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RESEARCH ARTICLE

The bi-lobe-associated LRRP1 regulates Ran activity inTrypanosoma brucei

Anaıs Brasseur1,`, Shima Bayat1,`, Xiu Ling Chua1, Yu Zhang1, Qing Zhou3,*, Boon Chuan Low2 andCynthia Y. He1,§

ABSTRACT

Cilia and flagella are conserved eukaryotic organelles important

for motility and sensory. The RanGTPase, best known for

nucleocytoplasmic transport functions, may also play a role in

protein trafficking into the specialized flagellar/ciliary compartments,

although the regulatory mechanisms controlling Ran activity at the

flagellum remain unclear. The unicellular parasite Trypanosoma

brucei contains a single flagellum necessary for cell movement,

division and morphogenesis. Correct flagellum functions require

flagellar attachment to the cell body, which is mediated by a

specialized flagellum attachment zone (FAZ) complex that is

assembled together with the flagellum during the cell cycle. We

have previously identified the leucine-rich-repeat protein 1 LRRP1

on a bi-lobe structure at the proximal base of flagellum and FAZ.

LRRP1 is essential for bi-lobe and FAZ biogenesis, consequently

affecting flagellum-driven cell motility and division. Here, we show

that LRRP1 forms a complex with Ran and a Ran-binding protein,

and regulates Ran–GTP hydrolysis in T. brucei. In addition to mitotic

inhibition, depletion of Ran inhibits FAZ assembly in T. brucei,

supporting the presence of a conserved mechanism that involves

Ran in the regulation of flagellum functions in an early divergent

eukaryote.

KEY WORDS: Bi-lobe, Flagellum, LRRP1, RanBP, Ran

INTRODUCTIONProtein trafficking into the membrane-bound flagellum or cilium

is tightly controlled by polarized vesicular trafficking as well as a

diffusion barrier located at the base of the structure (Hsiao et al.,

2012), creating a privileged compartment crucial for flagellar

biogenesis and functions. In recent years, several studies have

described a role of the small GTPase Ran – best known for its

biological function in nucleocytoplasmic protein transport (Sazer

and Dasso, 2000) – in the regulation of ciliary protein targeting in

various eukaryotes (Dishinger et al., 2010; Fan et al., 2011;

Ludington et al., 2013). Whereas the regulation of Ran activity at

the nucleus is relatively well-understood, how Ran activity is

regulated at the ciliary base is not yet clear.

In Trypanosoma brucei, the etiological agent for human african

trypanosomiasis (HAT; also known as sleeping sickness), a single

flagellum is present in each parasite cell, required for cell

locomotion, immune evasion, cell division, organelle positioning

and cell length regulation (Absalon et al., 2008; Absalon et al.,

2007; Broadhead et al., 2006; Engstler et al., 2007; Kohl et al.,

2003; Ralston et al., 2006). The normal function of T. brucei

flagellum requires flagellum attachment to the cell body, through

the flagellum attachment zone (FAZ), a unique feature found in T.

brucei and related trypanosomatid parasites (Vaughan, 2010).

Disruption of flagellum attachment in T. brucei leads to defects in

flagellum-driven cell motility and cell division as well as changes

in cell morphology, often resulting in cell death (LaCount et al.,

2002; Sun et al., 2013; Vaughan et al., 2008; Woods et al., 2013).

Little, however, is known about the protein composition of the

FAZ complex and how it is assembled together with the flagellum.

A bi-lobe structure is present at the proximal base of the single

flagellum, partially overlapping with the proximal tip of the FAZ

(Morriswood et al., 2012; Shi et al., 2008) and adjacent to the

single Golgi complex (He et al., 2005). Previously thought

to be required for Golgi duplication, recent studies suggest a

more fundamental role of the bi-lobe in FAZ assembly that,

consequently, affects flagellum functions in organelle

inheritance, cell division and cell morphogenesis (Zhou et al.,

2010; Zhou et al., 2011). Depletion of the bi-lobe proteins

Centrin2 (gene accession number Tb927.8.1080) and LRRP1,

both leads to defects in FAZ assembly, flagellum inheritance,

flagellum-driven cell motility and cell division (Shi et al., 2008;

Zhou et al., 2010). To better understand the bi-lobe and the

molecular function of LRRP1 and to identify LRRP1-interacting

proteins, we carried out yeast-two-hybrid screens. The results

showed that LRRP1 forms a complex with Ran GTPase and

a previously uncharacterized Ran-binding protein. Further

functional characterization suggested a role of LRRP1 as a Ran

regulator specific for flagellar functions.

RESULTSYeast-two-hybrid screening for LRRP1-interacting proteinsT. brucei LRRP1 has previously been identified in a comparative

proteomics screening for flagellum-associated proteins (Zhou et al.,

2010). The 713-aa protein lacks sequence homology to other

organisms but is conserved in trypanosomatids. LRRP1 is stably

associated with the bi-lobe structure in T. brucei, shown by stable

expression of YFP-tagged LRRP1 or by immunostaining with

antibody against LRRP1 (Zhou et al., 2010). LRRP1 comprises an

N-terminal leucine-rich repeat (LRR) domain followed by a 50-aa-

long coiled-coil region within the C-terminal part (Fig. 1A). LRR

1Department of Biological Sciences, Centre for BioImaging Sciences, NationalUniversity of Singapore, 14 Science Drive 4, S1A #06-04, Singapore 117543,Singapore. 2Singapore Mechanobiology Institute, National University ofSingapore, T-Lab, #10-01, 5A Engineering Drive 1, Singapore 117411,Singapore. 3University of Texas Medical School.*Present address: Department of Microbiology and Molecular Genetics,University of Texas Medical School, Houston, TX 77030, USA.`These authors contributed equally to this work

§Author for correspondence (dbshyc@nus.edu.sg)

Received 13 December 2013; Accepted 14 August 2014

� 2014. Published by The Company of Biologists Ltd | Journal of Cell Science (2014) 127, 4846–4856 doi:10.1242/jcs.148015

