorting may stem
from defects in the translocation of polytopic membrane proteins. In the absence of antibody reagents to T. brucei multispanning membrane proteins, we could not examine the effect
of SRP54 silencing on the translocation of such proteins.
The T. brucei SRP54 is highly related to its homologous
proteins in other organisms. It is more related to its mammalian counterpart than to yeast, as was observed for the 7SL
RNA (53). Like all SRP54 proteins, the trypanosome protein
carries the conserved N, G, and M domains. However, three
amino acid changes were observed in the most conserved positions (circled in Fig. 1). Interestingly, all these changes convert
negatively charged amino acids to uncharged amino acids.
Functional data are necessary to determine whether these
changes contribute to the peculiar behavior of the signal peptide-containing proteins in the in vitro mammalian translocation system (47).
The Cellular Implications of SRP54 Depletion in T. brucei—
This work joins an increasing number of studies that have
utilized RNAi in trypanosomes to elucidate the function of
essential genes. In all cases, when dsRNA production was
induced, the target mRNA decreased after 24 h and the protein
levels dropped to ⬃10% after 24 to 48 h of induction (35, 36). In
this study, the mRNA dropped to less than 10% after 2 days.
This reduction in mRNA level was accompanied by a similar
reduction in the level of the protein. The depletion of SRP54
resulted in the inhibition of growth, similar to growth defects
that were observed for other essential genes (35, 36). In all
cases reported, growth continued for the first few days upon
tetracycline induction. However, when essential genes were
47355
silenced, as in this study, the cells stopped growing (after 3
days) and the number of cells either remained constant or was
reduced thereafter. Microscopic examination of cells after 10
days of induction (as shown in Fig. 3B) indicated that all cells
had aberrant morphology, either multinucleated or anucleated
and eventually died. These results suggest that like topoisomerase II (36) and many of the exosome genes (35), SRP54
is also an essential gene.
Note that the dramatic morphological changes observed here
were not observed in any other organisms where SRP function
was previously disrupted. However, this is the first study that
was carried out in an organism that lacks a tight cell wall. In
the absence of cell wall depletion of the surface coat, GPIanchored or polytopic membrane proteins that interact with
the subpellicular microtubules may change the shape of the cell
and lead to the production of monster and huge round cells
observed in this study.
How Does SRP Depletion Affect Protein Translocation in
Trypanosomes Compared with Other Organisms—The translocation defects observed during the silencing of the SRP54 in T.
brucei are different from those observed during SRP depletion
in S. cerevisiae. Four proteins were chosen to examine the
translocation defects: 1) DPAP-B, a type II integral membrane
protein; 2) the Kar2p, the ER resident chaperone (Kar2p ⫽
BiP); 3) invertase; and 4) the secreted protein carboxypeptidase. The translocation of DPAP and Kar2p was severely affected by SRP depletion but carboxypeptidase was normally
translocated to the ER. These data suggest that the proteins
differ in their dependence on the SRP pathway and some proteins can utilize the alternative chaperone pathway (18, 20).
Indeed, the translocation of carboxypeptidase was completely
dependent on the chaperone pathway, because it was blocked
in a chaperone pathway mutant, Sec63 (20). Likewise, in S.
pombe, SRP54 depletion severely affected the translocation of
acid phosphatase but had only minor effects on the secretion of
BiP (21). These results suggest that yeast process their proteins by two active pathways: the SRP pathway and the chaperone pathway. The choice between these pathways for any
given protein is unpredictable because the same protein BiP is
translocated in S. pombe by the chaperone pathway (21) and in
S. cerevisiae (18) and Y. lypolytica (23) by the SRP pathway.
The pathway specificity is conferred by the hydrophobic core of
signal sequences (54).
The presence of these two pathways for protein translocation
explains why depletion of the SRP pathway is not lethal in S.
cerevisiae. However, structural features of some preproteins
may preclude efficient translocation using a post-translational
route. These proteins must therefore depend on the SRP pathway for translocation. In S. cerevisiae, all essential proteins can
be translocated via the chaperone pathway; however, the cells
grow slowly and are defective. In contrast, in S. pombe the
pathway is essential, because essential proteins are dependent
on the SRP pathway for translocation.
