A Novel Protein Kinase Localized to Lipid Droplets Is ... · EUKARYOTIC CELL, Nov. 2010, p. 1702–1710 Vol. 9, No. 11 1535-9778/10/$12.00 doi:10.1128/EC.00106-10 Copyright © 2010,

EUKARYOTIC CELL, Nov. 2010, p. 1702–1710 Vol. 9, No. 111535-9778/10/$12.00 doi:10.1128/EC.00106-10Copyright © 2010, American Society for Microbiology. All Rights Reserved.

A Novel Protein Kinase Localized to Lipid Droplets Is Required forDroplet Biogenesis in Trypanosomes�‡

John A. Flaspohler,1,2† Bryan C. Jensen,1† Tracy Saveria,1 Charles T. Kifer,1 and Marilyn Parsons1,3*Seattle Biomedical Research Institute, 307 Westlake Ave. N., Seattle, Washington 981091; Department of Biology, Concordia College,

901 8th St. South, Moorhead, Minnesota 565622; and Department of Global Health,University of Washington, Seattle, Washington 981953

Received 2 May 2010/Accepted 3 September 2010

Ubiquitous among eukaryotes, lipid droplets are organelles that function to coordinate intracellular lipidhomeostasis. Their morphology and abundance is affected by numerous genes, many of which are involved inlipid metabolism. In this report we identify a Trypanosoma brucei protein kinase, LDK, and demonstrate itslocalization to the periphery of lipid droplets. Association with lipid droplets was abrogated when the hydro-phobic domain of LDK was deleted, supporting a model in which the hydrophobic domain is associated withor inserted into the membrane monolayer of the organelle. RNA interference knockdown of LDK modestlyaffected the growth of mammalian bloodstream-stage parasites but did not affect the growth of insect (procy-clic)-stage parasites. However, the abundance of lipid droplets dramatically decreased in both cases. This losswas dominant over treatment with myriocin or growth in delipidated serum, both of which induce lipid bodybiogenesis. Growth in delipidated serum also increased LDK autophosphorylation activity. Thus, LDK isrequired for the biogenesis or maintenance of lipid droplets and is one of the few protein kinases specificallyand predominantly associated with an intracellular organelle.

Trypanosoma brucei is a single-celled eukaryotic pathogenresponsible for human African trypanosomiasis (also knownas African sleeping sickness) and nagana in domestic ani-mals. More than 50,000 cases of human disease occur yearly,with over 70 million people at risk. No vaccine exists, andchemotherapy is difficult to administer and prone to patho-gen resistance. As T. brucei transits between the mammalianbloodstream and the tsetse fly vector during its life cycle, theorganism encounters and adapts to profoundly different en-vironmental conditions. The parasite undergoes dramaticchanges in both energy (7, 51) and lipid biosynthesis andmetabolism (39, 47, 49) as it shifts between these environ-ments.

Protein kinases function in numerous regulatory aspects ofthe cell, including control of the cell cycle and morphology,responses to stress, and transmission of signals from the extra-cellular environment or between compartments of the cell. Asis the case in other eukaryotes, protein kinases, particularlythose associated with membranes, are expected to play pivotalroles in the cell’s ability to sense and appropriately respond toits environment. Trypanosoma brucei possesses over 170 pro-tein kinases (16, 44). Most of these can be assigned to thestandard groups of protein kinases based on sequence similar-ity within the kinase domain. However, sequence similaritieswith kinases from more well-studied organisms are rarelystrong enough to allow one-to-one orthologous relationshipsto be determined (44), and even those which appear ortholo-

gous by sequence have sometimes shown functional divergence(46). Hence, an understanding of the roles of specific proteinkinases of trypanosomatids requires an individualized assess-ment. The initial genome analysis of the trypanosomatids (16)showed a lack of receptor tyrosine kinases, but nine T. bruceipredicted serine/threonine kinases were annotated as possess-ing transmembrane domains. One of these was recently shownto be strategically located at a key interface between the hostand parasite: the flagellar pocket (38). This eukaryotic trans-lation initiation factor 2� (eIF2�) family kinase was postulatedto play a sensory role in monitoring protein transport.

Only a very small number of protein kinases of variousorganisms have been observed to localize to the membranes ofintracellular organelles, most of them to the endoplasmic re-ticulum (ER) (14, 27, 50). Lipid droplets (also known as lipidbodies, adiposomes, or oil bodies in plants) are thought to arisefrom the ER, although the routes of protein localization tothem are not well understood. They are increasingly recog-nized as legitimate organelles due to their dynamic roles inenergy metabolism (40), lipid trafficking (41), and protectionagainst toxic effects of nonesterified lipids and sterols (18).Studies also suggest that they function as potential proteinstorage depots (12) and in antigen presentation (10). Althoughrecent efforts to expand the lipid droplet proteome have re-sulted in a vastly increased and in many cases surprising cata-logue of potentially associated proteins (3, 5, 11, 12, 23, 37),relatively little is known as to how these structures form andare regulated within the cell.

