A Minimal Anaphase Promoting Complex/Cyclosome
(APC/C) in Trypanosoma brucei
Mohamed Bessat, Giselle Knudsen, Alma L. Burlingame, Ching C. Wang*
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California, United States of America
The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome
segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C
subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in
yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the
corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic
checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex
equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1
fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared
their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex
throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3.
Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core
subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no
detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells
showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the
proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all,
a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex,
thus indicating a unique T. brucei APC/C.
Citation: Bessat M, Knudsen G, Burlingame AL, Wang CC (2013) A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei. PLoS
ONE 8(3): e59258. doi:10.1371/journal.pone.0059258
Editor: Ziyin Li, University of Texas Medical School at Houston, United States of America
Received January 10, 2013; Accepted February 13, 2013; Published March 22, 2013
Copyright: ? 2013 Bessat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health R01 grant R01 AI21786 to CCW. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Trypanosoma brucei, is an early divergent protozoan parasite that
causes African sleeping sickness in human and nagana in livestock.
It has a complex biphasic life cycle that allows the cells to multiply
in both the mammalian host and the insect vector tsetse flies .
There are many unique features in the cell cycle progression in T.
brucei when compared with that in metazoa [2,3]. For instance,
cytokinesis in the bloodstream-form T. brucei is controlled by
mitosis whereas that in the insect (procyclic) form is driven
primarily by the duplication and segregation of basal bodies and its
associated mitochondrial DNA complexes, the kinetoplasts [4,5,6].
Therefore, procyclic form cells can undergo cytokinesis in the
absence of mitosis, whereas a mitotic arrest in bloodstream form
cells inhibits cytokinesis with continued kinetoplast replication and
segregation and nuclear DNA synthesis [2,5,6], which implicates
fundamental differences in cell cycle controls between different life
cycle forms of T. brucei and potential absence of the key cell cycle
checkpoints. Cell division in both forms of T. brucei proceeds
longitudinally along the dorsal line from the anterior to the
posterior end of the cell , which contrasts significantly from that
in metazoa [8,9]. The mechanism of this distinctive form of cell
division in T. brucei is initiated by a trans-localization of the
chromosome passenger complex (CPC) from the midzone of
central spindle across the nuclear envelope to the flagellar
attachment zone (FAZ) during the final stage of mitosis .
The CPC then moves along the FAZ to the anterior end of the
dividing cell and slides back toward the posterior end along the
FAZ in an unzipping action to separate the dividing mother from
the daughter . The cell cycle of T. brucei has thus become one
of the most intriguing subjects for further investigation in recent
The progression from metaphase to anaphase during mitosis of
T. brucei appears, however, somewhat similar to that observed in
other eukaryotes . The chromosomes in the nucleus of T. brucei
are replicated during S-phase and attached to the mitotic spindle
and aligned in two closely associated parallel rows during
metaphase . The chromosomal duplexes are then pulled
apart by the mitotic spindle into two separate entities during
anaphase . Metaphase-anaphase transition and mitotic exit in
metazoa and yeast are controlled primarily by the anaphase
promoting complex/cyclosome (APC/C) regulated by periodic
association and dissociation with the mitotic checkpoint complex
(MCC). APC/C is a multi-subunit E3 ubiquitin ligase consisting of
13 core subunit proteins in yeast that is inactivated by association
with the effector proteins of the MCC complex in the
prometaphase [12,13,14,15,16]. When proper alignment and
attachment of the duplicated chromosomes to the mitotic spindle
PLOS ONE | www.plosone.org1 March 2013 | Volume 8 | Issue 3 | e59258
are achieved toward the end of metaphase, MCC and APC/C are
dissociated from each other leaving a single MCC subunit protein,
CDC20, with the APC/C to activate the latter to poly-
ubiquitinate securin/Pds1 for 26S proteasomal degradation
[17,18,19]. Securin/Pds1 inhibits the activity of a protease
separase by binding to the latter during metaphase. Upon its
destruction, separase is freed and activated and degrades the
cohesin protein binding the sister chromatids for successful
chromosome segregation and cell cycle progression to the
anaphase . Toward the late mitotic phase, a mitotic cyclin B
is targeted by the activated APC/C for poly-ubiquitination and
subsequent 26S proteasome-mediated degradation thus lowers the
activity of cyclin-dependent kinas 1 (CDK1) for mitotic exit
In our previous study, we identified seven APC/C subunit
homologues in T. brucei that include APC1, APC2, APC10/
DOC1, APC11, CDC16, CDC23 and CDC27 . There is also
a CDC20 and a separase homologue found in T. brucei genome
database (unpublished data and ). But no homologue of
securin/Pds1 or any of the MCC subunit proteins has yet been
identified in this organism. The separase homologue demonstrated
a conserved role in catalyzing chromosome segregation in T. brucei
, but an RNAi knockdown of CDC20 in procyclic form T.
brucei showed no apparent mitotic arrest (unpublished data).
Further RNAi knockdowns of each of the seven subunit proteins in
the apparent APC/C of T. brucei showed that only depletion of
APC1 or CDC27 resulted in mitotic arrest in both procyclic and
bloodstream form cells , whereas knockdowns of the rest of the
5 subunits showed no apparent phenotype . This outcome is
similar to that from Saccharomyces cerevisiae, in which mutants of
APC9 , MND2, SWM1  or CDC26  did not register
any phenotype and were classified as the nonessential subunits of
the APC/C in yeast.
There is thus a likely presence of functional APC/C in T.
brucei . But the previous results did not verify whether the
7 subunits constitute the entire core of a APC/C structure or if
more protein subunits are involved in constituting the complex.
Nor is it clear if the APC/C function is regulated by associating
with a functional MCC-like complex or whether a functional
homologue of securin/Pds1 and a functional homologue of
CDC20 may exist and play pivotal roles in metaphase-anaphase
transition in T. brucei.
In an effort to clarify these issues, we first used the method of
yeast complementation to examine if any of the putative T. brucei
APC/C subunits are capable of replacing those in S. cerevisiae,
and found that among those tested, none was capable of
substituting the yeast counterpart. We then used tandem affinity
purification (TAP) and mass spectrometry to identify the
composition of T. brucei APC/C and examined its potential
association-dissociation with other proteins during the metaphase-
anaphase transition. The outcome indicated that, throughout the
entire cell cycle, there was a constant presence of an apparent
APC/C complex consisting of 10 subunit proteins. Neither the
CDC20 homologue nor any other protein was found detectable
with the APC/C at any phase of the cell cycle, suggesting a unique
mechanism of APC/C regulation in T. brucei. The mitotic cyclin B
of T. brucei (cycB2/cyc6)  was, however, found poly-
ubiquitinated by the APC/C and degraded by proteasome for
mitotic exit as in other eukaryotes.
