Supplemental Material can be found at:

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 31, pp. 24131–24140, July 30, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

Target of Rapamycin (TOR)-like 1 Kinase Is Involved in the

Control of Polyphosphate Levels and Acidocalcisome
Maintenance in Trypanosoma brucei*□ S

Received for publication, March 5, 2010, and in revised form, May 3, 2010 Published, JBC Papers in Press, May 21, 2010, DOI 10.1074/jbc.M110.120212
Teresa Cristina Leandro de Jesus‡§1, Renata Rosito Tonelli‡, Sheila C. Nardelli‡, Leonardo da Silva Augusto‡,
Maria Cristina M. Motta¶, Wendell Girard-Dias¶, Kildare Miranda¶储, Paul Ulrich§2, Veronica Jimenez§2,
Antonio Barquilla**, Miguel Navarro**, Roberto Docampo§3, and Sergio Schenkman‡4
From the ‡Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo, São Paulo 04023-062,
Brazil, the §Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens,
Georgia 30602, the ¶Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21949-900,
Brazil, the 储Diretoria de Programas, Instituto Nacional de Metrologia Normalização e Qualidade Industrial, Duque de Caxias,
Rio de Janeiro 25250-020, Brazil, and the **Instituto de Parasitología y Biomedicina “López-Neyra,” Consejo Superior de
Investigaciones Científicas, Granada 18100, Spain

Target of rapamycin (TOR) kinases are highly conserved pro- tsetse fly vector (Glossina spp.), trypanosomiasis represents an

Downloaded from www.jbc.org at CAPES, on September 1, 2010

tein kinases that integrate signals from nutrients and growth important public health problem and has a strong impact in the
factors to coordinate cell growth and cell cycle progression. It economic development of that region. T. brucei has a complex
has been previously described that two TOR kinases control cell life cycle involving different morphological and functional
growth in the protozoan parasite Trypanosoma brucei, the caus- stages. These parasites alternate between insect and mamma-
ative agent of African trypanosomiasis. Here we studied an lian hosts. The adaptation to these diverse environments
unusual TOR-like protein named TbTOR-like 1 containing a requires rapid changes in gene expression to fulfill metabolic or
PDZ domain and found exclusively in kinetoplastids. TbTOR- morphological changes (1). This parasite also has the ability to
like 1 localizes to unique cytosolic granules. After hyperosmotic survive a wide range of environmental conditions as it
stress, the localization of the protein shifts to the cell periphery, progresses through its life cycle, including extreme fluctuations
different from other organelle markers. Ablation of TbTOR-like in external osmolarity (2).
1 causes a progressive inhibition of cell proliferation, producing Target of rapamycin (TOR)5 proteins are evolutionarily con-
parasites accumulating in the S/G2 phase of the cell cycle. served protein kinases that integrate information from energy
TbTOR-like 1 knocked down cells have an increased area occu- levels, mitogenic signals, and nutrient supplies regulating cell
pied by acidic vacuoles, known as acidocalcisomes, and are growth accordingly. Studies on mammalian cells and yeast sig-
enriched in polyphosphate and pyrophosphate. These results naling pathways have shown that nutrient starvation inhibits
suggest that TbTOR-like 1 might be involved in the control of TOR activities, which results in G1 cell cycle arrest, and triggers
acidocalcisome and polyphosphate metabolism in T. brucei. a stress response program leading to a blockade of translation
initiation. Similar stress responses can be observed in cells
treated with rapamycin, an immunosuppressant drug, which
African trypanosomiasis, or sleeping sickness, is a disease binds to FKBP12, a prolyl-isomerase, forming a complex with
caused by the parasitic protist Trypanosoma brucei that affects the TOR kinase (reviewed in Refs. 3 and 4).
a half-million people in sub-Saharan Africa. Transmitted by the Two distinct aspects of cell growth are regulated by two func-
tionally different TOR kinases in T. brucei, termed TbTOR1
* This work was supported, in whole or in part, by National Institutes of Health and TbTOR2. TbTOR1 and TbTOR2 function in two distinct
Grant AI-077538 (to R. D.). This work was also supported by grants from TOR-containing multiprotein complexes (TORC) conserved
Fundação de Amparo à Pesquisa do Estado de São Paulo, Fundação de along eukaryote evolution, named TORC1 and TORC2.
Amparo à Pesquisa do Estado do Rio de Janeiro, Conselho Nacional de
Desenvolvimento Cientı́fico e Tecnológico e Instituto Nacional de Ciência TbTOR1, located in the nucleus, controls the synthesis and
e Tecnologia de Vacinas do Conselho Nacional de Desenvolvimento Cien- accumulation of cell mass through TORC1 signaling, whereas
tı́fico e Tecnológico, Brazil. TbTOR2, associated with the mitochondrion or endoplasmic
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. S1–S5.
reticulum, controls actin cytoskeleton organization and endo-
1
Supported in part by a training grant from the Ellison Medical Foundation to cytosis through TORC2 (5). Only this latter is sensitive to rapa-
the Center for Tropical and Emerging Global Diseases. mycin, contrary to what is observed for mammalian cells and
2
Supported by a postdoctoral fellowship from the American Heart
Association.
yeast, in which TORC1, but not TORC2 is sensitive to rapamy-
3
To whom correspondence may be addressed: Center for Tropical and
Emerging Global Diseases University of Georgia, 500 D. W. Brooks Dr., Ath-
5
ens, GA 30602. Tel.: 706-542-8104; Fax: 706-542-9493; E-mail: rdocampo@ The abbreviations used are: TOR, target of rapamycin; TORC, TOR complex;
cb.uga.edu. PCF, T. brucei procyclic form(s); BSF, T. brucei bloodstream form(s); poly P,
4
To whom correspondence may be addressed: Universidade Federal de São polyphosphate; DIC, differential interference contrast; DAPI, 4,6- dia-
Paulo, R. Botucatu 862 8°A, 04023-062 São Paulo, SP, Brazil. Tel.: 55-11- midino-2-phenylidole; RNAi, RNA interference; PBS, phosphate-buffered
5575-1996; Fax: 55-11-5571-5877; E-mail: [email protected]. saline.