We examine here a novel T. brucei protein kinase with apredicted transmembrane domain. Surprisingly, this protein islocalized intracellularly in association with lipid droplets.RNAi-mediated knockdown of this newly identified kinase,dubbed LDK (for lipid droplet kinase), reveals a role in theformation or maintenance of lipid droplets in both mammalian

* Corresponding author. Mailing address: Seattle Biomedical Re-search Institute, 307 Westlake Ave. N., Seattle, WA 98109. Phone andfax: (206) 256-7315. E-mail: [email protected].

† F.A.S. and B.C.J. contributed equally to this study.‡ Supplemental material for this article may be found at http://ec

.asm.org/.� Published ahead of print on 10 September 2010.

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bloodstream-form (BF) and insect procyclic-form (PF) stagesof the parasite life cycle.

MATERIALS AND METHODS

Cell culture. The single marker BF line of T. brucei, which is derived from the427 strain (52), was grown in HMI-9 supplemented with 10% fetal calf serum and2.5 �g of G418/ml. PF T. brucei line 29-13, also a derivative of 427 (52), wasgrown in SDM-79 (JRH Biosciences) media and 15% fetal calf serum. G418,hygromycin, and phleomycin were added to final concentrations of 15, 50, and 2.5�g/ml, respectively. PF were induced for lipid droplet formation by growth in 1.5�M myriocin (Sigma) for 24 h as previously described (20) or by substituting 10%delipidized bovine calf serum (Equitech) in the indicated experiments. Tetracy-cline (Tet) was used at 1 to 2 �g/ml for inductions.

DNA constructs. The Tb11.01.0670 (LDK) ORF was obtained by PCR from T.brucei strain 29-13 genomic DNA by using the primers LDK 5� AvrII (5�-CGGCCTAGGATGTCTACGGGAAAGATAATTGGTG-3�) and LDK 3� HindIII(5�-CCCAAGCTTGTTCTTCTCCAGCCAACGGAGCAC-3�). The LDK openreading frame (ORF) was cloned into pGEM-T Easy and sequenced. The ORFwas then subcloned into the AvrII and HindIII sites in the inducible expressionplasmids pLew79-3V5(PAC), which contains the puromycin resistance marker inlieu of the phleomycin marker in pLEW79, as well as sequences encoding threeV5 epitopes. The hydrophobic domain at residues 432 to 455 was deleted bysite-directed mutagenesis in the T. brucei pLew79-LDK-3V5(PAC) expressionplasmid (primers 5�-CTCCAGATACACCTAAAGGTGATCGGTTTTTGTTTGACGTCCCTG-3� and 5�-CAGGGACGTCAAACAAAAACCGATCACCTTTAGGTGTATCTGGAG-3�). Lysine 41 was mutated to alanine by using thesense primer (5�-GTGGACTTGACAACAGACAAGGTATACGCGGTAATGGTTATAAGTAAATCGGTAATTG-3�) and antisense primer (5�-CAATTACCGATTTACTTATAACCATTACCGCGTATACCTTGTCTGTTGTCAAGTCCAC-3�).

RNA interference (RNAi) constructs were based on the plasmid p2T7TAblue

(PAC), in which the hygromycin gene of the original plasmid (1) was replaced bythe puromycin resistance gene. The plasmid contains cloning sites betweenopposing T7 promoters under the control of the Tet operator, as well as se-quences mediating integration into the ribosomal DNA intergenic spacer region.A region of the LDK ORF (nucleotides 1 to 528), which was predicted to havelittle homology to other T. brucei mRNAs, was amplified by PCR and using theprimers LDK 5� AvrII and LDK 3� XhoI RNAi (5�-GCGCTCGAGCGTGCCGCACGAATGGTGAAG) and cloned directly into Eam11051 linearizedp2T7TAblue(PAC).

RNAi. PF 29-13 and BF single-marker lines each contain integrated copies ofgenes encoding the T7 RNA polymerase and the tetracycline (Tet) repressor,allowing Tet-regulated expression of introduced sequences (52). p2T7TAblue-LDK was linearized with NotI and transfected into BF and PF parasites asdescribed previously (52). Subsequent multiwell plating and selection with pu-romycin produced stably transfected clonal T. brucei BF. Multiple independentlyderived clonal cell lines were assayed in BF RNAi experiments. Procyclic trans-fectants were selected with puromycin for 3 to 4 weeks prior to RNAi studies.Four independent transfectant populations were assayed in RNAi experiments.Cell densities were determined by using a Z1 Coulter Counter, and growthcurves were plotted using duplicate or triplicate cultures. Cultures in log phasewere diluted to 5 � 105 cells/ml (PF) or 105 cells/ml (BF) to begin the experi-ment. RNAi was induced by the addition of 2 �g of Tet/ml. Tet was subsequentlyadded every 24 h (BF) or 48 h (PF), and cultures were maintained below 2 � 106

(BF) or 1.5 � 107 (PF). To assess knockdown of mRNA, cells uninduced orinduced for RNAi, were collected on day 0 and day 5 (PF). Washed parasiteswere resuspended in TRIzol, and RNA preparation and Northern analyses werecarried out as described previously (30).