Materials and Methods
Yeast Complementation Assay
Temperature-sensitive (ts) S. cerevisiae mutants cdc16-1, cdc23-
1, cdc27-1 and apc1-1 were kindly provided by Dr. D. Toczyski of
UCSF . The apc10-1 ts mutant was purchased from Open
Biosystems (Thermo Scientific). A wild-type 303wt strain of S.
cerevisiae was from Dr. P. Walter of UCSF. The genetic
background among the yeast strains are all of Mat/a except for
the apc10-1 mutant and 303 wt strain, which are of Mat/a. For
the standard yeast propagation, cells were grown in Yeast extract/
Peptone/Dextrose (YPD) medium. Full-length open reading
frames (ORFs) of genes encoding CDC16, CDC23, CDC27,
APC1 and APC10/DOC1 were amplified from the yeast and the
T. brucei genomic DNAs by PCR using gene specific primers with
restriction sites for cloning into the yeast expression plasmid,
pRS416-ADH . Individual constructs were introduced into the
corresponding yeast mutants and expressed under the control of
yeast specific ADH promoter . The transformed cells were
selected on the synthetic-defined (SD) minimal selection medium
supplemented with the Drop-Out (DO) supplement–Ura (Clon-
tech). Yeast transformation and selection were by the protocol of
Gietz [31,32]. Replica plates of transformed cells were incubated
for 3 days at either the permissive (25uC) or the restrictive (37uC)
temperature for indication of genetic complementation by the
Trypanosome Cell Culture
Procyclic-form T. brucei cells of the wild type strain 427 and the
modified strain 29-13 for RNAi studies  were both cultivated
at 26uC in the Cunningham medium supplemented with 10% fetal
bovine serum (Hyclone. Thermo scientific, USA). For the 29-13
strain, 15 mg ml21G418 and 50 mg ml21hygromycin B were
added to the culture medium to maintain expression of T7 RNA
polymerase and tetracycline repressor, respectively . Cell
densities were maintained at the mid-log phase of ,56106cells
ml21through regular subculturings. Transfection and selection of
the procyclic T. brucei cells were carried out as described previously
Expression of Protein A-tobacco Etch Virus (TEV) Protease
Site-protein C Epitope (PTP)-tagged APC1 and
Purification of the APC1-associated Protein Complex
The 39-terminal fragment of T. brucei APC1 gene encompass-
ing nucleotide sequence #4,668–#5,510 was amplified by PCR
from the genomic DNA of the cells of 427 strain and cloned in
frame into the pC-PTP-NEO plasmid to produce the pC-APC1-
PTP construct . The plasmid was linearized with XcmI and
transfected into the strain 427 cells by electroporation for genome
integration through homologous recombination. The transfected
cells were selected under 40 mg ml21G418. Correct PTP tagging
of the endogenous APC1 gene in the transfected cells was
subsequently confirmed with PCR and DNA-sequencing. The
transfected cell cultures were further diluted and distributed to 24-
well plates at a calculated average of a single cell per well for
further cell growth to identify the cloned cell line . The cells
showing consistent and optimal growth rates were selected and
tested for expression of the PTP-tagged APC1 protein in Western
blotting (see below) and stored in liquid nitrogen.
For purification of the PTP-APC1-containing protein complex
from the cloned transfected cells, crude lysate was prepared from
,16109cells by sonication, and cleared by a brief centrifugation
. The cell lysate was then subjected to stepwise purifications
through immunoglobulin G (IgG) column binding, TEV protease
The APC/C of Trypanosoma brucei
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elution, protein C antibody column binding and EGTA chelating
elution according to the established procedure . Each step of
the purification was monitored with Western blotting and SDS-
PAGE stained with SYPRO Ruby (Invitrogen, CA). Individual
protein bands in the final purified sample fractionated in SDS-
PAGE and stained with SYPRO Ruby were sliced from the gel,
diced and processed for in-gel trypsin digestion by a standard
protocol (The UCSF Mass Spectrometry In-gel digestion pro-
cedure @ ms-facility.ucsf.edu/ingel.html) followed by mass
spectrometric analysis (see below).
Synchronization of the Cell Cycle Progression in T. brucei
Mid-log phase procyclic-form T. brucei cells expressing APC1-
PTP were treated with 0.3 mM of hydroxyurea and incubated at
26uC for 16 hr to become synchronized in the late S phase .
Hydroxyurea was then washed off, and the late S phase cells were
cultivated for synchronized cell cycle progression for 2.5 additional
hrs for a cell population highly enriched in metaphase, and 4.5 hrs
to enrich the cells in anaphase . The unsynchronized cells are
enriched in G1 phase. They and the other cell populations
enriched in S phase, metaphase and anaphase were each used for
the TAP purification of APC1-associated protein complexes and
for comparisons of their protein profiles in SDS-PAGE-SYPRO
Ruby gels and mass spectrometric analysis.
Liquid Chromatography-tandem Mass Spectrometry (LC-
Following in-gel trypsin digestions, samples from individual
protein bands were each analyzed using an LTQ-Orbitrap XL
(Thermo) mass spectrometer. It was equipped with a 10,000 psi
system nanoACUITY (Waters) UPLC instrument for peptide
separation by reversed phase chromatography with a C18 column
(BEH130, 1.7 mm bead size, 100 mm6100 mm). The LC was
operated at a flow rate of 600 nL/min, and the peptides were
separated using a linear gradient of acetonitrile from 2% to 30% in
solvent A (0.1% formic acid in water) and solvent B (0.1% formic
acid in 70% acetonitrile) over a period of 42 min. Survey scans
were recorded over 310-1600 m/z range, and MS/MS was
performed in data dependent acquisition mode with CID
fragmentation on the six most intense precursor ions, measured
in the ion trap.
Lists of mass spectrometric peaks were generated using the in-
house software PAVA, and data were searched using the Protein
Prospector software v. 5.10.0 . Database searches were
performed against the SwissProt database (downloaded March
21, 2012) to which was added the T. brucei subset of the
TriTrypDB database v. 4.1 (downloaded June 21, 2012) totaling
563,498 entries. For estimation of false discovery rates, the
database was concatenated with a fully randomized set of
sequence entries . Data were searched with mass tolerances
of 20 ppm for parent and 0.6 Da for fragment ions.
For database searching, peptide sequences were matched as
tryptic peptides with no missed cleavages, and carbamidomethy-
lated cysteines as a fixed modification. Variable modifications
included oxidation of methionine, N-terminal pyroglutamate from
glutamine, loss of methionine and N-terminal acetylation. Protein
Prospector score parameters were set at a minimum protein score
of 22, minimum peptide score of 15, and maximum expectation
values of 0.01 for protein and 0.001 for peptide matches, resulting
in a protein false discovery rate of 0.4%. Protein identification
results from specific TAP experiments were reported with
a spectral count as an approximation of protein abundance, along
with percent sequence coverage and an expectation value for the
probability of protein identification [40,41].
