Phosphoproteome dynamics reveal heat-shockprotein complexes specific to the Leishmaniadonovani infectious stageMiguel A. Moralesa, Reiko Watanabea, Mariko Dachera, Philippe Chafeyb,c, José Osorio y Fortéad,1, David A. Scotte,Stephen M. Beverleye, Gabi Ommenf, Joachim Closf, Sonia Hemg, Pascal Lenormandg, Jean-Claude Rousselleg,Abdelkader Namaneg, and Gerald F. Spätha,2

aInstitut Pasteur, G5 Virulence Parasitaire, 75015 Paris, France; Institut National de la Santé et de la Recherche Médicale AVENIR and Centre National de laRecherche Scientifique (Unité de Recherche Associée 2581), 75015 Paris, France; bInstitut Cochin, Université Paris Descartes, Centre National de la RechercheScientifique (Unité Mixte de Recherche 8104), 75014 Paris, France; cInstitut National de la Santé et de la Recherche Médicale, U1016, 75014 Paris, France;dInstitut Pasteur, Unité d’Immunophysiologie et Parasitisme Intracellulaire, 75015 Paris, France; eDepartment of Molecular Microbiology, WashingtonUniversity School of Medicine, St. Louis, MO 63110; fLeishmaniasis Research Group, Bernhard Nocht Institute for Tropical Medicine, D-20359 Hamburg,Germany; and gInstitut Pasteur, Plate-forme de Protéomique and Centre National de la Recherche Scientifique Unité de Recherche Associée 2185,Department of Structural Biology and Chemistry, Pasteur-Genopole Ile-de-France, 75015 Paris, France

Edited by Elizabeth Anne Craig, University of Wisconsin, Madison, WI, and approved March 23, 2010 (received for review December 23, 2009)

Leishmania is exposed to a sudden increase in environmental tem-perature during the infectious cycle that triggers stage differentia-tion and adapts the parasite phenotype to intracellular survival inthe mammalian host. The absence of classical promoter-dependentmechanisms of gene regulation and constitutive expression of mostof the heat-shock proteins (HSPs) in these human pathogens raiseimportant unresolved questions as to regulation of the heat-shockresponse and stage-specific functions of Leishmania HSPs. Here weused a gel-based quantitative approach to assess the Leishmaniadonovani phosphoproteome and revealed that 38% of the proteinsshowed significant stage-specific differences, with a strong focus ofamastigote-specific phosphoproteins on chaperone function. Weidentified STI1/HOP-containing chaperone complexes that interactwith ribosomal client proteins in an amastigote-specific manner. Ge-netic analysis of STI1/HOPphosphorylation sites in conditional sti1−/−

null mutant parasites revealed two phosphoserine residues essentialfor parasite viability. Phosphorylation of themajor Leishmania chap-erones at the pathogenic stage suggests that these proteins may bepromising drug targets via inhibition of their respective proteinkinases.

signaling | stress response

Kinetoplastid parasites of the genus Leishmania generatea variety of pathologies collectively termed leishmaniasis,

afflicting millions of people worldwide (1, 2). During the in-fectious cycle, these insect-borne parasitic trypanosomatids areexposed to a temperature increase following transmission fromthe invertebrate to the vertebrate host. The temperature changeprovides a crucial signal for developmental transition of thepromastigote insect form to the amastigote form that thrivesinside host phagocytes, generating the disease (3). Despite therelevance of heat-induced stage differentiation for pathogenesis,mechanisms underlying the parasite heat-shock response and itsrole in the development and survival of the amastigote stageremain poorly understood.Trypanosomatids express highly conserved members of heat-

shock and chaperone protein families, suggesting that the cel-lular response to heat stress is similar between parasite and host(4, 5). However, in contrast to other eukaryotes that regulateheat-induced expression of molecular chaperones and cytopro-tective proteins via a family of heat-shock transcription factors(HSFs) (6), trypanosomatid genomes do not encode for classicaltransacting nuclear factors (7). Gene expression in these organ-isms relies on highly parasite-specific mechanisms involving poly-cistronic transcription and transsplicing (8, 9). Expression of themajor heat-shock proteins (HSPs) is constitutive, even if heatshock may induce a transient increase in synthesis, which has

been shown to be regulated exclusively at the posttranscriptionallevel (10–13). In contrast to its vertebrate host, both constitutiveand inducible expression of Leishmania HSPs occurs from thesame set of genes, making constitutive and stress-induciblechaperones indistinguishable at the sequence level (11, 14). Thisimportant difference in host and parasite biology raises questionsconcerning the role of Leishmania HSPs at low temperatures inpromastigotes and regulation of their chaperone function upontemperature increase in differentiating and proliferating amas-tigotes. By combining approaches of quantitative phosphopro-teomics, systems biology, and mutagenesis, we have uncoveredseveral unique properties of Leishmania donovani HSPs withrespect to protein modifications, complex formation, and theimportance of chaperone phosphorylation in parasite viability.