Immunofluorescence analysis. Immunofluorescence was performed as previ-ously described (15, 32). BF parasites were prefixed in 4% paraformaldehyde for5 min on ice prior to placing on poly-L-lysine coverslips. The V5 epitope tag wasdetected by using mouse monoclonal anti-V5 (Invitrogen) at 1 �g/ml, followedby goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC)(Southern Biotechnology). Anti-BiP (4), a gift from Jay Bangs, was used at 1:400,followed by goat anti-rabbit immunoglobulin conjugated to Texas Red (SouthernBiotechnology). To detect lipid droplets, slides bearing paraformaldehyde-fixedcells were incubated in phosphate-buffered saline (PBS) or SDM-79 containing1.5 �g of Nile Red (Sigma)/ml for 30 min at 25°C as described previously (24).Slides were washed twice in PBS prior to mounting using Prolong Antifade(Molecular Probes) for fluorescence microscopy. DAPI (4�,6�-diamidino-2-phe-nylindole) staining was used to identify the nucleus and kinetoplast. Fluores-cence of Nile Red in the red channel (617 nm) is brighter but less specific to lipid

bodies (36); therefore, data were collected in the FITC channel (528 nm) exceptin colocalization studies. Slides were viewed on a Deltavision RT deconvolutionmicroscope with an Olympus UPlan/Apo 100� 1.35 NA objective. Deltavisionimages were deconvolved using standard parameters and the conservative ratioalgorithm. Single deconvolved planes are shown.

Extractions, immunoblots, and protein kinase assays. Samples were analyzedby SDS-PAGE (8 to 16% acrylamide gradient). Immunoblots were probed withmouse anti-V5 at 2 �g/ml, rabbit anti-T. brucei phosphoglycerate kinase (1:4,000)(43), rabbit anti-vacuolar H� pyrophosphatase (VP1, 1:5,000, a gift from Rob-erto Docampo) (35), or a mixture of two monoclonal antibodies against EPprocyclin (antibodies 16 and 418 at 1:10,000) (45). Bound antibodies were re-vealed with goat anti-mouse immunoglobulin (IRDye800) and goat anti-rabbitimmunoglobulin (IRDye700). Blots were scanned with a LiCor Odyssey.

For phase partitioning of LDK in Triton X-114 (9), 5 � 108 PF were pelleted,washed in PBS plus glucose, and resuspended in 200 �l of 1% Triton X-114, 150mM NaCl, and 10 mM Tris (pH 7.4). After 10 min on ice, debris was removedby centrifugation at 16,000 � g for 10 min. The supernatant was incubated at 30°for 3 min to condense the Triton-X114. The lysate was then centrifuged for 3 minat 12,000 � g at room temperature. The upper aqueous phase was removed. Thedetergent phase (�20 �l) was reextracted with 200 �l of the above buffer(without Triton X-114). All samples were brought to 200 �l and mixed withsample buffer for SDS-PAGE.

For treatment with carbonate (22), 5 � 108 PF were washed with PBS plusglucose as described above. The cells were resuspended in 2 ml of 10 �M Tris(pH 7.4) with protease inhibitors on ice for 2 min and Dounce homogenized toachieve hypotonic lysis. Cell debris was pelleted at 1,000 � g, and the supernatantwas transferred to a fresh tube. An equal volume of 0.2 M sodium carbonate(pH 11) was added to the supernatant and incubated on ice for 1 h. Themembranes were then pelleted at 100,000 � g for 1 h at 4°C. The solubleprotein fraction was removed, and the membrane fraction was resuspended inan equal volume of PBS.

Immunoprecipitation of LDK-V5 from PF cell lysates (108 cells) of Tet-induced (24 h) and uninduced cultures was performed as previously described(15). Kinase assays were performed as described elsewhere (42) using [32�-P]ATP and in some cases casein (5 �g/reaction) as substrate. Reactions wereseparated by SDS-PAGE, transferred to nitrocellulose, and labeled proteinsdetected by phosphorimaging. Signals were quantified by using ImageQuant(Molecular Dynamics). The same blots were probed with anti-V5 as describedabove to allow normalization of activity.