Target gene fragments were selected based on default settings of
the RNAit software . They were amplified from the strain 29-
13 genomic DNA with PCR using sense primer carrying flanking
BamHI/HindIII sites and antisense primer carrying flanking
XhoI/XbaI sites for cloning the individual fragments into the
Stem-loop pALC14 plasmid [24,43]. The constructs were
linearized with NotI and transfected into 29-13 procyclic cells
via electroporation . The transfected cells were cultivated for
15 to 18 days, and the stable transfectants were selected in the
presence of 1 mg ml21puromycin. Clonal cell lines were
established by limiting dilution and cultivation in 24-well plate.
The cloned stable transfectants that reached a constant growth
rate after at least three regular passages were selected and tested
for RNAi phenotypes. To induce RNAi, 1 mg ml21tetracycline
was added to the culture of a cell density of 26105cells ml21. Cell
growth was monitored by counting the cell numbers with
a haemocytometer at 24 hr intervals. For RNA extraction and
cell cycle analysis, 24 hr cell samples after RNAi induction were
collected, washed once with PBS and processed for either RNA
extraction using the TRIzolH reagent (Invitrogen) or cell cycle
analysis in flow cytometry (see below).
Quantitative Real-time RT-PCR (qRT-PCR)
The first strand cDNA was generated from the RNA samples
using iScript RT kits (Bio-Rad). qPCR was performed on the
cDNA using gene-specific primer sets that are different from the
primer sets for the RNAi DNA construct. It was performed using
the SsoFast SYBRH Green supermix (Bio-Rad) with the quanti-
tative analysis and statistical significance empirically calculated by
the CFX ManagerTMsoftware (Bio-Rad). For loading controls,
100-bp products of Paraflagellar rod-A (PFR-A) and a-tubulin
genes were amplified in the same qPCR assay.
Flow cytometry was performed on propidium iodide (PI)-stained
cells to determine the DNA contents of the cells. At each 24 hr
interval after RNAi-induction, cells were fixed, stained and
processed for fluorescence activated cell sorting scan (FACScan)
analysis according to the previously established protocol . The
DNA peaks of PI-stained cells were analyzed with the FACScan
analytical flow cytometer (BD Biosciences). FL2-A DNA peaks
were calculated using CellQuest software (BD Biosciences). Cells
were also harvested, washed once with PBS, attached to Poly-L-
Lysine coated cover slips and mounted in vectashield medium with
DAPI (Vector Lab) for microscopic examination of nucleus (N)/
kinetoplast (K) configurations. Percentages of cells with different
N&K configurations in each sample were determined by counting
at least 200 cells with an epifluorescence microscope.
Epitope Tagging of Endogenous Proteins in Procyclic-
form T. brucei
The 39-terminal fragment of CDC27, AP1 and AP2 genes were
amplified by PCR and cloned into the pC-PTP-NEO plasmid
. The three resulting DNA constructs, pC.CDC27.PTP,
pC.AP1.PTP and pC.AP2.PTP were linearized with AvaI, BbsI
and XhoI, respectively, and transfected into the strain 427 cells by
electroporation. Stable clonal transfectants were selected under
40 mg ml21G418. For 3HA-epitope tagging of the endogenous
mitotic cycB2/cyc6 gene, the C-terminal fragment of the gene (DB
accession number: Tb11.01.8460) was amplified from the genomic
DNA using gene specific primers and cloned into the pC.3HA.Bla
plasmid, which is a modified version of the endogenous targeting
The APC/C of Trypanosoma brucei
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plasmid pC.PTP.NEO , and was transfected into either 29-13
procyclic cells or 29-13 cells with a stably maintained AP2 RNAi
plasmid. Transfectant selection was carried out under 10 ug ml21
Cells were harvested, washed twice in phosphate-buffered saline
(PBS), lysed by sonication in SDA-PAGE laemmli sample buffer
and cleared by a brief centrifugation. Samples were fractionated
on SDS-PAGE and transferred onto PolyVinylidene DiFluoride
(PVDF) membrane (Bio-Rad, CA). After blocking in TBST
(20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.1% Tween 20)
containing 5% skim milk, the immuno-blot membrane was probed
with primary anti-Prot C HPC4 monoclonal antibodies (Roche
diagnostics, CA) and stained with the secondary anti-mouse IgG-
HRP conjugate (Sigma, MO).
Cells were harvested, washed once in PBS and the cells
extracted in the lysis buffer (25 mM Tris-Cl, pH 7.6, 100 mM
NaCl, 1% Nonidet P-40, 1 mM dithiothreitol, and protease
inhibitor cocktail) for 30 min on ice. After being cleared by a brief
centrifugation, the lysate was incubated with anti-HA mAb
(Sigma, MO) at 4uC for 60 min and then with IgG Sepharose
beads overnight. The beads were sedimented by a brief centrifu-
gation, washed in PBS, cooked in SDS-PAGE sample buffer and
fractionated on SDS-PAGE. The gel was immuno-blotted onto
PVDF membranes (Bio-Rad, CA), and the HA tag was probed
with anti-HA HRP-conjugated mAb (Sigma, MO).
Functional Divergence between Yeast and T. brucei APC/
It has been generally believed that a strong evolutionary
conservation of APC/C function exists among APC/C subunit
proteins in organisms as diverse as Drosophila, Caenorhabditis elegans,
human and yeast [44,45,46]. Although sequence identities among
the individual subunit genes are in the range of only 9.6 to 25.5%
and sequence similarities in the range of only 21.5 to 54.5% (data
not shown), Drosophila APC11 , C. elegans CDC26  and
human APC13  were found capable of complementing their
counterparts in yeast. T. brucei APC/C subunits have 13.4–
23.0% sequence identity and 35.1–42.3% sequence similarity with
the yeast counterparts, which are not significantly different from
the other metazoa. In order to clarify if some of the T. brucei
APC/C subunits are capable of replacing the essential functions of
their yeast counterparts, we performed yeast complementation
assays to cover a total of 5 S. cerevisiae temperature sensitive
mutants apc1-1, cdc27-1, cdc16-1, cdc23-1 and apc10-1  with the
corresponding T. brucei subunit homologues. Results observed at
the restrictive temperature showed that the growth of yeast mutant
cells was fully rescued by homologous expression of the
corresponding yeast genes (Figure 1). But heterologous expression
of the corresponding T. brucei genes failed to rescue yeast cell
growth in all 5 cases tested (Figure 1). Therefore, none of the five
T. brucei APC/C subunit homologues was capable of complement-
ing their counterparts in the yeast. There could be thus a significant
structure-function discrepancy between the APC/C’s of the two
organisms (see Discussion below).