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T. brucei TOR-like 1 Kinase

cin (6). Surprisingly, two other sequences encode putative BamHI and HindIII sites (underlined nucleotides, respectively)
kinases with high domain similarity to TOR kinases in T. brucei: sites at opposite ends of the DNA fragments. Fragments were
TbTOR-like 1 and TbTOR-like 2; one of them, TbTOR-like 1, is cloned into pCR 2.1 TOPO (Invitrogen) and subcloned into the
not found in TbRaptor- or TbAVO3-containing complexes (5). BamHI and HindIII sites of the p2T2-177 vector (20). The plas-
The presence of additional TOR kinases would suggest that in mids were linearized with NotI and introduced into PCF 29-13
T. brucei, these enzymes could have acquired additional roles to and BSF 90-13 by electroporation. Log phase PCF (2.5 ⫻ 107) or
those carried out for TORC1 or TORC2, possibly related to BSF (1 ⫻ 107) were washed with Cytomix buffer (21) and elec-
their adaptation to different hosts and environments. troporated with two pulses in the presence of 10 ␮g of purified
In this work we investigated the role of TOR-like 1 protein in plasmids in a 4-mm cuvette at 1.5-kV voltage and 25-micro-
T. brucei. Among Eukarya TOR kinases, TbTOR-like 1 pos- farad capacitance. After a 24-h recovery in medium, the cells
sesses a unique PDZ domain, which is thought to be involved in were selected with the addition of 2.5 ␮g/ml phleomycin. After
protein-protein interactions, mediating binding of a class of selection, the induction of double-stranded RNA was obtained
submembranous proteins to membrane receptors and ion by adding 1 ␮g of fresh tetracycline/ml of culture every time the
channels (7). We show that RNAi knockdown produces cells cells were diluted.
with enlarged acidocalcisomes rich in pyrophosphate (PPi) and Antibodies—Antibodies against Tb TOR-like 1 (fragment 2)
polyphosphate (poly P), which is a polymer of a few to hundreds were generated by immunizing mice with a recombinant pro-
of phosphate units bound by phosphoanhydride bonds (8). Aci- tein expressed in pET 14b containing a 513-bp fragment that
docalcisomes are organelles known to accumulate poly P in T. was amplified by PCR using the primers 5⬘-CTCGAGTCTGG-
brucei as well as most protists (9). Poly P is known to regulate TGGCGACGAGGCTG and 5⬘-AAGCTTAACTTCCCCCC-

Downloaded from www.jbc.org at CAPES, on September 1, 2010

the activity of mammalian TOR (10) and is important for cellu- CTTGTCGGAG. The recombinant protein was obtained in
lar adaptation to different stresses (11). Acidocalcisomes were inclusion bodies and purified by chromatography on nickel
first characterized in T. brucei (12) and Trypanosoma cruzi (13) chelating resin in the presence of 6 M urea followed by prepar-
and are electron-dense membrane-bounded vesicles rich in cal- ative SDS-PAGE. Fragment 3 antibodies were obtained as
cium, sodium, zinc, and magnesium, in addition to PPi and poly described (5).
P. These components and several membrane pumps and cation Preparation of Whole Cell Extracts and Western Blot Analysis—
exchangers present in the membrane of acidocalcisomes (14 – PCF and BSF were harvested by centrifugation (1,500 ⫻ g for 10
17) are implicated in the control of cell size in response to dif- min), washed twice in PBS, and resuspended in PBS with 1%
ferent osmotic stresses. We provide evidence that TbTOR-like SDS and proteinase inhibitors (2 mM benzamidine, 1 ␮g of leu-
1 is implicated in the osmotic responses through control of poly peptin/ml, 4 ␮g of aprotinin/ml, 10 ␮g of pepstatin A/ml, 1 ␮g
P levels and acidocalcisome size. of antipain/ml, 1 mM phenylmethylsulfonyl fluoride, 10 mM
sodium pyrophosphate, 1 mM NaF) and broken with three
EXPERIMENTAL PROCEDURES cycles of freezing and thawing. The samples were quantified
Trypanosome Cultures and Transfections—The procyclic using the BCA protein kit (Pierce), mixed with 2⫻ SDS-PAGE
forms (PCF) of T. brucei strains 427 and 29-13 (18) were main- loading buffer, and boiled for 5 min. The cell lysates were
tained at 27–28 °C in SDM-79 medium supplemented with 10% resolved by SDS-PAGE, and the proteins were transferred onto
fetal bovine serum. For the 29-13 strain 50 ␮g/ml hygromycin nitrocellulose membranes (Bio-Rad) using a Bio-Rad transblot
and 15 ␮g/ml G418 were added to the culture medium to pre- apparatus (Bio-Rad) in 500 mM Tris, 1 M glycine, 0.1% SDS
serve the integrated copies of T7 RNA polymerase and the tet- buffer, pH 8, containing 10% methanol. Nonspecific binding
racycline repressor, respectively. The bloodstream forms (BSF) was blocked with 5% nonfat milk in PBS-T (PBS containing
of T. brucei 90-13 (19) were cultured at 37 °C at 5% CO2 in 0.1% Tween 20) for 1 h at room temperature. The antibodies
HMI-9 medium supplemented with 10% fetal bovine serum and were also diluted in PBS-T containing 5% nonfat milk. The
10% Serum Plus (SAFC BiosciencesTM) in the presence of membranes were incubated with anti-TbTOR-like 1 fragments
hygromycin (5 ␮g/ml) and G418 (2.5 ␮g/ml). Cell densities 2 and 3 at a dilution of 1:300 overnight at 4 °C or with anti-␣-
were determined using a Neubauer chamber, and growth tubulin monoclonal antibody (Sigma) at a dilution of 1:15,000.
curves were performed using duplicate cultures, diluted to 1 ⫻ After three washes of 10 min with PBS-T, the membranes were
106 cells/ml (PCF) or to 1 ⫻ 105 cells/ml (BSF) to begin the incubated with horseradish peroxidase-conjugated goat anti-
experiment. rabbit IgG or anti-mouse IgG (Sigma) diluted at 1:15,000 and
RNAi Experiments—For RNAi, a 457-nucleotide fragment of 1:10,000, respectively, for 1 h. After three washes of 10 min each
TbTOR-like 1 (nucleotides 430 – 886) of the sequence depos- with PBS-T, bound antibodies were detected using a chemilu-
ited in the GenBankTM as XM_839137 was amplified by PCR minescence kit (Pierce) according to the manufacturer’s
using primers 5⬘-CGGATCCCTTCTTGGGAAGCATTT- instructions.
TGG and 5⬘-AAGCTTTATACCCTCCAGTCACG. This Northern Blot Analysis—Total RNA was purified from 2 ⫻
sequence encodes a portion of the TbTOR like 1 gene, coding 108 cells using TRIzol reagent (Invitrogen) according to the
fragment 1. For TbTOR2 RNAi, a 436-nucleotide fragment manufacturer’s protocol. Samples of RNA (20 ␮g) were sepa-
(nucleotides 85–520) from the sequence deposited in the Gen- rated on a 1% formaldehyde-agarose gel (22), and the gel was
BankTM as XM_839099, we used the primers 5⬘-GGGATC- stained with ethidium bromide. The RNA was transferred onto
CGCTGAGGTTGTTAAGCAGGC and 5⬘-AAGCTTACAT- a Hybond NX nylon membrane (GE Healthcare) by capillary
GTCGAGAATTCGC. Both pairs of primers incorporated transfer and fixed by UV irradiation. The membranes were pre-