Results and DiscussionTwo-dimensional differential gel electrophoresis (2D-DIGE)analysis of affinity-enriched phosphoextracts obtained from L.donovani LD1S promastigotes and axenic amastigotes (15, 16)revealed dramatic differences in protein phosphorylation profilesacross the major Leishmania infectious stages (Fig. 1A, Fig. S1B,and Dataset S1). A total of 831 protein spots were detected au-tomatically using the DeCyder Differential Analysis SoftwarePackage (GEHealthcare), and>700 spotsmatched between threegels representing independent biological replicates, indicatinglittle experimental variation between samples and highly re-producible two-dimensional gel electrophoresis (2DE) conditions(Dataset S1). A total of 171 proteins were identified by massspectrometry using the genome database of highly related Leish-mania infantum (WWW.GeneDB.org) (Fig. S1A andDataset S1),including 55 putative phosphoproteins not identified in our pre-vious study using fluorescent multiplex staining (17). Gene on-tology (GO) analysis of the Leishmania phosphoprotein datasetvia yeast ortholog mapping (Dataset S2) identified six statistically

Author contributions: M.A.M. and G.F.S. designed research; M.A.M., R.W., M.D., G.O.,S.H., P.L., and J.-C.R. performed research; P.C., J.O.y.F., D.A.S., and S.M.B. contributednew reagents/analytic tools; M.A.M., J.C., A.N., and G.F.S. analyzed data; and G.F.S.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1Present address: Limagrain Europe, Biometry Department, ZAC les portes de Riom, 63204Riom, France.

2To whom correspondence should be addressed. E-mail: gerald.spaeth@pasteur.fr.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0914768107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.0914768107 PNAS | May 4, 2010 | vol. 107 | no. 18 | 8381–8386

MICRO

BIOLO

GY

significant GO categories that were overrepresented in our anal-ysis (Fig. 1B and Table S1). Three of these processes—translationinitiation, protein folding, and protein catabolism—have beenimplicated previously in trypanosomatid differential gene ex-pression (9), emphasizing the importance of protein phosphory-lation in posttranslational control of this process.We previously analyzed affinity-enriched L. donovani phos-

phoproteins by qualitative 2DE analysis and demonstrated thespecificity of this procedure combining fluorescent phosphopro-tein staining and phosphatase treatment (17). In contrast to thisanalysis, which suggested only little stage-specific phosphoryla-tion, the quantitative 2D-DIGE analysis revealed a statisticallysignificant difference (P value <0.05) in protein abundance for318 spots corresponding to 38% of the detected phosphopro-teins, with 10.2% of the phosphoproteins showing a statisticallysignificant increase in abundance at the amastigote stage of ≥2-fold (Fig. S1B and Dataset S1). Significantly, amastigote phos-phoproteins with increased abundance and thus phosphorylationcompared to promastigotes were almost exclusively proteinchaperones, including several isoforms of HSP90 family memberHSP83 (hereafter referred to as HSP90), various HSP70 familymembers, stress-induced protein STI1/HOP (referred to here-after as STI1) (18), cyclophilin 40, and the L. donovani orthologof tetratricopeptide repeat (TPR) domain-containing peptidyl-prolyl-isomerase-like protein LinJ19_V3.1560 (Fig. 1C andTable S2). Previous proteomic studies that quantified changes in

protein abundance during axenic amastigote differentiation (Fig.S2) (14), along with quantitative Western blot analysis of pro-mastigote and amastigote total and phosphoextracts (Fig. 2A),demonstrate that the expression of these protein chaperonesacross the promastigote and amastigote stages is largely consti-tutive and not induced by elevated temperature. The increasedabundance of these HSPs and chaperones in the amastigotephosphoproteome therefore does not simply result from in-creased expression, but rather reflects a change in phosphory-lation stoichiometry with, for example, an 8-fold increase in thephosphorylation ratio of STI1 at the intracellular stage (Fig. 2A).Evidence for a potential regulatory role of HSP phosphory-

lation in Leishmania arises from MALDI-TOF/TOF mass spec-trometry analysis of the phosphorylation sites of HSP90 andHSP70. Phosphopeptides were isolated after in-gel digestion andpeptide extraction from the 2D gels by TiO2 enrichment (Fig. 2Band Fig. S3A). Manual analysis of the mass spectrometry spectraidentified three phosphorylation sites at HSP90 Thr223 andSer526 and at HSP70 Thr498. Conservation of the threonineresidues between Leishmania and human HSP90 and HSP70identifies this residue as a putative phosphorylation site in highereukaryotes as well (Fig. 2C and Fig. S3B). Significantly, whereasthe Thr223 residue in L. donovani HSP90 corresponds to serinein human HSP90 and thus may be regulated by phosphorylation,HSP90 Ser526 is unique to Leishmania despite the highly con-served sequence to the human homolog (Fig. 2C). The presence