TAP of the APC1-PTP Protein Complex from T. brucei
In order to identify the intact APC/C core from T. brucei, the
potential scaffold protein subunit APC1 in the complex [29,47]
was tagged with a PTP and expressed in the procyclic form of T.
brucei via homologous genetic recombination. The APC1-PTP
fusion protein was then gently isolated from the cell lysate using
a well-established TAP procedure for purifying the entire APC/C
from T. brucei [10,35].
A stably transfected procyclic-form cell line of T. brucei strain
427 expressing APC1-PTP was isolated and the expression of
APC1-PTP in the crude lysate was confirmed with Western
blotting (Figures 2A lane 2 and 2B, lane 1). During the TAP,
Western blots indicated that the fusion protein was successfully
bound to the IgG beads (Figure 2B, lane 2) and eluted after TEV
protease digestion in a somewhat reduced molecular mass as
anticipated (Figure 2B, lane 3). The cleaved fusion protein was
then adsorbed to a column of anti-protein C beads (Figure 2B,
lane 4) and eluted effectively with EGTA (Figure 2B, lane 5)
resulting in a final preparation of purified APC/C. Samples from
each step of purification were examined with SDS-PAGE stained
with SYPRO-Ruby (Figure 2C). The stained gels showed
consistent changes of protein profiles through each step of
purification with a significant purification of the protein sample
achieved after TEV protease digestion and elution from IgG-beads
(Figure 2C, lanes 2 & 3). Further enrichment and elimination of
individual proteins were achieved after adsorption to the anti-
protein C beads and elution with EGTA. The protein profile of
the final product analyzed with SDS-PAGE (Figure 2C, lane 5)
was highly reproducible among several preparations of cells
(comparing Figure 2C, lane 5 and Figure 3A).
The protein profile represents the likely APC/C composition
from an asynchronous cell population, which is a population
enriched with G1 cells up to 80% shown in flow cytometry
(Figure 3A). The cells were then synchronized with 0.3 mM
hydroxyurea by an established procedure  resulting in 100%
of the cells arrested in late S-phase (Figure 3B). They were then
released from hydroxyurea and allowed to grow synchronously for
2.5 hrs when 65% of the population was found to have reached
the metaphase and 15% in the anaphase in flow cytometry and
immunofluorescence assays using chromosome passenger complex
1 (CPC1) protein as the marker (Figure 3C, Figure S1) . When
the time period of synchronous growth was extended to 4.5 hrs,
65–70% of the cells were in the anaphase, whereas the rest of the
cells had apparently gone beyond cytokinesis and reached the G1
phase (Figure 3D, Figure S1). The method thus enabled us to
prepare large batches of procyclic-form T. brucei cells enriched in
G1-phase, S-phase, metaphase and anaphase, respectively, and
subject them to the TAP (Figure 3). The results from SDS-PAGE
of the purified APC1-PTP protein complexes from the cells
enriched in different phases of the cell cycle are presented in
Figure 3 for direct comparisons. The protein profiles of each
sample appear highly similar to one another, and the intensities of
individual protein bands stained with SYPRO-Ruby in the gels
appear also relatively unchanged among the four cell samples
(Figure 3). This preliminary observation provides an indication
that T. brucei APC/C remains relatively unchanged throughout
the cell cycle without any association or dissociation with other
protein(s). This tentative conclusion requires further verification
with LC/MS/MS, which is capable of also identifying the
individual APC1-PTP-assocaited proteins and compare them with
the 7 APC/C subunit homologues previously found in T. brucei
genome database .
LC-MS/MS Analyses of T. brucei APC/C Derived from
Different Phases of Cell Cycle
Samples of the APC1-PTP fusion protein complex purified from
duplicate or triplicate cell samples, each enriched in different
The APC/C of Trypanosoma brucei
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Figure 1. Functional divergence between T. brucei and yeast APC/C subunits. Yeast complementation assay. Temperature-sensitive (ts)
yeast mutants of APC/C subunit genes apc1-1, cdc16-1, cdc23-1, cdc27-1 and apc10-1 were transformed with pRS416-ADH plasmids expressing
corresponding yeast (y) or T. brucei (t) full-length APC/C genes. After transformation and selection, the cloned cells were grown either at the
permissive (25uC) or the restrictive (37uC) temperature. Wild-type W303 (WT) cells and yeast ts mutant cells transfected with the empty vector
(plasmid) were used as positive and negative controls, respectively.
The APC/C of Trypanosoma brucei
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phases of the cell cycle, were fractionated in SDS-PAGE (Figure 3).
Individual protein bands in the gels were sliced out, diced and
digested with trypsin by the well-established procedures. As
a control, lysate of 427 cells transfected with empty pC.PTP
plasmid was purified by the same procedure and individual protein
bands were sliced from the SDS-PAGE and processed for trypsin
digest as mentioned above. Each digest was then fractionated with
LC, and subjected to MS/MS analysis for peptide identification.
The results in protein identities versus the individual protein bands
in a SDS-PAGE gel, are represented in Figure 3A of a sample
purified from a cell population enriched in G1 phase. Thirteen
proteins are identified in the purified APC-PTP complex. Other
than the persistent presence of a-tubulin, b-tubulin and TEV
protease, which are also present in the control sample and are thus
likely common contaminants (data not shown), the rest of the 10
proteins in the purified complex are found to include the 7 APC/
C subunit proteins originally derived from T. brucei genome
database plus 3 un-identified proteins designated the APC/C-
Associated Proteins (AP); AP1, AP2 and AP3, respectively. The
rest of the undefined protein bands have also been analyzed, and
they were primarily degradation products from the 10 identified
proteins and the high abundance background proteins.
These 10 identified proteins all appeared in the purified APC/C
samples from the G1-phase (Figure 3A), S-phase (Figure 3B),
metaphase (Figure 3C) and anaphase enriched cells (Figure 3D) at
relatively unchanged quantities. There has been no other protein
detected in any of the 4 purified APC/C samples, which suggests
that there is no additional protein above the limit of detection that
is associated with APC/C that could sustain the mild TAP
experimental conditions during any phase of the cell cycle
progression of T. brucei. The 10 proteins may thus constitute
the core subunits of the APC/C in T. brucei.
More detailed data from the MS/MS analysis are presented in
Table 1 and Table S1. The estimated molecular masses of the
7 APC/C subunits all agree well with those derived from the
genomic database. All the proteins are each identified with
Figure 2. TAP of APC1-PTP and identification of APC1-associated proteins. (A) Lysates of strain 427 cells transfected either with empty
vector (control, lane 1) or pC.APC1.PTP (lane 2) were fractionated on 10% SDS-PAGE gel, Western blotted and simultaneously probed with anti-
protein C antibody (HPC4) and anti-tubulin. The blot was then detected with an anti-mouse HRP-conjugated secondary antibody. (B) Stepwise
Western blot monitoring of APC1-PTP during TAP. Samples were analyzed from 1. IgG input (1x), 2. IgG-Sepharose flow-through (1x), 3. The elute
from IgG-Sepharose after TEV-protease digestion (5x), 4. The flow-through from anti-protein C matrix (5x), and 5. The EGTA final elute (20x). The blot
was probed with HPC4 antibody and the values (in x) represent the relative amounts of samples analyzed. (C) Samples collected from TAP as in (B)
were fractioned on 10% SDS-PAGE gel and stained with SYPRO-Ruby. (**) on the top indicates the position of APC1-PTP fusion protein.