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T. brucei TOR-like 1 Kinase

hybridized and hybridized as described in Ref. 23 with probes Osmotic Stress under Constant Ionic Conditions—For
labeled using the Rediprime II random prime labeling system osmotic stress under constant ionic strength, the following
(GE Healthcare) in the presence of 50 ␮Ci of [␣-32P]dCTP buffers described previously (31) were used: isotonic (64 mM
(3000 Ci/mmol; New England Nuclear). The tubulin probe used NaCl, 4 mM KCl, 1.8 mM CaCl2, 0.53 mM MgCl2, 5.5 mM glu-
was a fragment of the gene cloned in the pZJM vector (24). The cose, 5 mM Na-HEPES, pH 7.4, and 150 mM mannitol; 282
membranes were exposed on a phosphorimaging screen for mOsm/liter), hypotonic (the same as isotonic but with reduced
24 h, and labeling was detected using a Typhoon 9200 image mannitol concentration to 50 mM; 177 mOsm/liter), and hyper-
scanner (GE Healthcare). tonic (the same as isotonic but with increased mannitol con-
Fluorescence Experiments—For immunofluorescence, the centration to 500 mM; 650 mOsm/liter). Samples of 2 ⫻ 107
cells were harvested by centrifugation at 1,500 ⫻ g for 10 min, cells were collected, washed twice with PBS, and resuspended
washed with PBS two times, fixed with 2% p-formaldehyde in in 10 ml of isosmotic buffer. To the resuspended cells 500 ␮l of
ice-cold PBS for 20 min, and settled onto poly-L-lysine-coated the desired buffer (isosmotic, hyposmotic or hyperosmotic)
slides for 15 min. The fixed cells were then washed three times were added, and the samples were fixed with an equal volume of
with PBS and permeabilized with 0.3% Triton X-100 in PBS for 8% paraformaldehyde in PBS after the indicated periods of
3 min, washed again with PBS three times, and blocked with time. The cells were collected by centrifugation, washed once
PBS with 3% bovine serum albumin for 1 h at room tempera- with PBS, and adhered to coverslips pretreated with 0.1% poly-
ture. The cells were incubated with mouse anti-TbTOR-like 1 L-lysine for 1 h and processed for immunofluorescence as

F2 (1:600 dilution), rabbit anti-HSP70 or anti-BiP (both of them described above.

gifts from James D. Bangs, University of Wisconsin-Madison Electron Microscopy—For thin section images, trypanosomes