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domains are generally required for protein–protein interactions(Bella et al., 2008; Kobe and Kajava, 2001). The LRR in LRRP1belongs to the ribonuclease inhibitor (RI)-like subfamily – with anExpect (E)-value of 1.27610223 – whose structure has been first

described in the porcine ribonuclease inhibitor (Kobe andDeisenhofer, 1993). The RI-like LRR domain is also found in allknown Ran–GTPase-activating proteins (RanGAPs) such as yeast

rna1 (Hillig et al., 1999), human RANGAP1 (Haberland and Gerke,1999) and plant RanGAP (Rose and Meier, 2001). In T. brucei, theshort coiled-coil domain alone was sufficient to mediate

bi-lobe localization of the YFP reporter (CC–YFP) (Fig. 1C;supplementary material Fig. S1A), possibly through its proteinoligomerization activity (Meier et al., 2010). The LRR domain,

however, does not contain bi-lobe-targeting information and LRR–YFP was found throughout the parasite cell (Fig. 1B;supplementary material Fig. S1A).

A yeast-two-hybrid screening was performed to search for T.

brucei proteins that interact with the LRR or the coiled-coil domain.One LRR-binding candidate, encoded by Tb927.10.8650, is a 337-aa hypothetical protein that is conserved in trypanosomatids but

has no homolog in other eukaryotes. It contains a putative Ran-binding domain (RanBD, with an E-value of 1.29610210) in the C-terminal region (Fig. 1D), suggesting that it binds to Ran in T.

brucei.

T. brucei contains a bona fide Ran that is essential fornuclear divisionThe highly conserved small GTPase Ran is a key regulator ofnucleocytoplasmic transport (Sazer and Dasso, 2000; Yudin and

Fainzilber, 2009). Relying on the combined action of RanGEF(RCC1) in the nucleus and RanGAP in the cytosol, a Ran gradientis established across the nuclear envelope, with high Ran–GTP levels in the nucleus and high Ran–GDP levels in the

cytosol.Ran has been identified in many protozoan parasites, including

Giardia, apicomplexans and trypanosomatids (Casanova et al.,

2008; Chen et al., 1994; Frankel and Knoll, 2008). In T. brucei, asingle Ran homolog is encoded by Tb927.3.1120 (Casanovaet al., 2008; Field et al., 1995), with its amino acid sequence

sharing an overall 78.5% similarity to yeast Ran. Stableexpression of a YFP fusion protein of Ran in T. brucei (YFP–Ran) resulted in distinct nuclear staining throughout the cell cycle

(Fig. 2A). This distribution in T. brucei is similar to that observedin yeast and mammalian cells, but appears different to the peri-nuclear localization observed for Ran in Leishmania major

(Casanova et al., 2008) and the evenly throughout the cell

distributed Ran in Toxoplasma gondii (Frankel and Knoll, 2008).The nuclear localization of Ran in T. brucei was further verifiedby using a monoclonal antibody that crossreacts with T. brucei

Ran on immunoblots (Fig. 2B,C). Ran was present in the nucleusat all times of the cell cycle, consistent with the ‘closed’ mitosisobserved in this parasite. Inducible silencing of Ran by RNA

interference (RNAi) led to rapid, albeit incomplete, Ran depletion(Fig. 2D) that was sufficient to arrest cell proliferation 24 h postinduction (Fig. 2E). The DNA contents of Ran-RNAi cells wasexamined by fluorescence microscopy and the results indicated a

rapid increase of cells that contain duplicated mitochondrial DNA(aka the kinetoplast) and a single, undivided nucleus (hereafter

Fig. 1. T. brucei LRRP1 contains an N-terminal LRR-domain thatinteracts with a putative Ran-binding protein and a coiled-coil thatmediates its targeting to the bi-lobe. (A) Domain organization ofLRRP1 and truncation mutants LRR–YFP and CC–YFP. Numbersrepresent the amino acid residues. (B,C) Procyclic T. brucei cellsstably expressing LRR–YFP or CC–YFP were labeled with anti-TbMORN1 antibody for the bi-lobe. (D) RanBPL, a 337-aa RanBD-containing protein was identified as a LRR-interacting protein throughyeast-two-hybrid screening. The RanBD of T. brucei RanBPL(TbRanBPL; Tb927.10.8650) was aligned with RanBD of otherRanBP1 proteins from Leishmania major (LmRanBP1; LmjF12.1480),Saccharomyces cerevisiae (ScYrb2; P40517), Homo sapiens

(HsRanBP1; P43487), and a previously identified T. brucei RanBP1(TbRanBP1; Tb927.11.3380) (Casanova et al., 2008).

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referred to as 2K1N cells). 2K1N cells rose from ,10% in

control populations to .20% at 12 h post RNAi targeting Ranand .40% at 24 h post RNAi targeting Ran, indicating efficientmitotic arrest (Fig. 2F). In later induction stages, the fraction ofzoids (cells that lack nuclei) and cells that contain other aberrant

DNA compositions (including 1K2N cells and multi-kinetoplastand multi-nuclei cells) also increased, consistent withdysregulated cell division after mitotic block. Together, these

results indicate that T. brucei contains a bona fide Ran with adistinct nuclear localization and a strong effect on mitoticprogression, similar to Ran GTPases characterized in higher

eukaryotes (Sazer and Dasso, 2000).