Fig. 1. Quantitative analysis of L.donovani stage-specific phosphopro-teome. (A) 2D-DIGE analysis. Extractsfrom promastigotes and host-freeamastigotes of three independent bi-ological repeat experiments were dif-ferentially labeled with the spectrallyresolvable CyDyefluors Cy3 and Cy5 andseparated by two-dimensional electro-phoresis (2DE) on 11-cm (pH 4–7) IPGstrips and 12.5% polyacrylamide gels.A merged image of Cy5-labeled amas-tigotes (red) and Cy3-labeled promasti-gotes (green) is shown. The molecularweights of marker proteins (kDa) andthe pH of the gradient (pI) are indicated.(B) Gene ontology analysis. Over-represented categories of GO biologicalprocesses identified with the BiNGOplugin and visualized with Cytoscapesoftware are shown. Gray levels indicatetype I error level (hypergeometric test Pvalue) after false discovery rate correc-tion. Branches of the network selectedfor statistical significance are repre-sented (P values <5e-02). The node areais proportional to the number of genesthat correspond to a given GO category.(C) Analysis of the amastigote phos-phoproteome. The readout of theDeCyder Biological Variation Analysis(BVA) module is shown for the mostabundant amastigote phosphoproteincyclophilin 40 (CYP40, spot 572); oneisoform of HSP90 (spot 258); stress-induced protein STI1 (spot 361); andhypothetical proteins LinJ15.0040 (spot767), LinJ32_V3.2410 (spot 683), andLinJ04.0240 (spot 685). Enlarged regionsof 2D-DIGE gels for Cy3-labeled pro-mastigotes (pro, green) and Cy5-labeledamastigotes (ama, red), and the corresponding 3D views, are represented. The Bottom shows a graphic representation of differences in abundance of theseproteins across three independent experiments. For normalization purposes, a Cy2-labeled internal standard was included, corresponding to a pool of proteinfrom all extracts used in the analysis (st, standard).

8382 | www.pnas.org/cgi/doi/10.1073/pnas.0914768107 Morales et al.

of this phosphorylation site opens up the possibility that thefunction of this major HSP is regulated in a parasite-specificmanner by changes in the protein ionic state through phosphor-ylation, which may affect protein conformation and interactionwith other chaperones, thus adding new regulatory features to L.donovani HSP90. Whereas this phosphorylation site is conservedin TrypanosomaHSP90 as judged by multiple alignment (Fig. 2C),this position is occupied in human and mouse HSP90 by asparticacid. Thus, the HSP90 configuration in higher eukaryotes may belocked into a conformation that mimics constitutive phosphory-lation. These findings indicate that regulation of HSP90 functionsthrough posttranslational modifications may substantially differbetween parasite and host despite the highly conserved sequenceof this protein from Leishmania to man.Biological network analysis using PathwayArchitect software

applied to the identified parasite phosphoprotein datasets re-vealed the presence of a protein network formed between sixamastigote phosphoproteins (Fig. 3A, Table S3). In othereukaryotes, this multimeric chaperone complex has been shownto provide an important signaling function through its interactionwith so-called “client proteins,” which include various steroidreceptors and protein kinases (19, 20). Cochaperone STI1 playsa crucial role in formation of this complex, acting as a scaffoldingprotein that mediates the interaction between HSP90 and theHSP70/client protein complexes through specific TPR-richdomains (21, 22). We investigated the presence of the predictedSTI1-containing protein complexes in Leishmania by Blue Native(BN) electrophoresis and Western blot analysis. Amastigote-specific phosphorylation of multiple protein chaperones corre-lated with the presence of numerous STI1-containing complexesranging from 66 to 480 kDa (Fig. 3B). As judged by coimmu-noprecipitation using anti-STI1 antibody, only the amastigoteSTI1/HSP90 complex interacts with HSP70/client protein com-plexes (Fig. 3C). Mass spectrometry analysis of the coprecipi-tated protein bands identified numerous client proteins impli-cated in the assembly of the protein translation machinery andthe control of protein translation (Fig. 3D). The specificity of thisinteraction has been controlled for by using an isotype-specificcontrol antibody (Fig. S3C) and is further supported by the ab-sence of client protein detection in promastigotes, despite theirconstitutive expression at both stages (23). Our data correlateSTI1 phosphorylation with complex formation linked to proteintranslation, which may affect resistance of ribosomal client pro-teins against proteasome-dependent degradation (24). Although