Downloaded from www.jbc.org at CAPES, on September 1, 2010

Medical School (25, 26)) (1:5,000), rabbit anti-aldolase (a gift were collected and fixed with 2.5% glutaraldehyde, 4% freshly
from Paul A. M. Michels, Université Catholique de Louvain, prepared paraformaldehyde, and 0.1 M sodium cacodylate
Belgium) (1:4,000 dilution), rabbit anti-Dhh1 (a gift from Dr. buffer, pH 7.3, on ice for 1 h and processed as described (32).
Samuel Goldenberg, Instituto Carlos Chagas-Fiocruz, Brazil The images were obtained in a Zeiss EM 902 microscope. For
imaging whole cells, PCF were washed twice with filtered buffer
(27)) (1:100), rabbit anti-TbVP1 (28) (1:1,000), or rabbit anti-
A and directly applied to Formvar-coated grids as described
dihydrolipoamide dehydrogenase (provided by Kevin M. Tyler,
previously (33). The images were observed in an energy-filter-
University of East Anglia, UK (29)) (1:1,000), all diluted in PBS
ing Zeiss EM 902 microscope. The number of acidocalcisomes
with 3% bovine serum albumin for 1 h at room temperature.
was obtained as in Ref. 33.
After three washes with PBS, the cells were incubated with
Quantification of PPi, Long Chain, and Short Chain poly P—
Alexa-Fluor 546 or 488 conjugated either to goat anti-mouse
PPi and poly P were extracted from 2.5 ⫻ 107 PCF and 1 ⫻ 108
IgG or Alexa-Fluor 594-conjugated goat anti-rabbit IgG
BSF collected by centrifugation and washed three times with
(Invitrogen) at a dilution of 1:1,000 for 40 min. The cells were
buffer A (described above). For short chain poly P and PPi
washed and counterstained with 1 ␮M 4,6-diamidino-2-pheny-
extraction, the cell pellet was resuspended in 100 ␮l of buffer A
lidole (DAPI) before mounting with Vectashield anti-fading
and 200 ␮l of ice-cold 0.5 M perchloric acid and left on ice for 30
medium (Vector Laboratories). The cells were visualized with min. The suspension was centrifuged at 10,000 ⫻ g for 1 min,
an Olympus IX-71 inverted fluorescence microscope. Serial and the supernatant was neutralized by the addition of 0.72 M
images (0.2 ␮m) were acquired with a Photometrix Cool- KOH, 0.6 M KHCO3. Precipitated KClO4 was removed by cen-
SnapHQ charge-coupled device camera driven by DeltaVision trifugation at 10,000 ⫻ g for 1 min, and the supernatant was
software (Applied Precision). Alternatively, serial images used for measuring the phosphorus and PPi released. Long
(0.2-␮m Z-increment) were collected using a 100⫻ objective chain poly P was extracted as described in Ref. 33.
1.40 NA using the Cell^M software (Olympus Europe) in a Enzymatic assays of poly P and PPi were performed in tripli-
motorized Olympus IBX81 microscope. The images were pro- cate. The assays for short chain poly P were incubated at 35 °C
cessed by blind deconvolution using Autoquant X 2.1. for 30 min in 20 mM Tris-HCl, pH 7.5, 100 mM ammonium
For observation of acridine orange accumulation, the cells acetate, 5 mM magnesium acetate containing the sample, and
were prepared as described in Ref. 28. After incubation with 6 ⬎50 units of yeast exopolyphosphatase (34). Background phos-
␮M of acridine orange in a buffer containing 116 mM NaCl, 5.4 phate contamination of each sample was measured in triplicate
mM KCl, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM K-HEPES, pH wells not containing the exopolyphosphatase, and positive con-
7.2 (buffer A), for 10 min at 28 °C, the cells were centrifuged at trol reactions were included on each plate containing a final
2,000 ⫻ g, resuspended in PBS, adhered to coverslips pretreated concentration of 20 ␮M sodium triphosphate. The reactions
with 0.1% poly-L-lysine, and observed on an Olympus IX-71 were stopped by the addition of freshly mixed, malachite green
inverted fluorescence microscope. reagent (3 parts 0.045% malachite green and 1 part 4.2% ammo-
Cell Cycle Analysis—Cell samples for flow cytometry analysis nium molybdate, 4 M HCl), and absorbance at 660 nm was
were prepared as described previously (30). The cells were ana- immediately read on a M2e microplate spectrophotomer
lyzed with a FACSCalibur using CellQuest software (Becton (Molecular Devices). Release of phosphate from PPi was per-
Dickinson). For image analysis, the cells were fixed, adhered, formed for 10 min at 35 °C in 50 mM Tris, pH 7.5, containing 5
and permeabilized as described above, stained with 10 ␮g/ml mM MgCl2 and 0.1 unit of yeast pyrophosphatase (Sigma).
of DAPI for 10 min, and visualized with a fluorescence Background phosphate controls (no pyrophosphatase) and
microscope. positive control (20 –25 ␮M potassium pyrophosphate) wells

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T. brucei TOR-like 1 Kinase

itive controls. Calculations of con-
centrations of short and long chain
poly P and PPi were based in the
number of cells used in the extrac-
tion (amount of Pi or PPi/106 cells).

RESULTS
TbTOR-like 1 Sequence Contains
an Unusual PDZ Domain—The T.
brucei genome data base contains
sequences for four open reading
frames encoding for proteins with
sequences similar to mammalian
TOR: TbTOR1, TbTOR2, TbTOR-
like 1, and TbTOR-like 2. TbTOR1
and TbTOR2 proteins were previ-
ously described by Barquilla et al.
FIGURE 1. Identification and localization of TbTOR-like kinase in PCF and BSF of T. brucei. A, schematic
representation of TbTOR-like 1 and its structural domains. The portion used for RNAi (F1) and those used for (5). The TbTOR-like 1 gene is
antibody generation (F2 and F3) are indicated. B, immunoblot analysis of whole cell extracts of PCF (strain located in chromosome 4 and has

Downloaded from www.jbc.org at CAPES, on September 1, 2010

29-13) with antibodies generated against TbTOR-like 1 (F2 and F3) and a preimmune serum (PI). The molecular 7788 base pairs in length encoding
mass markers are indicated on the left. C, immunofluorescence analysis showing localization of TbTOR-like 1 in
29-13 PCF and 90-13 BSF with anti-TbTOR-like antibodies (F2, in red). The figure also shows DAPI staining (blue), for a 291.11-kDa protein with an
the merged fluorescence images, and DIC images of the same field. Scale bars, 10 ␮m. isoelectric point of 5.92. The pre-
dicted protein contains the con-
served sequence domains found in TbTOR1/2, ScTbTOR1/2,
and mTOR: HEAT repeats (repeats of an amino acid sequence
motif that was first identified in Huntingtin, in elongation fac-
tor 3, in the regulatory subunit of Protein Phosphatase 2A, and
in the TOR, which function in transport processes) (35), the
FAT domain (found in FRAP, ATM, and TRRAP proteins
involved in protein-protein interactions) (36), the FKBP12-rapa-
mycin binding domain (37), the kinase domain (catalytic domain
with homology to phosphatidylinositol kinases), and the FATC
domain (found at the extreme carboxyl terminus, always in com-
bination with the FAT domain (36) (Fig. 1A). The main difference
between the TbTOR-like 1 domain structure and the canonical
structure of the TOR kinases is the presence of a PDZ domain (38)
between the HEAT repeats and the FAT domain.
Subcellular Localization of TbTOR-like 1—PDZ domains are
usually present in proteins that interact marginally with mem-
branes. Thus, we investigated the subcellular localization of
TbTOR-like 1. In immunoblots anti-F2 and -F3 antibodies
reacted with a band of 291 kDa, the expected size for TbTOR-
FIGURE 2. T. brucei shows decreased mRNA and protein levels of TbTOR- like 1 (Fig. 1B). Immunofluorescence using anti-F2 antibodies
like 1 after ablation by RNAi. A, Northern blot analysis of PCF (29-13) con-
taining an integrated copy of the RNAi construct p2T7-177 TbTOR-like 1. RNAi revealed a granular pattern, which is distributed through the
was induced by the addition of 1 ␮g/ml of tetracycline for 7 days. Total RNA of cytosol in PCF and BSF (Fig. 1C). To initially confirm the cellu-
RNAi-induced cells (⫹Tet) and noninduced cells (⫺Tet) were extracted, sepa-
rated in formaldehyde-agarose gel, and analyzed by Northern blot. 20 ␮g of
lar localization of TbTOR-like 1, RNAi experiments were made
protein were loaded per lane, and the gel was stained with ethidium bromide in procyclic forms using a vector that generates double strand
to quantify the ribosomal RNA (rRNA). Blotted nylon membranes were then RNA from a single construct containing the gene segment cor-
hybridized with two different probes (F1 or F2) of TbTOR-like 1 or with a
tubulin probe as a loading control. B, immunoblot analysis after RNAi of responding to F1. Northern blot analysis shows that RNAi spe-
TbTOR-like 1. Untreated (⫺Tet) and tetracycline-treated (1 ␮g/ml) (⫹Tet) PCF cifically ablated the expression of TbTOR-like 1 (Fig. 2A). Sim-
containing the integrated p2T7-177 TbTOR-like 1 vector were collected after ilarly, the levels of protein recognized by immunoblots using
6 days and processed for immunoblot analysis using anti-TbTOR-like 1 anti-
body (F2). The molecular mass markers are shown on the left. anti-F2 or F3 antisera against TbTOR-like 1 decreased. Fig. 2B
shows the results for anti-F2, but identical decreases were
were included in triplicate. Phosphate was measured with mal- observed when using anti-F3 antibodies. Knockdown of
achite green as described above. On all occasions, standard TbTOR-like 1 is already observed 24 h after RNAi induction
curves of potassium phosphate were included on each plate. with tetracycline, without an immediate effect in cell growth for
Both phosphate content released from poly P and pyrophos- PCF (Fig. 3A) or BSF (Fig. 3B). Importantly, the immunofluo-
phate were normalized to reaction yield as determined by pos- rescence analysis showed a convincing decrease in labeling in