Tb927.10.8650 encodes a Ran-binding proteinInteraction of the RanBD-containing Tb927.10.8650 with T.

brucei Ran was investigated by using a pull-down approach. His-fusion to the full length Tb927.10.8650 or the C-terminal RanBD-containing region was purified and immobilized to Ni2+-NTA

beads and incubated with GST–Ran purified from Escherichia

coli. Both full length Tb927.10.8650 and the C-terminal RanBDinteracted specifically with GST–Ran but not GST alone

(Fig. 3A,B). Interactions were GTP-dependent and drasticallyinhibited when GST–Ran had been pre-loaded with GDP. Pre-loading of GST–Ran with non-hydrolyzable GTPcS did not

significantly enhance Ran binding to the beads, possibly becauseof a large amount of GST–Ran already in GTP-bound form.Furthermore, His-Tb927.10.8650 did not interact with T. brucei

Rab1, another small GTPase (Dhir et al., 2004), regardless of its

GTP- and/or GDP-bound states (Fig. 3C). Together, these resultsindicate that Tb927.10.8650, despite the sequence variationwithin the RanBD (Fig. 1D), binds specifically and selectively

to GTP-bound Ran, just like other Ran effectors (Coutavas et al.,

1993).The protein product of Tb927.10.8650 was thus named RanBP-

like protein (hereafter referred to as RanBPL), in order to distinguishthis protein from T. brucei RanBP1 (Casanova et al., 2008), the only

other RanBD-containing protein found in T. brucei genomedatabase. Although both contain a RanBD region (Fig. 1D), theprimary sequence of RanBPL lacks similarity to RanBP1. Using a

polyclonal antibody specific against RanBPL, T. brucei RanBPLwas found to be present on the nucleus (supplementary material Fig.S2B); this was similar to the localization observed for YFP–RanBP1

in T. brucei (supplementary material Fig. S2A) and in Leishmania

major (Casanova et al., 2008). However, unlike RanBP1, which isessential for T. brucei survival (Casanova et al., 2008), depletion of

RanBPL had only a moderate effect on proliferation of T. brucei

cells in culture (supplementary material Fig. S2C).Interactions of RanBP1 and RanBPL with Ran from T. brucei

cell lysates were also evaluated in pull-down assays (Fig. 3D).

His–RanBP1 and His–RanBPL both were able to bind Ran andpulled down endogenous Ran protein. Interestingly, whereas thebinding of RanBPL to Ran was GTP-dependent – with increased

binding in the presence of GTPcS and inhibited binding in thepresence of GDP – the binding of RanBP1 to Ran was lessaffected by the presence of GTPcS or GDP (Fig. 3E). It is not

clear what may account for this less discriminative binding of T.

brucei RanBP1 to GTP- and GDP-bound Ran. Only RanBPL,however, was investigated in further studies.

T. brucei Ran forms a complex with RanBPL and LRRP1The interaction between RanBPL and LRRP1, initially identifiedin the yeast-two-hybrid screen, was verified by using pull-down

Fig. 2. T. brucei contains a bona fide Ran.(A,B) T. brucei cells stably expressing YFP–Ran(A), and cells immunolabeled with an anti-Ranmonoclonal antibody (B), were stained with DAPIfor nuclear DNA (large blue structures) andkinetoplast (small blue dots). Early in the cellcycle, each cell contains a single kinetoplast anda single nucleus. Kinetoplast DNA duplicates anddivides before nuclear division. (C) Monoclonalanti-Ran antibody reacts specifically to T. brucei

Ran. Lysates of T. brucei 29.13 cells and E. coli

expressing GST–Ran were fractionated by SDS-PAGE and immune-probed with anti-Ranantibody. (D,E,F) Stable Ran-RNAi cells werecultured in the absence or presence oftetracycline to induce RNAi. Ran depletion wasmonitored by immunoblotting using anti-Ranantibody (D). Proliferation of the Ran-depletedcells ceased ,24 h post-induction (E), and 2K1Ncells that contained two kinetoplasts and a singlenucleus accumulated ,12–24 h post-induction(F), indicating a mitotic inhibition.

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analysis. As shown in Fig. 4A, His–RanBPL-coated beads wereincubated with parasite cell lysates. His–RanBPL was able to pull

down endogenous LRRP1, supporting the hypothesis thatRanBPL associates with LRRP1. Interestingly, the interactionbetween RanBPL and LRRP1 relied on the presence ofendogenous Ran. Neither full-length RanBPL nor C-terminal

RanBD were able to pull down LRRP1 from cell lysates that hadbeen induced for RNAi targeting Ran, although expression ofLRRP1 was not affected in Ran-depleted cells (Fig. 4A).

We next asked whether T. brucei Ran interacts with LRRP1 viathe LRR domain that was used as prey in the yeast-two-hybridsscreen (see above). To test this, GST–Ran or GST only were

immobilized on agarose beads and incubated with T. brucei celllysates expressing LRR–YFP or CC–YFP (Fig. 4B). As expected,LRR–YFP but not CC–YFP was pulled down with GST–Ran.Like the Ran–RanBPL interaction (c.f. Fig. 3), the interaction

between the LRR domain and Ran was also GTP-dependent andinhibited in the presence of GDP (Fig. 4C). As further control,LRR–YFP did not interact with GST–Rab1 in the presence of

GTPcS or GDP (Fig. 4D).Direct interaction between Ran and the LRR domain was

verified next by using bacterially expressed GST–LRR and His–

Ran. GST or GST–LRR were purified and immobilized onagarose beads, then incubated with E. coli lysates expressing His–Ran (Fig. 4E). His–Ran was pulled down by GST–LRR but not

GST alone, confirming direct interaction between Ran and the

LRR domain of LRRP1. Taken together, the above experimentssuggest that RanBPL and LRRP1 each interact directly with Ran

through the RanBD and the LRR domain, respectively, and –together – form a complex (Fig. 5A).

To further test whether endogenous Ran, LRRP1 and RanBPLforms a complex, co-immunoprecipation was performed by using

anti-Ran, anti-LRRP1 or anti-RanBPL antibodies. Neither anti-Ran nor anti-LRRP1 antibodies worked for immunoprecipitation,because Ran and LRRP1 did not immunoprecipitate following

the use of their respective monoclonal antibodies (Fig. 5B).Polyclonal antibody against RanBPL, however, was able toimmunoprecipitate RanBPL (Fig. 5C). Interestingly, Ran and

LRRP1 only co-immunoprecipitated with antibody againstRanBPL when cell lysates were supplemented with GTPcS,possibly stabilizing the GTP-dependent interactions.