we detected these complexes only in amastigotes, our data do notrule out the presence of similar complexes in promastigotes,which may have escaped our analysis due to their low abundance.These results are reminiscent of the observation that phos-phorylation of murine STI1 affects localization of this proteinand thus the types of client proteins that interact with STI1/HSP90/HSP70 (25).We next used a genetic approach to investigate the biological

significance of STI1 phosphorylation by mutagenesis. Three STI1phospho-site mutants were generated on the basis of previouslyidentified STI1 phosphoresidues in Trypanosoma brucei (26),mouse (27), and human (28) (Fig. 4A). As STI1 appears to beessential, we tested these mutants using a conditional knockoutsystem (29). In this approach, to guard against the lethal phe-notype, both chromosomal STI1 alleles were inactivated in thepresence of an episomal plasmid expressing WT STI1, yieldingthe mutant sti1−/−/pXNG-STI1 (Fig. 4B and Fig. S4). The epi-somal plasmid pXNG4SAT (29) additionally carries botha fluorescent (GFP) and a negative selectable thymidine kinase(TK) marker rendering parasites susceptible to the antiviral drugganciclovir (GCV). Thus by FACS or drug selection sti1−/−/pXNG-STI1 parasites could be tested for their requirement tomaintain the ectopic STI1 gene copy. The functionality of mu-tated STI1 genes was then tested in a “plasmid shuffle” (30), byintroducing a second plasmid, and asking whether the WT STI1borne on pXNG could be lost. As expected for an essential gene,it was not possible to segregate away pXNG-STI1 in the chro-mosomal sti1−/− null mutant, even in the presence of GCV (Fig.4C, parental line). The presence of an additional copy of func-tional STI1, however, allowed for efficient elimination of pXNG-STI1 during negative selection as judged by the substantial re-duction in GFP fluorescence intensity (STI1_WT, Fig. 4 C andD Left).This binary readout allowed us now to test for the functionality

of the STI1 phosphorylation site mutants. The mutants wereexpressed in independent sti1−/−/pXNG-STI1 clones, and theireffect on negative selection against pXNG-STI1 was analyzed.Expression of the T217A mutant fully compensated for pXNG-STI1, which was efficiently eliminated during negative selection(Fig. 4 C and D). This result suggests that the T217A mutationdoes not affect the functional properties of STI1 (case 2, Fig.4B). In contrast, expression of neither S15A nor S481A was ableto compensate for pXNG-STI1, which was maintained at levelscomparable to the sti1−/−/pXNG-STI1 parental line in both

A B

Lmajor Linfantum Tbrucei Tcruzi Human Mouse

KEI Yeast

EGV HFE E S EEEK QQR EE E K EGV HFE E S EEEK QQR EE E K EGV HFE E T EEEK KQR EE E K EGV HFE E T EEEK KQR EE E K EGL ELP E D EEEK KKQ EE K K EGL ELP E D EEEK KKQ EE K K D-FELE E TD EEK AER E KEI

*

HSP90

400800

12001600evitale

Rytis

netni

pro ama pro amaCE Phospho

evitaleR

ytisnet

ni

01000200030004000

tub

STI1

tub

C

1:1 1:2

1:0.5 1:4

Fig. 2. Analysis of phosphoprotein stoichiometryand phosphorylation site determination. (A) Quanti-tative Western blot. Promastigote (pro) and amasti-gote (ama) crude (CE) and phosphoprotein (Phospho)extracts were analyzed by Western blotting usingpolyclonal anti-Leishmania HSP90 and STI1 andmonoclonal anti-tubulin (tub) antibodies. The blotswere revealed using ZyMax Cy3-conjugated anti-rab-bit and ZyMax Cy5-conjugated anti-mouse secondaryantibodies (Invitrogen) and signals were detectedusing a Typhoon variable mode imager. Relative in-tensities correspond to quantified signals of HSP90and STI1 after normalization to α-tubulin (Image-Quant software; GE Healthcare). (B) Phosphopeptideanalysis. MALDI-TOF/TOF spectrum of one HSP90peptide isolated from 2D-DIGE gels after tryptic di-gestion and TiO2 enrichment is shown. Peptide519EGVHFEESEEEKQQR533 (m/z 1940.00) of HSP90contains y7, y8, and y8* ions, enabling identification ofSer526 as the phosphorylated residue. *, fragmentions arising from loss of phosphoric acid (−98 Da);[MH]+, precursor ion; [MH-P-18]+, precursor ion withloss of one phosphoric acid (−98 Da); pS, phosphorylated serine. (C) Multiple alignments. Sequence elements encompassing HSP90 phosphorylation sites ofthree trypanosomatids, human and yeast, were analyzed with ClustalXv2. The phosphoresidue is marked by the asterisk.