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T. brucei TOR-like 1 Kinase

calcisome marker), anti-dihydroli-
poamide dehydrogenase (a mito-
chondrial marker), and anti-BiP (an
endoplasmic reticulum marker) local-
ization (supplemental Fig. S1). Cell
fractionation studies revealed that,
different from TbTOR2, which is
associated with the parasite mito-
chondria or with the endoplasmic
reticulum (5), TbTOR-like 1 is found
in the soluble fraction and not asso-
ciated with membranous cell com-
partments (supplemental Fig. S2).
Phenotypic Analysis of TbTOR-
like 1 RNAi Cell Line—It can be
noted from Fig. 3 (A and B) that
RNAi of TbTOR-like 1 progres-
sively decreased cellular prolifera-
tion, whereas protein expression

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FIGURE 3. TbTOR-like 1 ablation by RNAi causes a progressive cell growth inhibition in PCF and BSF of T. disappears rapidly in PCF and BSF.
brucei. A and B, growth curve of tetracycline induced (⫹Tet) or noninduced (⫺Tet) PCF (A) and BSF (B). At days The effect of proliferation is more
4 and 3 the cells were diluted again to the initial dilution (1 ⫻ 106/ml and 3 ⫻ 105/ml for PCF and BSF,
respectively). The values are the means ⫾ S.D. of triplicate experiments. The differences were statistically
pronounced several days following
significant for PCF at days 6, 7, and 8 (p ⫽ 0.011, 0.003, and 0.025, respectively) and for BSF at days 3 and 4 (p ⫽ knockdown. In addition, TbTOR-
0.023 and 0.038, respectively). Lower panels, immunoblot of whole cell extracts of the same cells treated with like 1-depleted PCF (but not BSF)
tetracycline collected at the indicated days and revealed with anti-TbTOR-like 1 (F2) or with anti-tubulin anti-
bodies as a loading control. C, immunofluorescence analysis of PCF and BSF TbTOR-like 1 RNAi cell lines, appear larger than the noninduced
induced (⫹Tet) or not (⫺Tet) with tetracycline and using anti-TbTOR-like 1 (F2, red). The figure also shows DAPI controls (Fig. 3C, DIC). Morpho-
staining (blue) and the merged fluorescence images of the same field. Corresponding DIC images are shown as metric analysis of a large number of
well. Scale bars, 10 ␮m.
cells confirmed this observation.
The projected area taken from two-
dimensional images revealed that RNAi-induced cells were larger
than the noninduced controls (Fig. 4).
TbTOR-like 1-depleted PCF Are Arrested in the S/G2 Phase of
the Cell Cycle—The increased size of cells lacking TbTOR-like
1 could be due to a delayed progression in the cell cycle, which
would also explain the decrease in the proliferation rate. There-
fore, the cellular DNA content/cell was quantified by flow
cytometry after propidium iodide staining, 4 days after RNAi
induction with tetracycline. As shown in Fig. 5A, the number
of PCF in the G1 phase of the cell cycle was reduced concom-
itantly with an increase of cells in the S phase. No effect of
tetracycline was observed in control cells (29-13 procyclics)
as shown in Fig. 5B. We also quantified the number of cells
FIGURE 4. TbTOR-like 1 ablation causes an increase in cell size. PCF were containing one nucleus and one kinetoplast (1N1K), 1N2K,
induced (⫹Tet, black bars) or not (⫺Tet, white bars) with tetracycline for 4 and 2N2K and abnormal cells (0N1K) in the population
days, fixed, and adhered to coverslips as described under “Experimental Pro-
cedures,” and the images were taken using DIC. Binaries were generated using DAPI staining. Cells with one nucleus and an elon-
using the ImageJ software, and the area of each cell image was quantified. gated kinetoplast corresponding to cells in the S phase were
The graph shows the distribution of T. brucei projected surface area. The two also counted as 1N2K. A decrease in 1N1K cells was found
distributions were statistically different (p ⬍ 0.05) based on a nonparametric
Student’s t test (n ⫽ 98 and 100 for noninduced and induced RNAi, respec- with an increased number of other patterns when comparing
tively). No significance difference was observed for PCF 29-13 in the presence depleted TbTOR-like 1 cells with control cells, confirming
compared with the absence of tetracycline.
the cell cycle delay with an increase in S/G2 (1N2K) cells
observed by fluorescence-activated cell sorter analysis
both PCF and BSF (Fig. 3C), confirming the cellular localization (Fig. 5C).
of TbTOR-like 1. Ablation of TbTOR-like 1 Increases the Size of Acidocalci-
We found that TbTOR-like 1 is in the cytosol and is not somes in Electron Microscopy Images—RNAi-induced and
associated with major organelles. Its labeling pattern shows noninduced populations of PCF were fixed and processed for
some colocalization with anti-HSP70 (a cytosolic marker) but transmission electron microscopy. As shown in Fig. 6A,
differs significantly with respect of anti-aldolase (a glycosomal knocked down cells had an apparently higher number of
marker), anti-Dhh1 (a P-body marker), anti-TbVP1 (an acido- electron-dense vesicles when compared with control cells.