RESULTS

Tb11.01.0670 was identified by searching GeneDB(www.genedb.org) for T. brucei genes encoding proteins pos-sessing a protein kinase catalytic domain, as well as a predictedtransmembrane domain(s). The Tb11.01.0670 protein is 554amino acids (aa) long; the protein kinase domain begins 12 aafrom the N terminus (see schematic in Fig. 1A). It contains allof the essential motifs and residues required for protein kinaseactivity (see Fig. S1 in the supplemental material) (26). Thekinase domain is followed by a 267-aa extension, which con-tains a single predicted transmembrane domain of 24 aa, sug-gesting potential membrane localization. This extension showslittle homology with any known predicted proteins except withthe orthologous kinases of trypanosomatids (see Fig. S1 in thesupplemental material for alignment and systematic names).The orthologues in the related genus Leishmania are highlysimilar within the kinase domain, and also have a similarlyplaced predicted transmembrane domain within the C-termi-nal extension, but the extension is longer and shows reducedsequence similarity (see Fig. S1 in the supplemental material).These trypanosomatid kinases belong to the calmodulin-de-pendent kinase (CAMK) group of protein kinases and appearto be most closely related to the CAMKL (for CAMK-like)family (44). However, there is no obvious orthologous relation-ship to any specific eukaryotic protein kinase outside of thetrypanosomatids. Our previous genome-wide microarray anal-

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ysis showed that the corresponding mRNA was expressed tolow levels in both BF and PF T. brucei (31). Based on thepresence of a protein kinase domain and the data below, wenamed the protein lipid droplet kinase (LDK).

To assess whether LDK has protein kinase activity, we ex-pressed LDK tagged with three V5 epitopes at its C terminusin PF. Induction of LDK-V5 expression with Tet resulted inexpression of an immunoreactive species migrating at �75kDa, slightly larger than the predicted mass of 68 kDa (Fig.1B). In this experiment and some others, LDK-V5 appeared asa doublet, but this was not consistently observed. We immu-noprecipitated LDK-V5 from Tet-induced PF lysates and per-formed in vitro kinase reactions. As shown in Fig. 1C, immu-noprecipitates from cells induced for LDK-V5 expressionshowed a significant amount of phosphorylation of a speciescomigrating with LDK-V5, which likely represents autophos-phorylation. The LDK-V5 immunoprecipitates also phosphor-ylated the upper casein band, although some casein phosphor-ylation from control immunoprecipitates was also observed.The latter may result from casein kinases such as CK1.2,CK2�, and CK2��, each of which is ranked among the eightmost abundant kinases at the RNA level (31). Both LDK andcasein phosphorylation were seen with purified TAP-taggedLDK compared to a mock purification from untransfected cells(not shown). We observed modest protein kinase activity di-rected toward a small synthetic peptide that is a favored sub-strate for other kinases in the CAMKL kinase family and alower level of phosphorylation on myelin basic protein, an-other exogenous substrate (data not shown).

With rare exceptions, a lysine in subdomain II is required foractivity. Mutation of this residue (K41A, see Fig. S1 in thesupplemental material) strongly reduced phosphorylation ofLDK, but the phosphorylation of casein was not decreased(Fig. 1D). This finding suggests that LDK possesses kinaseactivity which autophosphorylates in cis. The activity of thekinase-dead LDK-V5 immunoprecipitates toward casein mayresult from association with the native kinase or another ki-nase; hence, casein phosphorylation was not considered fur-ther. Autophosphorylation was similar in 50 to 100 mM NaCland was dependent on Mg2� (Mn2� could not substitute).Ca2� (with or without calmodulin) did not increase activity.

LDK protein localizes to the periphery of lipid droplets. Wethen assessed the potential localization of LDK to specificmembrane locations in both PF and BF parasites. The expres-sion plasmid uses a promoter that is highly active in PF asopposed to BF, as well as a 3� untranslated region that iscompromised for expression in BF (52). However, LDK-V5was detected in both developmental stages upon immunoflu-orescence analysis of stably transfected parasites. In inducedcells, a distinctive ringlike pattern, apparently marking theperiphery of multiple intracellular structures (Fig. 2A to D)was observed, and this ring was absent in uninduced cells. Incolocalization studies with antibodies specific for glycosomes(43) and acidocalcisomes (17, 35), we eliminated these well-characterized organelles as the location of LDK-V5 (data notshown). Nile Red, a lipophilic fluorescent dye, was then used tospecifically stain intracellular lipid droplets (24). LDK-V5 wasfound to primarily reside at the periphery of Nile Red-stainedorganelles in both developmental stages, and we conclude thatLDK-V5 resides in or is associated with the monolayer mem-