Morales et al. PNAS | May 4, 2010 | vol. 107 | no. 18 | 8383

MICRO

BIOLO

GY

promastigote and amastigote stages (Fig. 4 C and D). Togetherthese data suggest that the STI1 residues S15 and S481 are es-sential phosphorylation sites (case 1, Fig. 4B) required for L.donovani viability in culture and emphasize the importance ofchaperone phosphorylation in parasite biology. Further analysisof the role of these phosphorylation sites in STI1 complex for-mation was precluded by the lethal sti1−/− phenotype and therequirement to maintain WT STI1.The manner in which Leishmania regulates the response to

stress is fundamentally different from that of other eukaryotes,including the mammalian host. In most organisms, stress-inducedexpression increases stress tolerance through protection of basiccell functions, without a major impact on cellular morphologyand phenotype. In Leishmania, however, stress signals such as lowpH and nutritional starvation, or high temperature, induce de-velopmental programs that lead to differentiation of metacyclic

promastigotes and amastigotes and adapt the parasite for trans-mission and intracellular survival (3, 31, 32). This reinterpretationof the stress response likely translates into unique regulatorymechanisms and interactions of Leishmania chaperones. Ourdata provide important insights into these parasite-specificmechanisms, which may depend on (i) stage-specific chaperonephosphorylation during environmentally induced parasite differ-entiation; (ii) phosphorylation of unique residues in parasitesHSP70, HSP90, and STI1; and (iii) formation of chaperonecomplexes. The emphasis on posttranslational regulation of thestress response in Leishmania through phosphorylation and otherprotein modifications may represent an evolutionary adaptationof trypanosomatid parasites to constitutive expression and theabsence of transcriptional regulation (9). By analogy to stressregulation through HSFs in other eukaryotes (33), our data in-dicate that the absence of these factors in trypanosomatids mayhave been compensated for by the evolution of protein kinasesthat regulate chaperone function. In resting cells, HSF1 is inac-tivated in other eukaryotes through its association with HSP70and HSP90, but is released and activated under stress conditions(34). In a similar fashion, Leishmania chaperones may tetherprotein kinases that are released and activated during environ-mentally induced stage differentiation. Indeed, binding of Leish-mania MAP kinases to HSP70 (35) and the direct implication ofHSP90 in parasite differentiation established in geldanamycin-treated parasites (36) suggest a uniquemodel in which chaperone/kinase interactions would regulate phosphotransferase activities,which in turn might directly feed back on chaperone functionsand differentiation.

Materials and MethodsCell Culture and Differentiation of L. donovani. The L. donovani strain 1S2D(MHOM/SD/62/1S-CL2D), clone LdB was cultured and axenic amastigoteswere differentiated as described (16, 37).

Preparation of L. donovani total and phosphoprotein extracts. Axenic pro-mastigotesor axenic amastigotes 48hafter inductionofdifferentiationbypHandtemperature shift were harvested from logarithmic cultures. For phosphoproteinpurification,proteinconcentrationwasadjustedto0.1mg/mL,and2.5-mgextractswere applied onto equilibrated affinity columns of the phosphoprotein purifica-tion kit (Qiagen) according to manufacturer’s instructions.

Western Blot Analysis. Proteins were revealed using the following antibodies:polyclonal anti-STI-1 (18) and anti-HSP83, mouse monoclonal anti-α-tubulinantibody (Sigma), and anti-rabbit or anti-mouse ZyMax Cy3 or Cy5 conju-gated secondary antibodies (Invitrogen). In some experiments, NativePAGENovex 4–16% Bis-Tris Gels (Invitrogen) were transferred onto PVDF mem-branes, and proteins were revealed using the antibodies described aboveand anti-rabbit HRP-conjugated secondary antibodies (Pierce).

Immunoprecipitation. Cells were lysed and incubated for 45 min at 4 °C withSTI-1 polyclonal antibody (10 μL, 200 μg/mL) and protein A MicroBeads(50 μL; Miltenyi Biotec). Mixtures were loaded on μMACS columns (MiltenyiBiotec). Eluates were separated by denaturing SDS/PAGE, gels werestained with SyproRuby, and bands of interest were excised and furtheranalyzed by MS-MS/MS.