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T. brucei TOR-like 1 Kinase

These electron-dense vesicles appear to correspond to aci-
docalcisomes, which appear in conventional electron micro-
scopy as electron-dense or partially empty vesicles (32). In
normal cells the electron density corresponds to cations
associated with PPi and poly P, and these components are
lost during the conventional processing for transmission
electron microscopy (9, 32). Indeed, more stained acidocal-
cisomes were detected in TbTOR-like 1-depleted PCF when
compared with control cells fixed and analyzed by energy-
filtering transmission electron microscopy (Fig. 6B), a suita-
ble method to observe acidocalcisomes (32). Quantitative
analysis indicated that although the number of acidocalci-
somes did not increase significantly when the total popula-
tion of cells was analyzed, the volume and area occupied by
acidocalcisomes increased significantly (Table 1). RNAi of
TbTOR-like 1 also promoted a larger accumulation of acri-
dine orange, a weak base that is incorporated into acidic
compartments, such as the acidocalcisomes (Fig. 6C).
Ablation of TbTOR-like 1 Increases Polyphosphate and Pyro-

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phosphate Levels—Taking into account that knockdown of
TbTOR-like 1 seems to increase the size of acidocalcisomes, we
determined the levels of short and long chain poly P and PPi. As
shown in Fig. 7 (A and C), the knockdown cells (PCF and BSF)
contained higher levels of both types of poly P when compared
with control (noninduced) cells, although the increase in
short chain poly P was more evident than that of long chain
poly P. The levels of PPi also increased significantly when
RNAi was induced (Fig. 7, B and D). In contrast to these
results, RNAi of TbTOR2 did not significantly affect the lev-
els of short chain poly P (supplemental Fig. S3), as compared
with control (noninduced) cells. Previous transmission elec-
tron microscopy data did not reveal the presence of more
electron-dense vesicles in TbTOR2 or TbTOR1 knockdowns
(5). These results indicate that acidocalcisome size and poly
P levels are specifically increased in cells lacking TbTOR-like
1 kinase.
Localization of TbTOR-like 1 Change after Hyperosmotic
Stress—Acidocalcisomes are organelles involved in osmoregu-
lation (9, 39). Therefore, the increase in acidocalcisomes and
poly P levels in cells having less TbTOR-like 1 could therefore
suggest that this kinase participates in a signaling cascade
affecting acidocalcisome function. To obtain some insight
about a possible mechanism of signaling activation by TbTOR-
like 1, we subjected PCF (strain 29-13) to hyposmotic and
hyperosmotic shocks under constant ionic conditions. Subse-
quently the cells were fixed and processed for immunofluores-
cence with anti-TbTOR-like 1 antibody. Upon hyposmotic
shock the cells rapidly swell and then recover. Upon hyperos-
motic stress, cells rapidly shrink and then recover their volume.
These processes occur in less than 5 min. As shown in Fig. 8,
FIGURE 5. A decrease expression of TbTOR-like 1 causes a partial arrest of only under hyperosmotic conditions do TbTOR-like 1 granules
the cell cycle in S/G2. A and B, after 4 days, untreated (⫺Tet) and 1 ␮g/ml
tetracycline-treated (⫹Tet) PCF of TbTOR-like 1 RNAi cell line (A) or control have a distinct distribution, becoming localized aligned at the
29-13 cells (B) were stained with propidium iodide and subjected to fluores- cell periphery, whereas the localization of TbTOR-like 1
cence-activated cell sorter analysis for DNA content or fixed and processed
for DAPI staining and microscopic observation under a fluorescence micro-
remains spread through all the cytosol in control or hyposmotic
scope. C, number of cells (n ⫽ 200 for each control or TbTOR-like 1 RNAi cell conditions. The relocalization pattern of TbTOR-like 1 was not
line, induced or noninduced, as indicated) with different numbers of nuclei observed for aldolase, a glycosome marker (supplemental
(N) and kinetoplasts (K). The differences between induced and noninduced
TbTOR-like 1 were statistically different for G1 (p ⫽ 0.003), G2 (p ⫽ 0.0023), and Fig. S4A), and BiP, an endoplasmic reticulum marker (sup-
zoids (p ⫽ 0.012). The other differences were not statistically significant. plemental Fig. S4B). A partial relocalization to the surface was

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T. brucei TOR-like 1 Kinase

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FIGURE 6. TbTOR-like RNAi cell line presents acidocalcisomes with increased size. A, transmission electron microscopy images of thin sections of resin-
embedded PCF previously induced (⫹Tet) or not (⫺Tet) for 4 days with tetracycline. The arrowheads indicate some acidocalcisomes. N, nucleus. B, transmission
electron microscopy of whole PCF attached to Formvar grids after 4 days of treatment without or with tetracycline. In both A and B the dark granules are
acidocalcisomes. Scale bars, 2 ␮m in A and B. C, fluorescence images showing the accumulation of acridine orange in RNAi for TbTOR-like 1 cells (⫹Tet)
compared with noninduced cells (⫺Tet). The three top rows correspond to PCF, and the bottom two rows correspond to BSF. Red pseudo color was generated
using Adobe Photoshop. Scale bars, 10 ␮m.