FIG. 1. Lipid droplet kinase structure and kinase activity. (A) Sche-matic of LDK, showing location of protein kinase (PK domain) andhydrophobic region (Hyd), which lies in the C-terminal extension.Between the kinase domain and the hydrophobic region, the protein isenriched for acidic aa (�), whereas distal to the hydrophobic region itis enriched for basic aa (�). The region included in the RNAi con-struct is marked. The annotated sequence is shown in Fig. S1 in thesupplemental material. (B) Immunoblot. Lysates were prepared fromPF induced (�Tet) or uninduced (�Tet) for LDK-V5 expression.After SDS-PAGE, samples were transferred to nitrocellulose. The blotwas incubated with anti-V5 and goat anti-mouse immunoglobulinIRDye800 and scanned. The migration of molecular mass markers (inkilodaltons) is indicated in this and other figures. (C) Kinase assay.Anti-V5 immunoprecipitates from cell expressing (Tet�) or not ex-pressing (Tet�) LDK-V5 were collected and assayed for protein ki-nase activity in the presence of casein as a candidate exogenous sub-strate. The gel containing the samples was blotted and subjected toovernight phosphorimaging, as well as Western analysis with anti-V5.Left section (32P), kinase assay. Filled arrowhead, LDK-V5; openarrowhead, upper band of casein. Below is the relevant portion of thesame blot stained with Ponceau S to reveal casein. Right section(anti-V5), Western analysis. Asterisks mark the heavy and light chainof the antibodies used for immunoprecipitation, which were detectedby the second step antibodies. (D) Phosphotransfer activity of LDKK41A. After immunoprecipitation and kinase assay of wild-type andK41A LDK-V5, 32P associated with the LDK-V5 band and the caseinbands was quantitated by phosphorimaging. LDK-V5 was quantitatedby immunoblotting of the same gel lanes, allowing normalization ofactivity. The graph depicts the relative level of phosphorylation byLDK K41A immunoprecipitates compared to the wild type.

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brane of lipid droplets. A similar ringlike staining pattern hasbeen seen for proteins localized to the lipid droplet surface inother eukaryotes (see references 6 and 8 for examples). In theculture conditions used, images of cultured PF showed anaverage of four lipid droplets, whereas cultured BF typically

showed two smaller lipid droplets. Staining of the ER, inaddition to lipid droplets, was seen in some cells, mostnoticeably in those with a higher level of expression (Fig.2E). This is consistent with the concept that lipid dropletsderive from the ER.

FIG. 2. Localization of LDK-V5 to lipid droplets. Images of control parasites (uninduced, Tet�) and or those induced for expression ofLDK-V5 (Tet�) were collected using the same parameters and identically processed. Transfectants expressing LDK-V5 were stained with NileRed to identify lipid droplets and costained with anti-V5 antibodies (green). Nile Red was collected on the Texas Red filter, while LDK-V5 wasvisualized in the green channel. Cells were costained with DAPI (blue). All bars, 5 �m. (A) Localization of LDK-V5 to lipid droplets in PF. Left,induced for LDK-V5 expression (�Tet); right, uninduced (�Tet). (B) Localization of LDK-V5 to lipid droplets in BF. (C) Deletion of thehydrophobic region of LDK leads to cytosolic localization in PF. Due to the lower maximum signal intensity, the scaling for brightness is twice thatof the images showing wild-type protein. (D) Threefold enlargement of lipid droplet staining of panel cell immediately above. (E) Tet-induced PFcell showing LDK-V5 localization to the ER and lipid droplets (green). Anti-BiP was used as a marker of the ER (red).



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Unlike other subcellular organelles, lipid droplets arebounded by a membrane monolayer in which the chargedmoieties face outwards. The presence of a single hydrophobic,predicted transmembrane domain suggested that this regionmight be critical for localization of LDK to lipid droplets. Wetherefore expressed a mutant version of the kinase lacking the24 aa comprising the hydrophobic region (LDK432-455) inPF. The mutant kinase was found throughout the cell, with noapparent association with lipid droplets (Fig. 2C). We exam-ined the association of LDK-V5 with the lipid droplet usingTriton X-114 and carbonate extractions (Fig. 3). In TritonX-114 extractions, LDK-V5 partitioned to the aqueous phase,although a C-terminal degradation fragment predicted to con-tain the hydrophobic region partitioned equally between theaqueous and detergent phases. EP-procyclin, a heteroge-neously glycosylated glycosylphosphatidylinositol-anchoredprotein, was predominantly in the detergent phase, whereasthe cytosolic and glycosomal phosphoglycerate kinase werepresent in the aqueous phase. When hypotonically lysed cellswere extracted with carbonate, pH 11, control proteins frac-tionated as expected: phosphoglycerate kinases were in thesupernatant, whereas the acidocalcisomal H� pyrophos-phatase (VP1), an integral membrane protein, was presentonly in the pellet. Most LDK-V5 remained in the integralmembrane protein fraction (pellet), but some was released intothe supernatant. The partial resistance of LDK to carbonateextraction indicates that the protein is strongly associated withthe lipid droplet membrane.