Blue Native PAGE. Native extracts were centrifuged at 20,000 × g for 30 min at4 °C. Twenty or 40 μg of protein, containing 0.025% Coomassie Blue G-250,were separated on NativePAGE Novex 4–16% Bis-Tris Gels (Invitrogen) at150 V for 3 h and 250 V for 1 h at 4 °C.

Sample Preparation and DIGE Labeling. Phosphoprotein pellets were resus-pendedinDIGEsamplebuffer(7MUrea,2MThiourea,4%CHAPS,30mMTris,pH8.5) toafinal protein concentrationof 5.0mg/mL. Phosphoprotein extracts frompromastigotes and amastigotes were differentially labeled with the spectrallyresolvable Cy3 and Cy5, and a pool of both extracts was labeled with Cy2 fornormalization purposes, following the manufacturer’s recommendations (GEHealthcare). Three independent biological replicates of promastigote andamastigote phosphoextracts were prepared and resolved by 2D-DIGE. In addi-

Fig. 3. Chaperone phosphorylation in amastigotes is linked to formation ofa stage-specific multiprotein complex. (A) Biological network analysis.Analysis was carried out with PathwayArchitect software (version 3.0.1;www.stratagene.com). An input set of 43 yeast IDs was used to build thebiological interaction network from annotations extracted from scientificliterature (automatic scanning of abstracts from PubMed database: www.ncbi.nlm.nih.gov/pubmed). Shaded levels indicate the fold increase inamastigote phosphoprotein abundance compared to promastigote phos-phoextracts. (B) Blue-Native PAGE. Native extracts from promastigotes (P) oramastigotes (A) were separated on NativePAGE Novex 4–16% Bis-Tris gels(Invitrogen) and complexes were revealed by colloidal Coomassie staining(Left). Replica gels were electroblotted onto PVDF membranes and proteinsdetected with anti-STI1 antibody (Right). Molecular weight (MW) of nativeprotein marker (M) is shown. (C) Coimmunoprecipitation. Pro- (P) andamastigote (A) crude extracts were incubated with STI1 polyclonal antibodyand protein A MicroBeads (Miltenyi Biotec). Eluates were separated by de-naturing SDS/PAGE and stained with SyproRuby. MW of marker proteins inkDa is shown. (D) STI1-associated proteins isolated by immunoprecipitationwere identified by MS/MS analysis.

8384 | www.pnas.org/cgi/doi/10.1073/pnas.0914768107 Morales et al.

Fig. 4. STI1 phosphorylation is essential for L. donovani viability. (A) Multiple alignment of STI1 homologs from Leishmania major (Lmajor, CAJ02290.1),L. infantum (Linfantum, CAM65800.1), Trypanosoma brucei (Tbrucei, CBH11274.1), Trypanosoma cruzi (Tcruzi, EAN97552.1), Mus musculus (Mouse,AAC53267.1), Homo sapiens (Human, AAA58682.1), and Saccharomyces cerevisiae (Yeast, CAA60743.1) using ClustalXv2 is shown. The number indicates theL. major STI1 amino acids targeted for analysis by mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Phosphorylation sites atserine 15 (S15) and serine 481 (S481) have been experimentally identified in human and T. brucei, respectively (26, 28). Threonine 217 (T217) has been selectedon the basis of experimentally identified threonine phosphorylation upstream of the yeast STI1 nuclear localization signal (NLS, dotted line) and the presenceof a casein kinase II signature sequence S/T-X-X-D/E (27). The shading indicates the level of amino acid conservation. (B) Generation of L. donovani sti1−/−

conditional null mutants for analysis of phosphorylation site-specific mutants. Heterozygous sti1+/− null mutants (STI1/Δsti1::PAC ) were transfected withepisomal vector pXNG carrying the STI1 wild-type ORF (STI1/Δsti1::PAC[pXNG-STI1]), before replacement of the second STI1 allele yielding homozygous sti1−/−