The APC/C of Trypanosoma brucei
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adequate numbers of unique peptides and sufficient percentages of
coverage in the two tables with but one exception with APC11;
data on this protein are apparently missing in the samples from
G1-phase and anaphase enriched cells. This is most likely
attributed to the relatively low molecular weight of APC11
(10 kD) that facilitates diffusion of the protein from the gel during
SDS-PAGE [48,49]. This small protein is thus still considered an
integral component of T. brucei APC/C.
To further verify that there is no significant fluctuation of the
intracellular levels of APC/C subunits during cell cycle pro-
gression, APC1, CDC27, AP1 and AP2 were each tagged with
PTP at the C-termini, integrated into chromosomes via homol-
ogous recombination and expressed in transfected cells as
previously described. The transfected cells were then synchronized
with hydroxyurea into late S-phase  and released for
synchronized growth for 8 hrs, which covers the entire cell cycle
of T. brucei . Hourly samples were taken during the incubation
and examined on Western blots. The results, presented in Figure
S2, indicated little changes of the protein levels in the four samples,
suggesting limited fluctuation in the level of APC/C subunits
during cell cycle progression of procyclic-form T. brucei.
Tentative Identification of AP1 as APC4
In NCBI BLAST analysis, the sequence of AP1 was successfully
aligned with the protein fragments from the core APC/C subunit
APC4 from several eukaryotic organisms including Schizosacchar-
omyces pombe and Arabidopsis thaliana (data not shown). By searching
the domain database of Pfam for specific motifs, the most
significant structural similarity between AP1 and the APC4’s was
identified in the Apc4-WD40-like domain. The Pfam-A (CL0186)
domain database identified a bit score of 11.2 and an E value of
0.18 for AP1. The WD40 domain in APC4 is an N-terminal
propeller-shaped domain that serves to stabilize the interaction
with APC5 in the APC/C complex . All known APC4 proteins
have been found to contain a single N-terminal WD40-like
domain except for the A. thaliana and T. brucei homologues, each of
which contains two WD-40-like domains (data not shown). This
finding was further confirmed by a reciprocal BLAST analysis
Figure 3. The T. brucei APC/C profiles during different phases of the cell cycle progression. The final EGTA elutes from TAP of APC1-PTP
from (A) the G1 phase enriched cells; (B) The late S-phase cells; (C) The metaphase enriched cells and (D) The anaphase enriched cells were each
fractioned in 10% SDS-PAGE and stained with SYPRO-Ruby. The individual protein bands identified by subsequent LC-MS/MS analysis are indicated
on the right-hand side of each gel panel with the molecular masses (in kDa) indicated on the left-hand side. Tub and TEV denote contaminating
tubulins and TEV protease, respectively. The cell samples were also analyzed by flow cytometry and the histograms of cellular DNA contents are
displayed below the corresponding SDS-PAGE panels.
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searching the TriTryp database with yeast and vertebrate APC4
sequences. AP1 is thus re-designated APC4.
AP2 and AP3 remain unidentified after extensive BLAST and
domain search analysis. But their orthologues have been found in
the genomic databases of Trypanosoma cruzi and Leishmania major
(data not shown), suggesting that they could be the common core
subunits of APC/C among all Kinetoplastidae.
RNAi Knockdown of AP2 Arrested the Cells in G2/M
Expression of the three newly identified APC/C subunits was
each knocked down with RNAi for potential cell phenotype. By
inducing target-specific RNAi in procyclic-form cells , specific
mRNAs were significantly depleted. Data from qPCR showed that
APC4 mRNA was almost totally depleted (Figure S3A), AP2
transcript was reduced more than 80% (Figure 4A), whereas AP3
mRNA was also knocked down by 80% (Figure S4A). Only the
knockdown of AP2, however, turned out to demonstrate a clear
phenotype; the cells continued to grow for two more days
following the RNAi induction, but apparently ceased growing
thereafter (Figure 4B). Microscopic examination of DAPI-stained
cells showed a gradual increase of cells containing 1 nucleus and 2
kinetoplasts (1N2K) and a slow reduction in the number of 2N2K
cells when compared with the control (Figure 4C). The elongated
nucleus and two well segregated kinetoplasts in the 1N2K cells
suggest that the cells are arrested in metaphase [3,51,52]
(Figure 4C, left lower panel). There was also a dramatic increase
of 0N1K cells (zoids) (Figure 4C, right lower panel) up to 25-30%
of the total cell population on day 4 of the RNAi induction. This is
a hallmark of the procyclic cells arrested during mitosis, in which
cytokinesis and cell division still carries on, generating anucleated
cells [4,6]. All these data thus provide a strong indication that the
procyclic-form cells are arrested during mitosis, likely in the
metaphase, following a knockdown of AP2. Flow cytometry
indicated a diminished 2C cell numbers and an increase of 4C cell
population plus a prominent band of cells with less than a 2C
DNA content after 4 days of RNAi, most likely the zoids
(Figure 4D). Thus all the experimental data point to a blocked
mitosis, which are highly similar to the data from a knocking down
of APC1 or CDC27 in our previous study, showing that the
procyclic-form T. brucei was arrested in the metaphase .
Being a likely subunit of APC/C, the knockdown of AP2 may also
result in a blocking of metaphase-anaphase transition.
APC4 and AP3 were also knocked down, but the results,
presented in Figures S3 and S4, indicated no clear phenotype.
They are thus classified as the APC/C subunits like APC2,
CDC16, CDC23, APC10 and APC11; whose knockdowns from
T. brucei did not register a phenotype .
AP2 Depletion Stabilized the Mitotic Cyclin CycB2/cyc6
Following our tentative identification of the composition of
APC/C in T. brucei, the next question concerned the potential
function of this protein complex. Since a homologue of securin/
Pds1 has not yet been found in T. brucei, we focused our attention
on another commonly known substrate of APC/C; the mitotic
cyclin B, whose destruction by the combined actions of APC/C
and 26S proteasome toward the late phase of mitosis is required
for mitotic exit in yeast and metazoa [22,53]. A functional
homologue of mitotic cyclin B, cycB2/cyc6, has been identified in
T. brucei [4,28]. It is involved in activating the mitotic CDK cdc2-
related-kinase 3 (CRK3) during mitosis . An RNAi knockdown
of cycB2/cyc6 arrested T. brucei in the G2/M phase , though
a potential disappearance of cycB2/cyc6 from T. brucei in the late
phase of mitosis has not yet been studied.