The intriguing finding that the translocation of four proteins
from different locales (ER, surface of the parasite, lysosome,
and flagellar pocket) may suggest that in trypanosomes all
signal peptide-containing proteins do not utilize the SRP pathway. The SRP pathway may exclusively translocate polytopic
membrane proteins. This is indeed the case in E. coli. In E. coli
the essentiality of the SRP pathway stems from the absolute
dependence on the SRP pathway for processing of polytopic
membrane proteins (15, 16). If this is also the case in trypanosomes and because trypanosomes diverged very early from the
eukaryotic lineage (24) this may suggest that the primordial
function of the SRP particle was to translocate polytopic membrane proteins and only later in the course of SRP evolution
47356
RNAi of the Signal Peptide-binding Protein SRP54
was it adjusted to translocate signal peptide-containing proteins. It will therefore be of great interest to describe all the
proteins that are severely affected during SRP54 silencing by
comparing the nascent translated proteins in uninduced and
SRP54-silenced cells by two-dimensional gels. Such a proteomic approach will define all the trypanosome proteins that
are dependent on the SRP pathway, and that their translocation cannot be compromised by the chaperone alternative
pathway.
We cannot exclude the possibility that all signal peptidecontaining proteins are properly translocated to the ER during
SRP54 silencing. However, the fact that four different signal
peptide-containing proteins were translocated to the ER during
silencing may suggest that the major deficiency in the silenced
cells originates from defects in polytopic membrane protein
biogenesis. Defects in the processing of such membrane proteins could change the composition of both intracellular membranes such as the ER and Golgi apparatus and the plasma
membrane and affect the intracellular trafficking. Such secondary defects may cause the accumulation of the post-ER
membranous structures observed for p67, EP, and CRAM. The
mislocalization observed for these proteins is therefore a secondary effect that may result from sorting defects. Interestingly defects in secretion of signal peptide-containing proteins
(␤-lactamase) were also observed in E. coli SRP receptor, FtsYdepleted cells but these effects were secondary and appeared
only after the clear effects on the membrane proteins (16, 55).
At present there are no antibody reagents to examine the
processing of polytopic membrane proteins in T. brucei and this
analysis awaits the development of a reporter fusion protein
carrying multispanning membrane segments. Interestingly,
however, membrane proteins carrying an N-terminal signal
peptide and a single TM, such as the CRAM and p67, were
translocated to the ER under SRP54 depletion but were
mislocalized.
The Trypanosome SRP Complex—At present, we know very
little about the T. brucei SRP protein composition. We have
recently identified three additional SRP protein homologues,
SRP19, SRP72, and SRP68 in the T. brucei genome project.3 It
is currently unknown whether the trypanosome 7SL RNA, like
in yeast and mammals, is first assembled with five of the SRP
proteins in the nucleolus (56, 57). Experiments are in progress
to silence the genes encoding for SRP19 and SRP72. These
experiments will enable us to determine whether, like in yeast,
the assembly of the trypanosome particle takes place in the
nucleolus (56). Interestingly, the silencing of SRP54 did not
affect the level of 7SL RNA. In addition, when SRP complex
from the silenced cells was analyzed on sucrose gradient only a
marginal difference was observed in its S value compared with
uninduced cells (data not shown). These results indicate that
the SRP subparticle can be stably formed in the absence of
SRP54. Srp54p depletion in S. cerevisiae also did not significantly change the S value of the particle. However, no SRP
complex was observed upon depletion of Srp68p, Srp72p,
Srp21p, and Srp14p (20). These differences stem from the fact
that these four proteins are essential for the assembly of the
preparticle in the nucleolus, whereas SRP54 joins the preparticle in the cytoplasm (56).
The most intriguing property of the trypanosome particle is
its RNA composition. The trypanosome SRP complex carries a
tRNA-like molecule that is a genuine constituent of the particle
(28).2 The function of this unique tRNA-like molecule is unknown. We proposed that it may carry the arrest function of the
particle and substitute for the truncated domain I of the
trypanosomatid 7SL RNA. However, we cannot exclude the
possibility that this RNA may also be involved in the transport
of the particle to the cytoplasm, because domain I of 7SL RNA
was shown to be involved in the transport of SRP from the
nucleolus to the cytoplasm (58).
This is the first study to decipher the in vivo function of SRP
in trypanosomes. The unique RNA composition of the SRP and
the essentiality of the SRP translocation pathway make it a
very attractive drug target to fight the diseases caused by this
important family of parasites.
Acknowledgments—We thank Paul Englund for providing the constructs to perform the RNAi silencing. We especially thank James
Bangs for valuable comments and advice during the course of this
research, for excellent comments on the manuscript, and for the generous gift of anti-BiP and anti-p67 antibodies. We thank Peter Walter
for the anti-Srp54p antibodies, and Mary Lee for the CRAM antibodies.
We thank Leonid Roitman for help in the confocal microscopy.
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