LDK RNAi parasites show minimal growth defects but aredepleted of lipid droplets. To further examine the role of LDKand ascertain its importance for survival of T. brucei, we per-formed RNAi knockdown analysis. A segment of the LDKORF was cloned into the p2T7TAblue vector between Tet-inducible T7 promoters. Addition of Tet initiates RNAi knock-down. We isolated several clonal lines of BF transfectants, aswell as nonclonal lines of PF transfectants. RNAi knockdownof LDK mRNA in PF was verified by Northern analysis andphosphorimaging, which demonstrated ca. 8% of the unin-duced control level when normalized to levels of -tubulinmRNA (Fig. 4A). The average decrease of LDK mRNA in BFwas 70% by quantitative PCR. However, the reduction in LDKmRNA did not alter the growth rate significantly under the

FIG. 3. Extraction of LDK. (A) Triton X-114 extraction. Afterincubation with Triton X-114 the samples were separated into aqueous(Aq1) and detergent phase by centrifugation extraction, and the de-tergent phase was re-extracted (Aq2 and Det). Cell equivalents wereanalyzed by Western blotting with anti-V5, anti-procyclin, and anti-phosphoglycerate kinase (which detects both the 45-kDa cytosolic andthe 56-kDa glycosomal matrix isoforms). (B) Carbonate extraction.Cell equivalents of the carbonate supernatant and pellet in SDS-PAGE sample buffer were loaded onto SDS-PAGE gels and blotted.The sample used for detection of the integral membrane protein VP1was not boiled but heated to 45°C. Blots were probed with anti-V5,anti-phosphoglycerate kinase (anti-PGK), and anti-VP1. TCL, totalcell lysate; P, carbonate pellet; S, carbonate supernatant.

FIG. 4. RNAi targeting LDK in PF and BF does not abrogateparasite proliferation. (A) Northern analysis of PF transfectants bear-ing an RNAi cassette targeting LDK. The abundance of full-length3.32-kb mRNA seen in the uninduced condition (Tet�) is dramaticallyreduced upon induction of RNAi (Tet�). The small species in theTet� lane corresponds to the double-stranded RNA produced uponinduction. At right is the control hybridization with �-tubulin. Migra-tion of molecular mass markers is shown (in kb). (B) Growth curve forPF, induced (Tet�) or uninduced (Tet�) for RNAi. Standard devia-tions of cell counts are obscured by markers. (C) Growth curve for BF(Tet�) or uninduced (Tet�) for RNAi. Standard deviations of cellcounts are obscured by markers.



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conditions examined (Fig. 4B). Several independently derivedclonal BF lines showed a small change in growth rate uponinduction of RNAi; the results obtained with one of these linesare shown in Fig. 4C.

We next analyzed BF and procyclic RNAi transfectants fornumbers of lipid droplets, utilizing Nile Red staining and mi-croscopic analysis. Parasites were induced with Tet for RNAiand then examined on day 5 (PF) and day 2 (BF) for thepresence of Nile Red staining bodies. The numbers of lipiddroplets were enumerated by examining at least 50 randomlyselected cells for each condition. In both developmental stages,LDK RNAi knockdown resulted in a 90% reduction in thenumber of lipid droplets (Fig. 5). In addition, the remaininglipid droplets appeared to be somewhat smaller and stainedmore faintly than those seen in the control samples. In controlexperiments with the parental 29-13 PF line and an irrelevantBF RNAi line (CK2��), Tet treatment gave rise to a smallincrease in lipid droplet numbers (7 and 16%, respectively),indicating that neither Tet nor the process of RNAi itself areresponsible for the reduction in lipid droplets seen upon LDKRNAi. These data show that LDK is required for maintainingnormal numbers of lipid droplets in vitro.