The formation of a complex between LRRP1, Ran and

RanBPL (Fig. 5A) is, thus, reminiscent of the RanGAP–Ran–RanBP1 ternary complex that has previously been identified inyeast and mammalian cells (Seewald et al., 2002; Seewald et al.,

2003). In this ternary complex, Ran interacts with RanBD ofRanBP1 on one side and the LRR domain of RanGAP on theother side; and these interactions allow rapid and irreversible

hydrolysis of Ran–GTP to Ran–GDP. T. brucei LRRP1, whichcontains an LRR domain that interacts with RanBPL in a Ran-dependent manner and interacts with Ran in GTP-dependent

manner, might thus play a role in Ran regulation.

Fig. 3. T. brucei RanBPL binds specifically to Ran through the C-terminal RanBD in a GTP-dependent manner. (A,B) RanBPL selectively interacted withGTP-bound Ran through the RanBD. His-fusion to the full-length RanBPL (His–RanBPL) or the C-terminal region containing the RanBD (His–RanBPL/C) wasimmobilized to agarose beads and then incubated with purified GST–Ran or GST. (C) RanBPL selectively interacted with Ran but not Rab1. His–RanBPL-coated beads were incubated with lysates of E. coli cells expressing GST–Ran or GST–Rab1. 10% of the purified protein samples or cell lysates used for pulldown (+) were loaded as input control (–). (D,E) RanBPL but not RanBP1 selectively bound to Ran–GTP. His–RanBP1- or His–RanBPL-coated beads wereincubated with T. brucei cell lysates, with GTPcS, GDP or without. Uncoated beads were also incubated with T. brucei lysates as a negative control. 8% ofthe cell lysates used for pull down (+) was loaded as input control (–). The amount of Ran protein under each pull-down condition was quantified relative tothe amount of Ran in the input control. Increased Ran binding in the presence of GTPcS and decreased Ran binding in the presence of GDP werenormalized against Ran pull down from control cell lysates without GTPcS or GDP. Quantification is shown as the mean6s.d. from three independentexperiments.

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LRRP1 facilitates Ran–GTP hydrolysisThe insolubility of T. brucei Ran expressed in E. coli preventeddirect in vitro enzymatic assay (data not shown). We, therefore,examined the effect of LRRP1 on Ran–GTP hydrolysis in an ex

vivo Ran activation assay. In this assay, His-tagged T. brucei

RanBPL immobilized on agarose beads was incubated with T.

brucei cell lysate that had been depleted of LRRP1 or with lysate

of cells that had been overexpressing LRR–YFP. The LRRdomain of RanGAP is sufficient to mediate Ran binding andRan–GTP hydrolysis within the ternary complex (Seewald et al.,2002; Seewald et al., 2003). Unlike full-length LRRP1–YFP,

whose expression suppresses the level of endogenous LRRP1

Fig. 4. LRRP1 interaction with RanBPL is mediated by Ran. (A) His–RanBPL or His–RanBPL/C-beads were incubated with lysates of control orRan-RNAi cells. Neither His–RanBPL nor His–RanBPL/C could pull down endogenous LRRP1 in the absence of endogenous Ran. (B) LRRP1 interactedwith Ran through the LRR domain. GST–Ran beads were incubated with lysates of cells that stably expressed LRR–YFP or CC–YFP. Beads bound to GSTonlywere used as negative control. GST–Ran pulled down LRR–YFP but not CC–YFP. (C) The LRR domain interacted with Ran in a GTP-dependent manner.GST–Ran beads were incubated with LRR–YFP cell lysates that had been pre-loaded with GTPcS or GDP. Lysates of T. brucei 29.13 cells were included asnegative control. (D) The LRR domain did not interact with Rab1. GST–Rab1 beads were incubated with LRR–YFP lysates that had been pre-loaded withGTPcS or GDP. (E) The LRR domain interacted directly with Ran. GST–LRR was purified from E. coli, immobilized to agarose beads and incubated withbacterial lysates expressing His–Ran. 10% of the cell lysates used for pull down were loaded as input controls (–).

Fig. 5. Endogenous LRRP1, Ran and RanBPLform a complex. (A) Schematic presentation of theinteraction between LRRP1, Ran and RanBPL, whereLRRP1 interacts with Ran through the N-terminal LRRdomain and Ran interacts with RanBPL through the C-terminal RanBD. (B) Neither anti-LRRP1 nor anti-Ranmonoclonal antibodies worked for immunoprecipitation.Input, 10% of T. brucei 29.13 cell lysates used for theimmunoprecipiation; IP, immunoprecipitated products.(C) Anti-RanBPL co-immunoprecipitated both LRRP1and Ran only in the presence of GTPcS. Co-IP usinganti-RanBPL was performed on T. brucei 29.13 celllysates in the absence or presence of 1 mM GTPcS.Input: 10% of cell lysates used for IP.

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(Zhou et al., 2010), stable expression of LRR–YFP did not affectendogenous expression of LRRP1 (supplementary material Fig.

S1B). The selective binding of His–RanBPL to Ran–GTP (c.f.Fig. 3D,E) was utilized to specifically pull down Ran–GTPpresent in the cell lysates, allowing quantification of Ran–GTPlevels in cell lysates of parasites by immunoblotting (Fig. 6A).

The total level of Ran was not affected by LRRP1 depletion orLRR–YFP overexpression, evidenced by similar Ran levelsin non-induced control and LRRP-RNAi cells (Fig. 6B).

Significantly more Ran–GTP was pulled down from LRRP1-RNAi lysates – and less from LRR–YFP overexpression lysates(Fig. 6B) – than from the non-induced controls. This was verified

by quantification (Fig. 6C). These observations suggested thatRan–GTP hydrolysis is inhibited in cells that lack LRRP1 andenhanced in cells that express LRR–YFP, supporting a role of

LRRP1 in Ran–GTP hydrolysis.