Table 1. LC-MS/MS analysis of the APC/C samples purified from different phases of T. brucei cell cycle progression.
The number of unique peptides and percent sequence coverage are summarized across the replicate analysis as indicated. For a complete analytical report, please refer to Table S1.
The APC/C of Trypanosoma brucei
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Since an APC2 knockdown in mice and an APC16 depletion in
human cells were reported to stabilize the mitotic cyclin B [48,54],
we knocked down the expression of AP2, and monitored the fate of
CycB2/cyc6 thereafter. To do this, one of the two endogenous
alleles encoding CycB2/cyc6 was tagged with 3HA through
homologous recombination and the expression of CycB2/cyc6-
3HA was verified on Western blot (Figure 5A).
The cells expressing CycB2/cyc6-3HA were treated with
0.3 mM hydroxyurea for 16 hrs for synchronization of the cells
in late S-phase, and were released for synchronous growth. Hourly
cell samples were taken for Western blot analysis. The results
(Figure 5B) showed that following an initial increase in the
beginning 3 hrs, when the cells entered from the late S-phase into
the metaphase (see Figure 3 and Figures S1 & S5), there was
a steady decrease in the level of CycB2/cyc6 thereafter. The
decrease continued up to 8 hrs when a full cell cycle was
completed . CycB2/cyc6 is thus confirmed to disappear like the
other mitotic cyclins in the late phase of mitosis.
To verify if the APC/C in T brucei is involved in regulating the
turnover of CycB2/cyc6, the same experiment was repeated using
the AP2 RNAi cell line. In the control cells without RNAi
induction, the time course of CycB2/cyc6-3HA level change in the
synchronized cells paralleled that observed in Figure 5B except
that the heightened level of the cyclin was extended a little longer
to 4 hours before the decline (Figure 5C), which could be
attributed to slightly different physiological conditions between
different batches of synchronized cells. When AP2 RNAi was
induced for 48 hrs (see Figure 4B) and the cells were then
subjected to hydroxyurea synchronization and released for
synchronous growth, expression of AP2 was knocked down as
(Figure 5C). The level of CycB2/cyc6 reached the high plateau
within 3 to 4 hrs, reduced by about 30% after 5 to 6 hrs, but
restored to the original high level after 7 to 8 hours (Figure 5C).
Comparing with the no RNAi induction control, it is clear that
a knockdown of AP2 expression resulted in stabilization of CycB2/
cyc6 in the late mitotic phase of T. brucei. The progression of
synchronized AP2 knockdown cells was also analyzed with flow
cytometry and the data are presented in the lower right panel of
Figure 5C. When these data are compared with those from the
progression of synchronized wild-type cells in Figure S5, it is clear
that the progression of AP2 knockdown cells is arrested in the G2/
M phase. Since AP2 is a core subunit of APC/C, it is most likely
that the function of APC/C is required for the destruction of
CycB2/cyc6 and passage of cells beyond mitosis. From the
knowledge derived from other eukaryotes, it is reasonable to
Figure 4. RNAi knockdown of AP2 in T. brucei procyclic cells. (A) Specific AP2 mRNA depletion was assayed with qPCR after RNAi induction for
72 hours. Data are derived from two independent experiments with standard deviation (6SD) error bars displayed. (B) The rate of cell growth after
RNAi induction. The mean 6SD values are derived from two independent experiments. (C) Time samples (d, days) of the RNAi-induced cells were
stained with DAPI and analyzed with a microscope for numbers and configurations of nucleus (N)/kinetoplast (K) among individual cells. Data are
presented as percentages of cells from a total number of ,200 cells at each time point from two independent induction experiments. Error bars
represent the standard deviation (SD) are presented. Representative DAPI-stained 1N2K and 0N1K cells after 4 days of RNAi induction are shown
(lower panel). (D) Time-dependent (d, days) changes of DNA profiles in the RNAi-induced cells were analyzed by flow cytometry.
The APC/C of Trypanosoma brucei
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postulate that the APC/C of T. brucei functions as an E3 ligase
that poly-ubiquitinates CycB2/cyc6 and subjects it to 26S
The Mitotic Cyclin in T. brucei is Likely Poly-ubiquitinated
by APC/C and Degraded by 26S Proteasome during
The presence of 26S proteasome and the structure and function
of this protein complex in T. brucei have been thoroughly
investigated and identified by us in our previous studies [34,55]. In
order to verify if the turnover of CycB2/cyc6 depends on the
function of proteasome in T. brucei, the latter was treated with
a reversible inhibitor of proteasome, MG132, at 20 mM known to
totally inhibit the proteasome activity in T. brucei . Cells
expressing CycB2/cyc6-3HA and synchronized with hydroxyurea
were released for synchronous cell cycle progression in the
presence of MG132. Hourly cell samples were taken for
immunoprecipitation with anti-HA followed by analysis on
Western blot stained with the antibodies to HA or ubiquitin.
The results showed that while there is a drop of CycB2/cyc6 level
in the cells after 3 hrs without MG132 treatment as anticipated
(Figure 6A, upper panel), it keeps increasing up to 6-fold of the
original value after 8 hrs of growth in the presence of MG132
(Figure 6B, upper panel). Apparently, the proteasome function is
required for the degradation of CycB2/cyc6 during mitosis of T.
brucei. The time-dependent accumulation of ladders of higher
molecular mass bands in the MG132 treated samples suggests also
formation of poly-ubiquitinated CycB2/cyc6 (Figure 6B, upper
When the Western blot of the anti-HA immunoprecipitate was
stained with anti-ubiquitin, there was little specific staining for
poly-ubiquitinated proteins while MG132 was not present
(Figure 6A, middle panel). In the presence of MG132, however,
there was a steady increase in the intensity of a ladder of protein
bands on top of the CycB2/cyc6 band beyond the mitotic phase of
the cells (Figure 6B, middle panel). They are most likely the poly-
ubiquitinated CycB2/cyc6 that cannot be degraded when the
Figure 5. Effect of AP2 knockdown on the expression of CycB2/cyc6 in T. brucei. (A) Western blot analysis of the cell lysates of; 1. wild-type
cells; and 2. the cells expressing CycB2/cyc6-3HA. The blot is stained with anti-HA mAb and the anti-tubulin antibody was used as a loading control.