TABLE 1
Morphometric analysis of the acidocalcisomes in T. brucei TOR-like 1 mutants
The results are expressed as the means ⫾ S.D. (n ⫽ 100). The mean area, mean diameter, and mean volume differences have significant differences according to Student’s
t test (p ⬍ 0.05).
Number of
Mean area Mean circularity Mean diameter Mean volume
acidocalcisomes/cell
nm² nm nm nm3
Without tetracycline 26 ⫾ 8 58.0 ⫾ 35.5 ⫻ 10 3
0.92 ⫾ 0.03 286.2 ⫾ 76.6 20.4 ⫾ 10.4 ⫻ 106
With tetracycline 31 ⫾ 9 162.0 ⫾ 74.0 ⫻ 103 0.90 ⫾ 0.01 477.4 ⫾ 106.3 81.2 ⫾ 57.5 ⫻ 106

observed for the dihydrolipoamide dehydrogenase mitochon- the effect of knocking down TbTOR-like 1 occurs earlier than
drial marker (supplemental Fig. S4C). in regular medium (supplemental Fig. S5). These results sug-
A possible link between the hyperosmotic stress and gest that TbTOR-like 1 kinase participates in the control of
polyphosphate was proposed earlier based on a previous obser- osmotic stress response as a consequence of changes in the poly
vation that adding hyperosmotic solution to T. cruzi increased P and acidocalcisomes.
the levels of poly P (11). We found that cultivating T. brucei in
medium supplemented with 0.3 M mannitol reduced growth DISCUSSION
and also increased poly P levels (Fig. 9A and B). Importantly, In this is study we characterized a novel type of TOR-like
upon RNAi induction for TbTOR-like 1, poly P starts to protein present in T. brucei and not previously found in any
increase soon after induction, before growth arrest (Fig. 9, C other Eukarya. Ablation of this TbTOR-like by RNAi leads to a
and D). As shown before for T. cruzi, poly P is high in the lag progressive inhibition of cell proliferation, with increases in the
phase and decreases when the cells grow exponentially. size of acidocalcisomes and in the content of poly P and PPi.
These results would suggest that cells lacking TbTOR-like 1 This protein kinase is found in cytosolic granules dispersed
would be more sensitive when growing at higher osmolarity. through the cytosol, which specifically relocalizes toward the
Indeed, this seems to be the case. In the presence of mannitol, cell periphery under hyperosmotic conditions. Hyperosmotic

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T. brucei TOR-like 1 Kinase

normal cell growth. The absence of this protein kinase seems to
affect proliferation after several rounds of cell division, suggest-
ing that the growth defect is due to a cumulative effect. A par-
allel accumulation of poly P and PPi inside acidocalcisomes,
which increase in size and become heavily stained at the elec-
tron microscopy level, is observed when compared with control
cells. Cellular proteins remain at the same level as judged by
quantitative profiles of two-dimensional gel electrophoresis
(data not shown).
It is possible that the accumulation of poly P in acidocalci-
somes is a consequence of the growth arrest determined by
TbTOR-like 1 knockdown. In this regard, conditions that favor
growth arrest, such as hyperosmotic stress, also result in
increased poly P synthesis and in potentiation of the effects of
TbTOR-like 1 knockdown (Fig. 9). The mechanism, by which
FIGURE 7. TbTOR-like 1 ablated cells have higher levels of poly P and PPi.
the absence of TbTOR-like 1 leads to an increased content of
The levels of short chain (SC) and long chain (LC) poly P and the levels of PPi poly P, is presently unknown, but it could be due to a direct
were measured in noninduced (⫺Tet) and induced (⫹Tet) PCF (A and B) or BSF effect on enzymes affecting the turnover of poly P, as occurs in
(C and D) after 6 and 4 days with tetracycline, respectively. The figure shows
the mean values ⫾ S.D. of three experiments measured in triplicate. The dif- bacteria submitted to amino acid starvation (40). Amino acid

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ferences between noninduced and induced cells were found significant by starvation in bacteria leads to coupled cessation of protein syn-
using Student’s t test (p ⬍ 0.005). thesis and stable RNA synthesis, a process known as the “strin-
gent response” that is accompanied by accumulation of poly P.
Poly P accumulation is mediated by alarmone guanosine
5⬘-diphosphate 3⬘-diphosphate synthesis, which ultimately
inhibits an exopolyphosphatase, an enzyme that degrades poly
P (40). Together with the cessation of nucleic acid synthesis and
continued assimilation of Pi from the medium, this results in
large accumulations of poly P. Although guanosine 5⬘-diphos-
phate 3⬘-diphosphate has been found in plants (41), it is not
known whether it is present in early branched eukaryotes. In
support of the notion that TOR kinases could be involved in
poly P metabolism, it has been shown that poly P stimulates
mammalian TOR kinase activity in vitro and most likely in vivo
(10). It is important to mention that knockdown of T. brucei
TOR2 kinase does not cause poly P accumulation, although a
substantial growth inhibition and an increase in cell size occur
(5), indicating that poly P accumulation is specific for TbTOR-
like 1 knockdown.
Poly P has been shown to be essential for adaptation to vari-
ous stresses and for survival of bacteria in the stationary phase
(43– 45). Similar studies have been reported in eukaryotic cells,
such as yeast (46), fungi (47), and algae (48). It has long been
recognized that the poly P content is low during rapid growth
and increases under conditions of nutritional imbalance unfa-
FIGURE 8. TbTOR-like 1 relocalizes to the cell periphery in PCF submitted vorable for growth in different organisms from bacteria to yeast
to hyperosmotic conditions. T. brucei PCF (29-13) at exponential growth
were collected by centrifugation and resuspended with isosmotic (Iso), (49). Knockdown of TbTOR-like 1 might have a phenotype
hyposmotic (Hypo), and hyperosmotic (Hyper) buffers as described under similar to that of nutritional imbalance.
“Experimental Procedures.” Ten seconds after different buffer treatments, the Studies of TOR signaling in yeast, Drosophila, and mammals
cells were fixed and processed for immunofluorescence with anti-TbTOR-like
1 (F2) antibody (green) in the presence of DAPI (blue). The merged fluores- have demonstrated that TOR kinases participate directly in cel-
cence images and the merged fluorescence and DIC images are shown as lular growth and size control. In these organisms ablation of
well. Scale bars, 10 ␮m.
TOR leads to a decrease in cell size by several mechanisms (4,
51). One mechanism appears to involve a balance between sig-
conditions increase poly P levels, arrest cell growth, and sensi- naling pathways interfering with cell cycle control and growth
tize parasites to TOR-like 1 dependence. These results suggest (52). In the case of TbTOR-like 1, knocked down PCF are larger
a link between TOR-like kinase 1 signaling pathway and cell than control cells, similar to what is observed for TbTOR2 but
responses to osmotic stresses. opposite to the TbTOR1 knockdown phenotype (5). In addi-
TbTOR-like 1 is expressed as a cytosolic 291-kDa protein in tion, when mammalian TORC1 and T. brucei TOR1 expression
BSF and PCF of T. brucei and in both forms is required for are reduced, cells arrest in the G1 phase of the cell cycle (5, 53),