Unlike Ran, LRRP1 has only a moderate effect on the nucleusThe nucleocytoplasmic Ran–GTP gradient is crucial for protein

trafficking across the nuclear membrane. LRRP1-depletionsubstantially inhibits flagellum-driven cell motility and celldivision, but has no obvious effects on nuclear division (Zhou

et al., 2010). This lack of nuclear effects in LRRP1-depleted cellsappears to contradict the above observations that LRRP1 affectsRan–GTP hydrolysis and intracellular Ran distribution, and is

also in contrast to the rapid mitotic arrest observed in Ran-RNAicells (c.f. Fig. 2).

To further confirm the lack of effect LRRP1 depletion has on

the nucleus, the localization of the RNA-binding protein La wasmonitored by using antibody against La in LRRP1-RNAi andRan-RNAi cells (Fig. 7). La contains a nuclear localization signal(NLS) and is normally found in the nucleus (Fig. 7A) (Marchetti

et al., 2000). La expression levels were not affected followingRNAi targeting either LRRP1 or Ran (Fig. 7H); however, itsnuclear localization was greatly inhibited in Ran-RNAi cells

(Fig. 7E) but only moderately affected in LRRP1-RNAi cells

(Fig. 7C). The change in La localization was quantified byintensity profiling across the nucleus (Fig. 7B,D,F) and by

comparing the relative intensity of La and DNA staining overthe nuclear region (Fig. 7G). The moderate effect LRRP1 has onnuclear protein import is consistent with the previous observationthat RNAi that targets LRRP1 does not affect nuclear division

(Zhou et al., 2010), which supports a primary, non-nuclear effectof this Ran regulator.

T. brucei Ran affects FAZ assemblyRecent studies have found a role of the Ran/importin system inflagellar protein trafficking (Dishinger et al., 2010; Fan et al.,

2011; Ludington et al., 2013). In T. brucei, the presence ofLRRP1, a Ran regulator at the bi-lobe essential for flagellumfunctions, suggested a conserved mechanism of Ran in flagellum

regulation in addition to its nuclear functions (c.f. Figs S2 andS7).

To examine the effects of Ran on flagellum or FAZ biogenesis,control and Ran-RNAi cells (24 h post induction) were scored for

new flagellum attachment, new flagellum length and new FAZlength during the cell cycle of T. brucei (Fig. 8). We chose tofocus on 2K1N-stage cells that contain two kinetoplasts and one

nucleus because this is the stage at which T. brucei cells undergoflagellum and FAZ formation. Besides, due to mitotic inhibition,2K1N cells accounted for ,10% of an asynchronous control

population and ,40% of the Ran-RNAi population (Fig. 2F). Incontrol cells, new FAZ and new flagellum elongate in acoordinated way, their lengths correlating strongly (R250.74).

As such, the majority (,90%) of new flagella were attached tothe cell body. By contrast, in Ran-RNAi cells .30% of the cellscontained new flagella that were partially detached, and thecoordinated assembly of new flagellum and new FAZ was

disrupted in these cells (R250.32) (Fig. 8A,B). Furthermore,whereas the length distribution of new flagella was similar incontrol and Ran-RNAi cells (Fig. 8B), that of new FAZs was

consistently shorter in Ran-depleted cells throughout the stages of

Fig. 6. LRRP1 expression level affects Ran–GTP levels in T. brucei. (A) A schematic presentation of the Ran activation assay used in B. His–RanBPL-coated beads were incubated with T. brucei cell lysates, allowing GTP-bound Ran to be specifically precipitated and quantified by immunoblots. (B,C) Ran–GTPlevels were assessed for lysates from LRRP1-RNAi cells induced for 60 h or cells stably overexpressing LRR–YFP. 8% of the cell lysates used for pull down(+) were loaded as input control (–). Ran–GTP levels (relative to Ran levels in the input lanes) were normalized against the relative Ran level in thecorresponding controls, and the results are shown as mean6s.d., n53 (independent experiments). P values indicated for each condition were calculated bycomparison to control.

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new flagellum synthesis (Fig. 8C–G). Together, these results

supported a role of T. brucei Ran in FAZ assembly.

DISCUSSIONThe leucine-rich-repeat-containing protein LRRP1 has previously

been characterized as a protein resident on the bi-lobe andinvolved in diverse cellular processes including coordinatedorganelle duplication, cell motility and cell division (Zhou et al.,

2010), all of which require normal function of flagellum andflagellum attachment zone (FAZ). Whereas assembly of theflagellum appeared unaffected in LRRP1-depleted cells, FAZ

assembly was significantly inhibited (Zhou et al., 2010).In this study, LRRP1 was found to interact with a previously

uncharacterized Ran-binding protein RanBPL (encoded by

Tb927.10.8650) in a Ran-dependent manner. The small GTPaseRan interacted directly with both the RanBD in RanBPL and theLRR domain in LRRP1. Although robust interactions werereadily detected with overexpressed proteins in T. brucei or in E.

coli, complex formation and colocalization of the endogenousproteins were difficult to observe. Whereas LRRP1 resides on the

bi-lobe, Ran and RanBPL both show primary nuclear

localization. Neither Ran nor RanBPL was found associatedwith the bi-lobe in immunofluorescence experiments or YFP-reporter assays. Consistently, formation of the complex byendogenous Ran, RanBPL and LRRP1 was not observed unless

GTPcS was also present to stabilize the GTP-dependentinteractions. The LRRP1–Ran–RanBPL complex is, thus,similar to the RanGAP–Ran–RanBP1 ternary complex found in

higher eukaryotes. Similarly, in yeast and mammalian cells, bothRanBP1 and RanGAP are cytosolic proteins, whereas Ran ispredominantly found in the nucleus (Quimby and Dasso, 2003).