Cells respond to an increase in fatty acids or to variousstresses in lipid homeostasis by increasing the number oflipid droplets (48). We therefore investigated whether stim-uli that normally induce lipid droplet biogenesis were stillable to do so when LDK was depleted. Two different con-ditions were tested, both of which were tolerated by PF,although they were much more toxic to BF. Each conditiondeprives cells of at least one type of lipid, presumably yield-ing a compensatory increase in lipid synthesis or uptake andsubsequent storage in lipid droplets. Treatment of PF with1.5 �M myriocin, a potent inhibitor of the first step insphingolipid biosynthesis, has been reported to increase thenumber of lipid droplets (20). Reduction of lipids in themedium has been previously shown to induce fatty acidsynthesis in T. brucei (34), and we observed that replacingfetal calf serum with delipidized calf serum for 24 h almostdoubled the number of lipid droplets in PF (see Table S1 inthe supplemental material). Both of these treatments wereapplied to PF that had been induced for LDK RNAi for 5days. Although the treated PF remained viable for the treat-ment period, both treatments increased the proportion ofparasites with two, three, or four nuclei as revealed by DAPIstaining. We therefore normalized the number of lipid drop-lets to the number of nuclei in these experiments to moreaccurately reflect any potential induction of lipid droplets(Fig. 6B; Table S1 in the supplemental material providesdetail by number of nuclei per cell). Myriocin increased thenumber of lipid droplets per nucleus from �4 to �10,whereas delipidated serum increased the number to �7.Under both inducing conditions, cells depleted of LDK av-eraged less than one droplet per nucleus (Fig. 6). The re-duction was apparent when only “normal” cells (one or twonuclei) were considered (Table S1 in the supplemental ma-terial). These findings indicate that LDK plays a critical rolein the biogenesis and/or maintenance of lipid droplets.

Induction of lipid droplets increases LDK autophosphory-lation. Given that LDK appears to be important for theinduction or maintenance of lipid droplets, we hypothesized

that the kinase might show increased activity after cells wereexposed to lipid droplet inducing conditions. Because thestudies with the K41A mutant suggested that autophosphor-ylation was a better reflection of LDK activity, we focusedon autophosphorylation. Cell lysates were prepared aftergrowing the cells in delipidated serum for 4 h. Immunopre-cipitates were prepared and assayed for kinase activity, and

FIG. 5. LDK RNAi depletes lipid droplets. Parasites induced forLDK RNAi were stained for lipids using Nile Red after 5 days (PF) or48 h (BF) and compared to parallel untreated cultures. (A) Represen-tative images of cells stained with Nile Red. Bar, 5 �m. (B) Thenumber of lipid droplets per cell under each condition was enumer-ated. The means and standard deviations are shown.



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the same blots were incubated with anti-V5 to quantitateLDK-V5. As shown in Fig. 7, growth in the absence of lipidsresulted in significantly increased phosphorylation of LDKin the assay. Because phosphorylation of LDK was shown to

require its catalytic lysine (Fig. 1D), the enhanced phosphor-ylation is likely attributable to increased activation of LDK.

DISCUSSION

We describe here a novel protein kinase, LDK, which local-izes to the periphery of lipid droplets in both BF and PF T.brucei. LDK was identified by searching T. brucei predictedproteins for those possessing both a protein kinase catalyticdomain and at least one putative transmembrane domain. Al-though we anticipated that our screen might yield genes en-coding proteins localized to the plasma membrane, this kinasewas instead localized to an intracellular organelle. Previouswork has shown some partial localization of mitogen-activatedprotein kinases to lipid droplets of leukocytes (53), as well asanonymous kinase activity associated with lipid droplets (33).However, these physical associations have not been tied todroplet function. Recently, a genome-wide RNAi screen ofDrosophila cells identified over 200 genes that modulate lipiddroplet morphology, affecting their size, subcellular distribu-tion, or number (25). Of those that resulted in a �fewer lipiddroplets’ phenotype, two were protein kinases: cdc2 and tlk.These protein kinases are involved in cell cycle regulation andare not localized to lipid droplets. Hence, their modulation oflipid droplet number likely results from pleiotropic effects.Neither of these kinases is closely related to LDK at the se-quence level. Thus, LDK is unique in that it is both positionedat the lipid droplet surface and is involved in lipid dropletmaintenance.

To our knowledge, LDK is the first protein kinase identifiedas localized to intracellular organelles of trypanosomatids andthe first protein shown to primarily reside on lipid droplets ofthese organisms under normal conditions. Lipid droplet mem-branes are unusual since they consist of a monolayer of phos-pholipids. The internal core of the lipid droplet is composedprimarily of neutral lipids, an environment unfavorable to al-

FIG. 6. LDK RNAi prevents lipid body induction. Parallel culturesof PF were treated with or without Tet to induce RNAi. After 4 days,the cultures were split into three conditions—control, myriocin (1.5�M), or delipidated serum—and cultured an additional 24 h. Theywere compared to parallel untreated cultures. (A) Representative im-ages of cells stained with Nile Red. Bar, 5 �m. Figure S2 in thesupplemental material provides additional images. (B) The numbers oflipid droplets per nucleus following the various treatments were de-termined. The means and standard deviations are shown. Occasionalcells with �4 nuclei were not included in the analysis. Detail is pro-vided in Table S1 in the supplemental material.