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T. brucei TOR-like 1 Kinase

mulation of poly P. Alternatively,
the delay in the cell cycle could be
directed by a similar mechanism
found in the fission yeast, in which
TOR 1 affects the mitogen-acti-
vated protein kinase (MAPK) con-
trol of cell cycle progression (52). In
this sense, it is relevant that the
osmotic stress in Schizosaccharo-
myces pombe cross-talks with TOR
signaling (57). A similar cross-talk
might occur for TbTOR-like 1.
One unique characteristic of
TbTOR-like 1 is the presence of the
PDZ domain. This domain is
involved in the scaffolding and
assembly of multi-protein com-
plexes at various subcellular sites,
which suggests their participation

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in several aspects of cellular physiol-
ogy such as receptor or channel
clustering, cell junction formation,
FIGURE 9. Hyperosmotic medium causes a similar increase of poly P as TbTOR-like 1 ablation. T. brucei PCF and intracellular signaling events
29-13 (A and B) and PCF containing the p2T7-177 TbTOR-like 1 vector (C and D) were grown in regular medium
(open symbols) or medium with 0.3 M mannitol for PCF 29-13 or with 1 ␮g/ml tetracycline for PCF TbTOR-like 1 (58). Our results unequivocally
(closed symbols). The number of parasites was counted daily, and the amount of short chain poly P was meas- demonstrate that TbTOR-like 1 has
ured in triplicate experiments. The values are the means ⫾ S.D. For all values, p ⬍ 0.05. The stars indicate the a cytosolic distribution in dispersed
ratios of poly P levels between cells incubated in the presence and absence of tetracycline.
granules because RNAi knockdown
abolishes immunofluorescence la-
whereas TbTOR-like 1-depleted cells accumulate at the S/G2 beling. This result is compatible with the formation of multi-
phase. The increased amount of zoids (0N1K) found here was protein complexes between the kinase and other unidentified
also observed for TbTOR-like 2 knocked down cells (53). How- proteins, and this is supported by the fact that upon gel filtra-
ever, in this case cells also accumulate in the G1 phase as found tion of soluble extracts, TbTOR-like 1 eluted with sizes corre-
for TbTOR1 RNAi. In contrast, TbTOR2 knocked down cells sponding to proteins from 200 to 650 kDa (data not shown).
accumulate in G1 with the formation of multinucleated and Moreover, TbTOR-like 1 does not colocalize with any of the
multiflagellated cells (5). Different from TbTOR-like 1, markers used to label specific organelles, suggesting that it
TbTOR2 RNAi resulted in multinucleated cells with an might form a new type of structure. Perhaps the presence of the
enlarged flagellar pocket without the appearance of dense aci- PDZ domain is involved in this different type of cytosolic com-
docalcisomes. TbTOR1 RNAi causes autophagy events without plex. In contrast, TbTOR 1 is nuclear, and TbTOR 2, which lacks
the appearance of dense acidocalcisome granules. All of these the PDZ domain, seems to be associated with the endoplasmic
findings indicate that TOR kinases of T. brucei act through reticulum or the mitochondrial membrane (5), as found for yeast
different mechanisms and might have different substrates and mammalian TORC complexes (42, 59–62). It is important to
and/or activators, probably affecting growth, proliferation, and mention that the identification of other partners of TbTOR-like 1
cell cycle control at different points as proposed earlier (54, 55). and its substrates, determination of whether TbTOR-like 1 affects
Decrease of TbTORs expression also differently affects PCF translation, and demonstration of kinase activity will be necessary
and BSF, which may reflect differences observed in cell cycle to characterize this protein.
control between these stages of the parasite (56). For example, A relevant finding of this work is that hyperosmotic stress
PCF form multinucleated cells, zoids, and other abnormal cell changes momentously the localization of TbTOR-like 1.
types when TbTOR 1 expression is reduced (53), whereas in Immunofluorescence labeling revealed that it becomes clus-
BSF these effects are not observed (5). When TbTOR-like 2 tered near the parasite surface, different from glycosomes,
expression is reduced, the cells accumulate at S/G2 in PCF, endoplasmic reticulum, and stress granules. Some cellular reor-
generating zoids and multinucleated cells, whereas no effect is ganization of the single trypanosome mitochondrion is noticed,
evident in BSF when the same protein is ablated (53). In the suggesting that it could participate in the same signaling path-
present case PCF delay in S/G2 is more evident than in BSF way. Because PDZ domains are found in several types of
(data not shown). The mechanism by which TbTOR-like 1 and dynamic protein-protein interactions (50), it is tempting to
other TbTOR affect T. brucei cell cycle requires further inves- speculate that changing protein localization during osmotic
tigation. Nevertheless, one possibility is that a delay in the S/G2 stress is important for TbTOR-like 1 function. The next step
phase might occur by inhibition of de novo nucleotide synthesis would be to identify proteins that interact with the PDZ domain
because of a decrease of free phosphate resulting from the accu- of TbTOR-like 1.

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T. brucei TOR-like 1 Kinase

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24140 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 31 • JULY 30, 2010