null mutants (Δsti1::BLEO/Δsti1::PAC[pXNG-STI1]). Independent clones were transfected with episomal vector pLEXSY expressing either an additional copy ofwild-type STI1 or one of the phospho-site mutants described in A. The ability of episomally expressed STI1 phospho-site mutants S15A, T217A, and S481A tocomplement for the loss of pXNG-STI1 during negative selection with gangciclovir (GCV) provides a genetic test to distinguish mutations that affect STI1function (case 1, pXNG-STI1 is maintained) from silent mutations (case 2, pXNG-STI1 is lost). Selections were performed using 25 μg/mL puromycin, 150 μg/mLnourseothricin, 5 μg/mL bleomycin, and 50 μg/mL hygromycin B, respectively. BLEO, bleomycin resistance cassette; GCV, ganciclovir; GFP, green fluorescentprotein gene; HYG, hygromycin B resistance cassette; PAC, puromycin resistance cassette; SAT, nourseothricin resistance cassette; TK, herpes simplex virusthymidine kinase gene. (C) Analysis of STI1 mutants by negative selection in conditional sti1−/− lines. Elimination of pXNG-STI1 was followed by FACS analysismonitoring the levels of GFP expressed from the same episome. GFP mean fluorescence of conditional sti1−/− null mutant promastigotes treated for threeculture passages with 50 μg/mL GCV (Left and Center) or axenic amastigotes treated for 72 h with GCV (Right) is shown. Data are means ± SD of a repre-sentative experiment analyzed in triplicate. (D) Histogram plots of one representative analysis of axenic amastigotes after 72 h of GCV selection (shadedhistograms). Dotted lines represent the background fluorescence of untransfected control parasites. Solid lines represent the GFP fluorescence levels ofparasites cultured without GCV.

Morales et al. PNAS | May 4, 2010 | vol. 107 | no. 18 | 8385

MICRO

BIOLO

GY

tion, a gel containing a pool of either pro- or amastigote extracts from allextractions was included for normalization purposes.

2DE. A total of 90 μg of protein sample containing 30 μg of Cy3- and Cy5-labeled samples was pooled together with 30 μg of Cy2-labeled control andadjusted with 150 μL Destreak rehydration buffer (GE Healthcare) contain-ing 0.5% IPG buffer 4-7 and 1.0% DTT. Samples were simultaneously sepa-rated in the first dimension by iso-electric focusing (IEF) overnight (see SIMaterials and Methods for details).

Staining Procedures and Image Analysis. After electrophoresis, gels werescanned on a Typhoon 9410 Variable Mode Imager (GE Healthcare) andanalyzed with DeCyder 6.5 software (GE Healthcare).

Protein Identification by Mass Spectrometry. Spots of interest were excisedfrom gels using the ProPic Investigator robotic system (Genomic Solutions).MS and MS/MS raw data for protein identification were obtained as pre-viously described (17) (see SI Materials and Methods for details).

Phosphopeptide Identification. Protein digests, obtained as described above,were diluted in loading buffer (80% ACN, 5% TFA) (38) and loaded on TiO2

microcolumns as described previously (39). All phosphorylated peptideswere first analyzed for the presence of the major fragment ion [MH-H3PO4]

+ = MH − 98 Da corresponding to the loss of the phosphate moietyand identified positively by MASCOT. In addition, all MS/MS spectra were

carefully curated manually for assignment of phosphorylation sites (see SIMaterials and Methods for details).

Bioinformatics Approaches. Retrieval of yeast orthologous sequenceswas carriedoutwithaBLASTPalgorithm(40)byqueryingtheSaccharomycescerevisiaeproteindatabase with the L. infantum protein sequences (default parameters; Refseqprotein database build 2.1 released May 17, 2005; www.ncbi.nlm.nih.gov/ge-nome/seq/BlastGen/BlastGen.cgi?taxid=4932). From 92 unique L. infantum IDsused in this analysis, 17 IDs were specific to Leishmania and 75 IDs mapped to 71yeast orthologs with expectation values ranging from E = 0 to E = 0.003 andalignment scores rangingfrom974to38.9bits. Functionalenrichmentanalysiswascarried out with the 71 yeast IDs as input using the BiNGO plugin (version 2.1) oftheCytoscape software (version2.5.1). Biological network analysiswas carriedoutwith PathwayArchitect software (version 3.0.1; www.stratagene.com). An inputset of 43 yeast IDs was used to build the biological interaction network from theannotations extracted from the scientific literature (automatic scanning ofabstracts from PubMed database; www.ncbi.nlm.nih.gov/pubmed/).

ACKNOWLEDGMENTS. We thank Dr. Zilberstein (Technion, Israel) for anti-HSP83 antiserum, Dr. Reed (Infectious Disease Research Institute, Seattle) foranti-STI1 antibody, and Malcolm McConville for critical reading of themanuscript. This work was supported by the Institut National de la Santé etde la Recherche Médicale AVENIR program (G.F.S., M.A.M., R.W.), NationalInstitutes of Health Grants AI-21903 and AI-29646 (to S.M.B.), the 7th Frame-work Programme of the European Commission through a grant to the LEISH-DRUG Project (223414), and the Fondation de Recherche Medicale EquipeFondation pour la Recherche Médicale program (DEQ20061107966).

1. Ashford R, Desjeux P, Raadt P (1992) Estimation of population at risk of infection andnumber of cases of Leishmaniasis. Parasitol Today 8:104–105.