(B) The Control Cells; Cells expressing CycB2/cyc6-3HA were synchronized in late S-phase with hydroxyurea and released for synchronous growth for
8 hrs to complete a cell cycle. Hourly cell samples were lysed and fractionated in SDS-PAGE, immuno-blotted and probed with anti-HA mAB for
CycB2/cyc6-3HA expression. The time course of level changes of CycB2/cyc6-3HA was quantified against tubulin control using the ImageJ software
and presented in the lower panel. The relative abundance of CycB2/cyc6 has a value set at 1 from the zero time point. Error bars represent the SD
from two independent experiments. (C) AP2 RNAi cells expressing CycB2/cyc6-3HA were induced (+Tet) for RNAi for 48 hours, and then synchronized
to late S-phase with hydroxyurea and released for synchronous growth for 8 hours while the RNAi was maintained. CycB2/cyc6-3HA expression was
monitored as described in (B). RT-PCR analysis of AP2 transcript levels in the hourly cell samples was performed. They and the levels of CycB2/cyc6
from the Western analysis are plotted versus time and presented in the lower left panel. Error bars represent the SD from two independent
experiments. The same time samples from AP2 RNAi induced culture (+ Tet) after hydroxyurea release were analyzed by flow cytometry and the data
are presented in the lower right panel.
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proteasome activity is inhibited. It is probable that the poly-
ubiquitinated CycB2/cyc6 is a product of the APC/C action.
In the present study, we indicated that among the five APC/C
subunit homologues in T. brucei; APC1, CDC16, CDC23,
CDC27 and APC10, none was capable of complementing the
function of their counterparts in yeast. Although APC/C has
a conserved presence and function among all the eukaryotes
examined thus far, the sequences of individual APC/C subunit
proteins do not appear to be highly conserved . Drosophila
APC11 , C. elegans CDC26  and human APC13 
have been, however, tested in the yeast complementation assays
and found capable of substituting the corresponding subunits in
yeast. It makes thus the negative outcome from testing all 5 T.
brucei subunits a little difficult to explain from a simple view on
protein sequence discrepancies. An alternative explanation could
be by postulating a distinctive mechanism of inter-subunit
interactions in constituting the APC/C in T. brucei, i.e., T.
brucei APC/C subunits may be incapable of incorporating into
The assembly and three-dimensional structure of APC/C
remain poorly understood for the time being . Three-
dimensional electron microscopic structural analysis of yeast
APC/C located APC1 in an L-shaped rod that links APC2 to
CDC23, whereas CDC23 is connected to APC5 with APC4
interconnecting APC1 and APC5 [58,59]. APC1, the largest
APC/C subunit, consists of 11 highly repetitive 35–40 amino acid
proteasome-cyclosome (PC) sequences at the C-terminus. This PC
motif is shared with the RPN1 and RPN2 subunits of the
proteasome 19S regulatory particle , and is assumed to be the
main binding sites for other subunits in forming APC/C. APC/C
purified from the yeast mutant apc1D was found lacking
association among the majority of other subunits . APC1 is
Figure 6. Polyubiquitination and proteasome degradation are involved in the turnover of CycB2/cyc6 during mitosis. Cells arrested in
late S-phase by hydroxyurea treatment were released for synchronous growth for 8 hours in the absence (A) or the presence (B) of 20 mM MG132.
Hourly cell samples were lysed, subjected to immunoprecipitation with anti-HA mAb and fractionated with SDS-PAGE. Western blotting was used to
analyze the time course of the level of CycB2/cyc6-3HA with anti-HA mAb (upper panels) or polyubiquitination staining with anti-Ubiquitin (Sigma,
U5379) antibody (middle panels). Anti-Bip was used as loading controls. A quantitative analysis of CycB2/cyc6-3HA relative abundance and ubiquitin
staining intensity for each experiment were plotted versus time in the bottom panels. Error bars represent the SD from two independent
The APC/C of Trypanosoma brucei
PLOS ONE | www.plosone.org11 March 2013 | Volume 8 | Issue 3 | e59258
thus classified as the major scaffold protein in yeast APC/C .
These 11 PC repeats are, however, absent from T. brucei APC1
(data not shown), which could provide a supporting evidence that
T. brucei APC/C may have a mechanism of assembly highly
distinctive from that in yeast. This distinction may explain why the
APC/C subunits from T. brucei are not complementary to the
corresponding subunits missing from the yeast.
The outcome from our TAP and LC-MS/MS analysis of
APC1-PTP protein complex indicates that T. brucei APC/C is
made of 10 core subunit proteins. The composition and level of
APC/C remain apparently unchanged throughout the entire cell
cycle of procyclic-form T. brucei. An even more intriguing finding
was that neither CDC20 nor MCC complex proteins were found
associated with APC/C during any phase of the cell cycle. This is
in contrast to that observed among the other eukaryotes. An intact
MCC complex with CDC20 protein had been co-purified with
APC/C subunits using a similar TAP procedure in human cells
. Also, CDC20 was detected in the mitotic-enriched APC/C-
TAP sample from the budding yeast and fission yeast . In
budding yeast, the MCC components MAD1, MAD2 and MAD3
throughout different stages of the cell cycle . A common
factor enabling all the complex formations mentioned above is
CDC20. The fact that the T. brucei CDC20 homologue is not
associated with APC/C at all during all phases of the cell cycle
shows that it is not performing the function of mediating a binding
of APC/C to MCC or activating APC/C to poly-ubiquitinate
mitotic cyclin CycB2/cyc6 . Our previous finding that an
RNAi knockdown of CDC20 showed no detectable cell phenotype
(data unpublished) tends to support this conclusion. The CDC20
homologue in T. brucei could be a structural homologue but not
a functional one.
Our findings that there is no apparent structural homologues of
MCC subunits in T. brucei genomic database (data not shown)
and that no detectable protein was found associated with APC/C
in the metaphase and dissociated from it in the anaphase of T.
brucei provide a strong indication that this organism may not have
a similar mechanism of regulating metaphase-anaphase transition
as observed in other eukaryotes [63,64]. A similar observation has
been also made in the budding yeast, in which neither the MCC
subunit protein MAD2 nor the spindle assembly checkpoint
complex is required for normal cell growth [65,66]. This
peculiarity was attributed to the persistent presence of mitotic
spindles throughout the yeast cell cycle . It may not require
specific spindle assembly prior to mitosis to facilitate capture of the
mitotic microtubules by the kinetochores in chromosomes and bi-
orientations of the chromosomes on the mitotic spindle . T.
brucei is an even more primitive organism than yeast and may not
maintain an active regulation of spindle assembly either. This
postulation may explain the apparent absence of a MCC-like
complex in T. brucei. But the mechanism of activation of APC/C
in triggering the metaphase to anaphase transition remains still
unclear. The question whether a functional homologue of securin/
Pds1 is present in T. brucei requires still an answer.
Other than the 7 subunits already identified in T. brucei APC/
C genome DB , three additional subunit proteins, APC4, AP2
and AP3, were identified in this protein complex. AP2 and AP3
cannot find homologous proteins in all the genomic databases
other than those of Kinetoplastidae. Searches for common motifs
in APC/C subunits such as PC repeats, cullin homology, TPR,
RING H2 and WD40/IR in these two proteins also turned out
negative results. AP2 and AP3 could thus be specific subunits of
only the APC/C’s among the Kinetoplastidae.