FIG. 7. Growth in delipidated serum increases LDK autophos-phorylation activity. (A) PF induced for expression of LDK-V5 weregrown for 4 h in medium containing delipidated or normal serum. Thetagged LDK was immunoprecipitated and subjected to a kinase assayas in Fig. 1, measuring autophosphorylation (32P) and the amount ofLDK-V5 by Western blotting (anti-V5) on the same lanes. U, un-treated; D, grown in delipidated serum. (B) Graph of the relativeactivity of LDK from cells grown in delipidated serum versus cells froma parallel culture grown in normal serum. Shown are the data from twoindependent isolates (Iso 1 and Iso 2), and two independent experi-ments for isolate 2 (Exp1 and Exp2).



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most all proteins. The topology of proteins associated with thelipid droplet membrane is therefore likely to be distinct fromtransmembrane proteins in lipid bilayers that make up othermembranes of the cell. Because deletion of the hydrophobicdomain led to diffuse cytosolic staining, we propose that LDKassociates with the monolayer membrane via its single hydro-phobic domain, leaving both the N-terminal kinase domainand the C-terminal portion extending toward the cytosol. Be-cause LDK-V5 is partially extractable with carbonate and par-titions to the aqueous phase upon Triton X-114 extraction (notshown), we suggest it is a tightly associated peripheral mem-brane protein. However, given the lack of studies on the be-havior of proteins in the lipid body monolayer, this topic bearsfurther study. Indeed, a previous publication showed mem-brane-dipping proteins may be more readily extracted by car-bonate than transmembrane proteins (28). Unlike some mem-brane-dipping domains (13), the LDK hydrophobic domain isnot predicted to form an amphipathic alpha helix. However, itis flanked by charged regions, especially basic amino acids.Basic amino acids have been shown to work in concert with thehydrophobic domain of caveolin to mediate its interaction withthe lipid body membrane when cells are treated with fatty acids(29).

The location of LDK at the lipid droplet surface suggests itcould function as a mediator of the lipidomic status in the cell,reminiscent of the sensing of AMP/ATP ratio by protein kinaseAMPK. Indeed, LDK is most similar to members of theCAMKL family, which includes AMPK. We propose that lo-calization to the lipid droplet surface may also be important formodulating activity, similar to what is seen for a key enzyme inphospholipid synthesis, CTP:phosphocholine cytidylyltrans-ferase. This enzyme shows a dramatic increase in kcat anddecrease in Km when associated with membranes and specifi-cally associates with lipid droplets as they enlarge (21).Changes in membrane curvature are thought to affect the con-formation and hence the activity of the molecule (13). Thelocation of LDK at the lipid droplet membrane suggests it, too,could be modulated by the composition or curvature of themembrane, allowing it to transmit signals regarding the lipidstatus of the cell. We observed an increase in autophosphory-lation activity of LDK when cells were incubated in a lipiddroplet inducing condition, indicating activation of the kinase.Phosphorylation of natural substrates by the kinase anchoredat the droplet surface might provide a more accurate measureof activation. Identification of the substrates would be a steptoward understanding the regulation of lipid droplet ho-meostasis in the parasite.

The observation that LDK RNAi knockdown causes littlegrowth phenotype despite a dramatic reduction in the numberof detectable lipid droplets indicates that droplet loss does notinevitably lead to parasite death. It is possible that small lipiddroplets, undetectable by the techniques we used, remained inthe cells or that survival is facilitated by other lipid storage andmobilizing mechanisms. The higher sensitivity of BF to knock-down of LDK may result from their high requirement formyristic acid (used to anchor the variant surface glycoproteinthat covers the parasite surface) or their upregulated exocyticand endocytic transport systems (2, 19). In either case, it ap-pears that LDK is not essential in vitro, but confirmation willrequire gene knockout approaches. Using conditional knock-

outs in studies that manipulate the lipids provided to andsynthesized by the cells may elucidate conditions in which theprotein provides important regulatory information. In addi-tion, such knockouts will provide a means to dissect the struc-tural features of LDK that are essential for lipid droplet bio-genesis and maintenance.

ACKNOWLEDGMENTS

We thank Jay Bangs, Roberto Docampo, and Terry Pearson for thegifts of antisera directed against trypanosome BiP, acidocalcisomal H�

pyrophosphatase VP1, and procyclin, respectively. We also thankNichelle Kunecke and Jennifer Wierman for technical assistance andSunil Laxman for the plasmid pLEW79-1V5(PAC).

This study was supported by grant NIH R01AI31077.The authors are solely responsible for the contents of this article.

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A Novel Protein Kinase Localized to Lipid Droplets Is ... · EUKARYOTIC CELL, Nov. 2010, p. 1702–1710 Vol. 9, No. 11 1535-9778/10/$12.00 doi:10.1128/EC.00106-10 Copyright © 2010,