In vivo, direct observation of the formation of the Ran–RanBP1–RanGAP complex is also challenging, unless overexpressedproteins are used. This is probably owing to the transient,

enzymatic nature of these interactions (Seewald et al., 2003).Whether LRRP1 acts as a RanGAP remains to be

experimentally confirmed. Owing to sequence divergence,identification of RanGAPs by using bioinformatics has been

challenging, particularly in early-branching eukaryotes (Frankeland Knoll, 2009). Several lines of evidence suggested that LRRP1

Fig. 7. RNAi targeting LRRP1 only moderately affects nuclear import of La. (A,C,E) Control T. brucei 29.13 cells, cells induced for RNAi targeting LRRP1 orRan for 48 h were fixed and labelled with anti-La and DAPI for fluorescence microscopy. All images were acquired and processed using identical conditions.(B,D,F) Anti-La intensity was measured over a 10-mm distance along the long axis of the parasites, centering at the single nucleus (as shown in B; onlycells containing a single nucleus were measured). Intensity plots of 10 randomly selected control, LRRP1-RNAi and Ran-RNAi cells were shown. (G) Therelative intensity of anti-La staining and DAPI fluorescence in the nuclear region was also measured, calculated and plotted in randomly selected, single-nucleated cells from control, LRRP1-RNAi and Ran-RNAi sample cells (80 cells each; each dot in the plot represented a cell). Total levels of La expressionremained constant in control and RNAi-induced cells (H; anti-PFR2 was used as a loading control), whereas RNAi targeting Ran inhibited import of La into thenucleus and RNAi targeting LRRP1 only moderately affected it.

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is a Ran regulator, possibly a RanGAP: (1) the LRR domain ofLRRP1 belongs to the RI-like family, like the LRRs of other

previously characterized RanGAPs; (2) Ran interacts directly withthe LRR domain of LRRP1 in a GTP-dependent fashion; (3)endogenous LRRP1, RanBPL and Ran form a complex; 4) LRRP1

protein expression in T. brucei affects Ran–GTP levels. Furtherconfirmation of the RanGAP activity requires biochemical and/orenzymatic analyses, which so far – mainly because of the insolubility

of T. brucei Ran expressed in E. coli – have not been successful.Unlike most Ran regulators identified to date that are either on

the nuclear envelope or in the cytosol and crucial for nuclearfunction (Rose and Meier, 2001; Frankel and Knoll, 2009;

Casanova et al., 2008), LRRP1 of T. brucei is present at the

bi-lobe structure – a cytoskeletal complex located at the proximalbase of the single flagellum (Zhou et al., 2010) – possibly through

oligomerization of the C-terminal coiled-coil domain. Despiteperturbation of Ran–GTP hydrolysis, depletion of LRRP1 hasonly modest effects on nuclear division and nuclear protein

import – the latter at least in the case of La. Rather, as previouslyreported (Zhou et al., 2010), RNAi targeting LRRP1 inhibitsduplication of the bi-lobe and assembly of the FAZ, consequently

inhibiting flagellum inheritance, flagellum-driven cell motilityand cell division. Thus, the role of LRRP1 as a Ran regulator islikely to be restricted to flagellum-associated structures, such asthe bi-lobe and the FAZ, both of which are crucial to flagellum

inheritance and flagellum functions.

Fig. 8. RNAi targeting Ran affects the length of FAZ. Stable Ran-RNAi cells were cultured in the absence or presence of tetracycline to induce RNAi. Controland Ran-RNAi cells (24 hr post induction) at 2K1N stage were monitored for new flagellum attachment, new flagellum and new FAZ length. (A) RNAi targetingRan led to partial detachment of newly formed flagella. Almost 90% of control cells (blue) had a new flagellum completely attached to the cell body;whereas .30% of Ran-RNAi cells had partially detached new flagellum (red). The results are presented as mean6s.d. from three independent experiments. Atotal of 248 2K1N cells was monitored for control cells and 276 for Ran-RNAi cells. (B) RNAi targeting Ran disrupted the coordinated FAZ-flagellum elongation.In non-induced control cells (n566), the new FAZ length correlated to the length of new flagellum (R250.63). This correlation was impeded in Ran-RNAicells (R250.32) (n567). (C) Measurements of new flagellum and/or FAZ lengths shown in B were categorized according to intervals of flagellum length.The results supported a consistent inhibitory effect of RNAi targeting Ran on elongation of FAZ in cells with variable flagellum length. *P,0.05; **P,0.01.(D–G) Representative images of control (D,E) and Ran-RNAi cells (F,G) used for the above quantifications. Cells were fixed with methanol and immunolabeledwith L3B2 for FAZ (red), anti-PFR2 for the flagellum (green) and DAPI for DNA (blue). White arrows mark the ends of the new FAZ. Triple arrows mark theunattached part of the new flagellum. NF, new flagellum; OF, old flagellum.

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The lack of effect on nuclear division in LRRP1-RNAi cells isin contrast to the rapid mitotic arrest observed in the Ran-RNAi

cells. It is possible that ‘closed’ mitosis and nuclear proteinimport in T. brucei function independently of the Ran–GTP/Ran–GDP gradient but require the presence of Ran. It is also possiblethat other RanGAP or Ran-regulators are present in the parasite,

that are sufficient to maintain nuclear function and division.For T. brucei, a putative RanGAP (encoded by Tb927.7.1430)that contains an RI-type LRR has previously been predicted

bioinformatically; however, so far there is no biochemical orfunctional evidence in support of its RanGAP activity (Casanovaet al., 2008).

The presence of LRRP1, a Ran regulator at the bi-lobesuggested a possible Ran-mediated function at the base of theflagellum. Indeed, in addition to nuclear functions, T. brucei Ran

was found to also have an effect on FAZ assembly. The FAZ is astructure uniquely observed in the trypanosomatids, whosecoordinated assembly with the flagellum is crucial for correctflagellar functions (Vaughan, 2010). It is interesting to note that,

although both LRRP1 and Ran affect FAZ assembly, neitherprotein has obvious effects on the biogenesis or structure of theflagellum (Zhou et al., 2010). How LRRP1 and Ran affect FAZ

assembly remains unknown. It is, however, possible that theobserved FAZ defect is a consequence of other effects, especiallyconsidering the diverse role of Ran in various cellular processes

including protein trafficking and cytoskeletal organization (Sazerand Dasso, 2000). Further characterization for Ran effectors in T.

brucei will be crucial to address this question.