Among the 10 core subunits identified in T. brucei APC/C,
only a knockdown of three of them, APC1 , CDC27  and
AP2, each resulted in an arrest of T. brucei procyclic-form cells in
the metaphase. In budding yeast, 8 of the 13 APC/C subunits
(APC1, APC2, APC4, APC5, APC11, CDC16, CDC23, CDC27)
were found indispensible for viability. Their depletion abrogates
the APC/C catalytic activity and blocks yeast cell cycle pro-
gression [25,50,67,68]. The rest of the subunits are either non-
essential (APC9, CDC26, SWM1, MND2) [25,46], or their loss
(APC10/DOC1) affects only the complex integrity or the rate of
substrate binding and processing [69,70]. SWM1 and CDC26
only have essential roles at restrictive temperatures in maintaining
structural stability of the complex , whereas SWM1 and
MND2 are essential during meiotic cell division . In S. pombe,
individual knockdowns of APC14 and APC15 did not display any
abnormal phenotype but the cells developed a temperature–
sensitive phenotype and chromosome segregation abnormalities
when two proteins were mutated simultaneously or depleted with
other APC components . The fact that 7 out of 10 T. brucei
APC/C subunits are dispensable for cell cycle progression could
mean that they have redundant structural and functional roles
with the other subunits. It may require double or multiple
knockdowns of these subunits to inflict a detectable phenotype.
Despite all the apparent structural and functional uniqueness, T.
brucei APC/C showed also some conserved functions as those
observed in the other eukaryotes. The knockdowns of APC1,
CDC27 and AP2 were each found to arrest the procyclic form T.
brucei cells in metaphase, suggesting that the APC/C function is
required for metaphase-anaphase transition. The APC/C function
is also needed for degradation of the mitotic cyclin CycB2/cyc6
during mitosis in T. brucei. The degradation, essential for mitotic
exit among the other eukaryotes [16,22], is mediated by the 26S
proteasome in T. brucei, which recognizes poly-ubiquitinated
CycB2/cyc6 as substrate. This ubiquitination-dependent degra-
dation is likely provided by the poly-ubiquitinating action of APC/
C on CycB2/cyc6, a function that apparently remains conserved
in T. brucei.
We have thus characterized an APC/C in T. brucei that
performs apparently both of the well-known functions during
mitosis. But its unusually simple composition and the apparent
functional redundancy among the 10 subunits distinguish it from
the other APC/C’s. The lack of an MCC mediated regulatory
mechanism and the apparent absence of securin/Pds1 and
CDC20 in T. brucei further demonstrates a significant discrep-
ancy between T. brucei APC/C and the others. The APC/C in T.
brucei could be thus easily classified as a potential target for anti-
cycle progression in T. brucei. (A) Western blotting of
TbCPC1-eYFP expression in TbAPC1-PTP cells. The same cell
extract was immuno-probed with HPC4 antibody for PTP
expression and anti-GFP antibody for eYFP expression. Asterisk
indicates a non-specific anti-GFP immune-reactive band and
upper and lower arrows indicate the positions of APC1 and CPC
fusion proteins, respectively. (B) Flow cytometric analysis of
hydroxyurea synchronized cells at 0, 2.5 and 4.5 hours after
release. (C) Fluorescence microscopic analysis of cells co-expres-
sing APC1-PTP and CPC1.eYFP at S-phase (0 hr), metaphase
(2.5 hr) and anaphase (4.5 hr) after hydroxyurea release. DIC,
DAPI and YFP filters are shown with merge composite.
Bars=2 mM. (D) Quantitative microscopic analysis of eYFP
Hydroxyurea (HU) synchronization of the cell
The APC/C of Trypanosoma brucei
PLOS ONE | www.plosone.org 12March 2013 | Volume 8 | Issue 3 | e59258
signals. Approximately 200 cells from each sample were counted
and data are presented as localization pattern of S-phase (0 hr),
metaphase (2.5 hr) and anaphase (4.5 hr) from two independent
AP1 and AP2 in synchronized T. brucei growth. Cells
expressing endogenous PTP fusion proteins of (A) APC1; (B)
CDC27; (C) AP1 and (D) AP2 were arrested in late S-phase after
16 hr treatment with 0.3 mM hydroxyurea. Samples of the
released cells were taken every hour and their lysates monitored
for the expression of individual fusion proteins by immunoblotting
using the HPC4 antibody with anti-tubulin antibody used as
Time courses of expression of APC1, CDC27,
assay of the level of AP1/APC4 mRNA 72 hrs after the induction
of AP1/APC4 RNAi. (B) The rate of cell growth was monitored
for 7 days after the RNAi induction. (C) N/K tabulations of the
AP1/APC4-depleted cells on days 0, 1, 3 and 5 after RNAi
induction. (D) Flow cytometric analysis of DNA contents in AP1/
APC4-depleted cells. Little distinction was observed in the results
from RNAi-induced and un-induced cells.
The RNAi knockdown of AP1/APC4. (A) qPCR
are as described in Figure S3.
RNAi knockdown of AP3. Panels A, B, C and D
in T. brucei with hydroxyurea. Strain 29-13 procyclic T.
brucei cells expressing CycB2/cyc6-3HA were treated with 0.3 mM
Synchronization of the cell cycle progression
hydroxyurea for 16 hours, washed twice in fresh medium and
allowed to progress synchronously for 8 hours. The hourly cell
samples were stained with propidium iodide, processed for flow
cytometry and the FL2-A DNA peaks are presented. DNA
contents (2C or 4C) were shown at the bottom.
unit proteins. Protein components of the APC/C complex were
identified by comparison of replicate LC-MS/MS peptide
sequencing analyses. To be considered a candidate APC/C
subunit, it was required that the protein was observed in all four
cell cycle stages (G1, S-phase, Metaphase and Anaphase), that at
least one experiment included a minimum of two unique peptides
for confident protein identification, and that the protein was not
observed in a control experiment.
Identification by LC-MS/MS of APC/C sub-
We thank Drs D. Toczyski, P. Walter and D. Morgan at the University of
California San Francisco for their generous gift of yeast wild type and
APC/C mutant strains. Mass spectrometry analysis was performed in the
Bio-Organic Biomedical Mass Spectrometry Resource at the University of
California San Francisco (A.L. Burlingame, Director). We would also like
to thank Chris Adams and the Stanford University Mass Spectrometry
Core, for data acquisition and analysis of one of the MS data sets.
Performed the LC-MS/MS: GK MB. Conceived and designed the
experiments: CCW MB. Performed the experiments: MB GK. Analyzed
the data: MB GK. Contributed reagents/materials/analysis tools: CCW
ALB. Wrote the paper: MB CCW.
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