In mammalian cells, several components of the nuclear porecomplex have been localized to the transition zone region at thecilium base (Kee et al., 2012). This, together with the discoverythat Ran/importin have a function in cilium biogenesis (Fan and

Margolis, 2011), suggest the presence of a highly conservednuclear-pore-like mechanism that operates at both cilium andnucleus to selectively target proteins into the ciliary or nuclear

compartments. In T. brucei, in addition to LRRP1, severalnuclear-pore components have been found in previouslypublished bi-lobe proteomes (Gheiratmand et al., 2013;

Morriswood et al., 2012; Zhou et al., 2010). Whether the bi-lobe contains or functions as a selective gate, regulating proteintrafficking to the FAZ or the flagellum by using a mechanism thatinvolves Ran, will be of great interest.

MATERIALS AND METHODSCells and vectorsAll experiments were performed on procyclic YTat1.1 (T. brucei

rhodesiense) (Ruben et al., 1983) or 29.13 cells (T. brucei brucei) that

were genetically engineered to allow tetracycline-inducible expression

and RNA interference (RNAi) (Wirtz et al., 1999). Cell concentration

was kept between 106 and 107 cells/ml by dilution with fresh medium and

growth curves were presented as cumulative cell density.

For LRRP1-truncation mutants, the LRR region (nucleotide 1–1074)

and the coiled-coil region (CC, nucleotide 1441–1594) were amplified

and fused to the N-terminus of YFP in the pXS2 vector (Bangs et al.,

1993). The full-length coding sequences of RanBP1 (Tb927.11.3380) and

Ran (Tb927.3.1120) were fused to the C terminus of YFP in pXS2 vector.

RNAi targeting LRRP1 has been reported previously (Zhou et al., 2010).

For RNAi targeting Ran or RanBPL, a 457 bp fragment (nucleotide 142–

598) or a 593 bp fragment (nucleotide 347–932), respectively, was

cloned into the p2T7 plasmid (Wickstead et al., 2002). Stable cell

transfection was achieved by electroporation with 15 mg of linearized

plasmid followed with serial dilution and antibiotic selection for clonal

lines. cDNA encoding the full-length RanBP1 (Tb927.11.3380), RanBPL

(Tb927.10.8650), the C-terminal RanBD (nucleotide 594–1014) or the

full-length Ran (Tb927.3.1120) were cloned into to the pET-28b(+)

vector (Novagen) to generate His–RanBP1, His–RanBPL, His–RanBPL/

C and His–Ran fusions. GST–Ran, GST–Rab1 and GST–LRR were

generated by cloning the full-length Ran cDNA (Tb927.3.1120), Rab1

cDNA (Tb927.8.4610) or LRR cDNA (Tb927.11.8950, nucleotide 1–

1074) into the pGEX-6p-1 vector (GE Healthcare).

Yeast-two-hybrid screeningProcyclic T. brucei (YTat1.1) cDNA library was generated using a

MatchmakerTM Library Construction kit (Clontech). To construct the bait

plasmid either the LRR domain (1–349 aa) or the coiled-coil domain

(480–530 aa) of LRRP1 was cloned into the multi-cloning site of the

pGBKT7 vector (Clontech), by using EcoRI and BamHI, which allowed

in-frame fusion of the LRRP1 fragments to the Gal4-DNA-binding

domain in the vector. The bait constructs were then tested for

transcriptional auto-activity and toxicity.

Yeast mating1 ml aliquot of the T. brucei cDNA library in AH109 and 5 ml (§109

cells/ml) of Y187 transformed with bait construct were combined in a

sterile 2 l flask containing 40 ml 26YPDA and 50 mg/ml kanamycin.

The contents were then incubated at 30 C for 20–24 h with gentle

swirling (30–50 rpm to allow mating). After mating, the mixture was

centrifuged at 1000 g for 10 min. Cell pellet was resuspended in 10 ml

0.5 XYPDA/Kan (50 mg/ml). After 5 days of incubation at 30 C, diploids

that express interacting proteins appeared on the plates. To eliminate the

most common group of false-positive colonies, Ade+/His+ colonies were

streaked out on fresh SD/2Ade/2His/2Leu/2Trp/XaGal master plates

and incubated for 4–5 days at 30 C in order to test Ade+/His+ colonies for

the third reporter; MEL1, which encodes yeast a-galactosidase. Positive

blue colonies were further analyzed by PCR.

Identification of cDNA inserts from positive coloniesTo identify the genes (and thus proteins) responsible for the positive two-

hybrid interactions, the cDNA inserts were rescued by PCR colony-

screening using the Matchmaker AD LD-insert screening amplimer set

(Clontech). PCR products were then analyzed by gel electrophoresis

and excised from the gel for DNA purification and sequencing. The

DNA sequences were blasted against the T. brucei genome database

(tritrypdb.org).

Immunofluorescence microscopyCells were washed and resuspended in phosphate-buffered saline (PBS

pH 7.4), attached to coverslips for 20 min and either fixed and

permeabilized 5 min in cold methanol (220 C) (anti-MORN, L3B2

and anti-PFR2) or 20 min in 4% PFA followed by 5 min in 0.25% Triton

X-100 in PBS. Fixed cells were then blocked with 3% BSA in PBS and

probed for 1 h with the corresponding antibodies. Monoclonal anti-

LRRP1 (1:1000) and anti-MORN1 (1:500) were used to mark the bi-lobe

(Morriswood et al., 2009; Zhou et al., 2010). A monoclonal anti-Ran

(1:100) antibody (Cell Biolabs) was used to label T. brucei Ran. Rabbit

anti-La antibody (1:1000) recognized the nuclear La protein (Marchetti

et al., 2000). FAZ was labeled with the monoclonal L3B2 antibody (1:25)

which is directed against FAZ1 (Vaughan, 2010). The flagellum was

labeled with a rat antibody against PFR2 (1:5000). Nuclear and

kinetoplast DNAs were stained with 10 mg/ml DAPI for 20 min. All

images were acquired using a Zeiss Axio Observer microscope equipped

with a CoolSNAP HQ2 camera (Photometrics). Images were processed

with ImageJ or Adobe Photoshop. Quantification of fluorescence

intensity and flagellum/FAZ length measurements were performed by

using ImageJ.

Antibody against RanBPLTo obtain anti-RanBPL antibody, His–RanBPL was expressed in E. coli and

affinity purified using Ni2+-NTA (Qiagen) following manufacturer’s

instructions. Purified His–RanBPL was used for the production of rabbit

polyclonal antibody (Abnova, Taiwan). Affinity-purified anti-RanBPL was

used at 1:200 for immunofluorescence assays and 1:1000 for immunoblots.

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Pull-down assaysImmobilization of His–RanBP1, His–RanBPL or His–RanBPL/C onNi2+-NTA beads10 ml of BL21 bacterial culture was induced for 2 h with 0.1 mM IPTG.

Pelleted bacterial cells were resuspended in 1 ml PBS supplemented

with protease inhibitor cocktail, and homogenized by sonication.

After solubilization with 1% Triton-X-100 at 4 C for 1 h, lysates were

cleared by centrifugation. The supernatant was incubated with 50 ml

Ni2+-NTA beads for 2 h at 4 C. Unbound proteins were removed by

washes with PBS containing 20 mM imidazole. Similar procedure was

used to immobilize GST–LRR, GST–Ran, GST–Rab1, or GST-only on

glutathione Sepharose 4B beads (GE Healthcare).

Preparation of cell lysates and pull downTypically, 10 ml bacterial culture or 16108 T. brucei cells were used for

pull-down assays. The cells were resuspended in 1 ml PBS, lyzed

by sonication and extracted with 1% Triton-X-100 for 1 h at 4 C. Beads

coated with recombinant proteins were then incubated with

centrifugation-cleared cell lysates for 1 h at 4 C to allow binding.

Unbound proteins were removed in three washes with PBS and proteins

bound to the beads were eluted by boiling 5 min in SDS loading buffer.

When GTPcS and GDP were used as controls, bacterial lysates or cell

lysates expressing GST–Ran or endogenous Ran were preloaded with

1 mM GDP or 0.1 mM non-hydrolysable GTPcS for 30 min at 30 C,

prior to the binding with the His–RanBPL beads.

Immunoprecipitation10 ml anti-RanBPL unpurified serum, 8 mg anti-Ran, or 10 ml anti-

LRRP1 was immobilized on 50 mg of protein G magnetic Dynabeads

(NovexH). Approximately 16108 parasite cells were lyzed as described

previously. When required, cleared lysates were pre-loaded with 1 mM

GTPcS for 30 min at 30 C, before incubation with the beads.

For immunoblots, proteins samples were separated on SDS-PAGE and

probed with the appropriate antibodies: anti-Ran (1:1000), anti-LRRP

(1:2000), anti-RanBPL (1:1000), anti-GFP (1:1000), anti-La (1:1000),

anti-His (1:5000) (GE Healthcare), anti-TUBa (1:5000) (Santa Cruz

Biotechnology) and anti-GST (1:5000) (Santa Cruz Biotechnology).

Ran-activation assayRanGTP hydrolysis in cell lysates was evaluated by the amount of

Ran–GTP pulled down with His–RanBPL, taking advantage of its

discriminative binding to GTP-bound Ran. To prepare parasite lysates

for each assay, 16108 of T. brucei cells were washed twice with ice-

cold PBS and resuspended in 1 ml ice-cold lysis buffer (25 mM

HEPES pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 1 mM

EDTA, 2% glycerol). Cells were lyzed by sonication and cell lysates

were then centrifuged at 14,000 g for 10 min at 4 C to remove cell

debris. The volume of each sample was adjusted to 1 ml with lysis

buffer.

To pull down Ran–GTP in the cell lysates, His–RanBPL-conjugated to

Ni2+-NTA beads were incubated with cell lysates prepared as described

above. The tubes were then incubated at 4 C for 1 h with gentle mixing.

The beads were washed thoroughly with lysis buffer before resuspended

in reducing SDS-PAGE sample buffer. Proteins bound to the beads were

eluted, denatured by boiling at 100 C for 5 min, fractionated by SDS-

PAGE and immunoblotted with appropriate antibodies. Imaging and

quantification of the immunoblots were performed using an ImageQuant

LAS 4000 mini (GE Healthcare). All Ran–GTP measurements were

normalized against the intensity of Ran–GTP pulled down from the

control cells performed at the same time of each experiment.

RT-PCRmRNAs from T. brucei cells before and after induction of RNAi targeting

RanBPL were extracted with Trizol (Invitrogen) and used as template for

reverse transcription with RT Superscript II (Invitrogen). The cDNAs

were then amplified by PCR with specific primers for the RanBPL and a-

tubulin mRNAs.

AcknowledgementsWe thank the Protein Expression Facility at the Singapore MechanobiologyInstitute for technical assistance in cloning and expression of some of therecombinant proteins described in this study.

Competing interestsThe authors declare no competing interests.

Author contributionsA.B., S.B., Z.Y., C.X.L. and Z.Q. did the experiments, analyzed the data and wrotethe paper. L.B.C. and C.Y.H. conceived the project, analyzed the data and wrotethe paper.

FundingThis project is funded by Singapore National Research Foundation Fellowship[grant number NRF-RF001-121].

Supplementary materialSupplementary material available online athttp://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.148015/-/DC1

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RESEARCH ARTICLE Journal of Cell Science (2014) 127, 4846–4856 doi:10.1242/jcs.148015

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