2. Bañuls AL, Hide M, Prugnolle F (2007) Leishmania and the leishmaniases: A parasitegenetic update and advances in taxonomy, epidemiology and pathogenicity inhumans. Adv Parasitol 64:1–109.

3. Zilberstein D, Shapira M (1994) The role of pH and temperature in the developmentof Leishmania parasites. Annu Rev Microbiol 48:449–470.

4. Clos J (1997) Heat Shock Proteins in Biology and Disease, eds Radons J, Multhoff G(Research Signpost, Kerala, India), pp 421–448.

5. Folgueira C, Requena JM (2007) A postgenomic view of the heat shock proteins inkinetoplastids. FEMS Microbiol Rev 31:359–377.

6. Morimoto RI (1998) Regulation of the heat shock transcriptional response: Cross talkbetween a family of heat shock factors, molecular chaperones, and negative regulators.Genes Dev 12:3788–3796.

7. Ivens AC, et al. (2005) The genome of the kinetoplastid parasite, Leishmania major.Science 309:436–442.

8. Clayton C, Shapira M (2007) Post-transcriptional regulation of gene expression intrypanosomes and leishmanias. Mol Biochem Parasitol 156:93–101.

9. Clayton CE (2002) Life without transcriptional control? From fly to man and backagain. EMBO J 21:1881–1888.

10. Argaman M, Aly R, Shapira M (1994) Expression of heat shock protein 83 inLeishmania is regulated post-transcriptionally. Mol Biochem Parasitol 64:95–110.

11. Brandau S, Dresel A, Clos J (1995) High constitutive levels of heat-shock proteins inhuman-pathogenic parasites of the genus Leishmania. Biochem J 310:225–232.

12. de Carvalho EF, de Castro FT, Rondinelli E, Soares CM, Carvalho JF (1990) HSP 70 geneexpression in Trypanosoma cruzi is regulated at different levels. J Cell Physiol 143:439–444.

13. Hunter KW, Cook CL, Hayunga EG (1984) Leishmanial differentiation in vitro:Induction of heat shock proteins. Biochem Biophys Res Commun 125:755–760.

14. Rosenzweig D, et al. (2008) Retooling Leishmania metabolism: From sand fly gut tohuman macrophage. FASEB J 22:590–602.

15. Barak E, et al. (2005) Differentiation of Leishmania donovani in host-free system:Analysis of signal perception and response. Mol Biochem Parasitol 141:99–108.

16. Goyard S, et al. (2003) An in vitro system for developmental and genetic studies ofLeishmania donovani phosphoglycans. Mol Biochem Parasitol 130:31–42.

17. Morales MA, et al. (2008) Phosphoproteomic analysis of Leishmania donovani pro-and amastigote stages. Proteomics 8:350–363.

18. Webb JR, Campos-Neto A, Skeiky YA, Reed SG (1997) Molecular characterization ofthe heat-inducible LmSTI1 protein of Leishmania major. Mol Biochem Parasitol 89:179–193.

19. Pratt WB, Galigniana MD, Harrell JM, DeFranco DB (2004) Role of hsp90 and the hsp90-binding immunophilins in signalling protein movement. Cell Signal 16:857–872.

20. Pratt WB, Morishima Y, Osawa Y (2008) The Hsp90 chaperone machinery regulatessignaling by modulating ligand binding clefts. J Biol Chem 283:22885–22889.

21. Dittmar KD, Banach M, Galigniana MD, Pratt WB (1998) The role of DnaJ-like proteinsin glucocorticoid receptor.hsp90 heterocomplex assembly by the reconstituted hsp90.p60.hsp70 foldosome complex. J Biol Chem 273:7358–7366.

22. Scheufler C, et al. (2000) Structure of TPR domain-peptide complexes: Critical elements

in the assembly of the Hsp70-Hsp90 multichaperone machine. Cell 101:199–210.23. Rosenzweig D, Smith D, Myler PJ, Olafson RW, Zilberstein D (2008) Post-translational

modification of cellular proteins during Leishmania donovani differentiation. Proteomics

8:1843–1850.24. Kim TS, et al. (2006) Interaction of Hsp90 with ribosomal proteins protects from

ubiquitination and proteasome-dependent degradation. Mol Biol Cell 17:824–833.25. Longshaw VM, Chapple JP, Balda MS, Cheetham ME, Blatch GL (2004) Nuclear

translocation of the Hsp70/Hsp90 organizing protein mSTI1 is regulated by cell cycle

kinases. J Cell Sci 117:701–710.26. Nett IR, et al. (2009) The phosphoproteome of bloodstream form Trypanosoma

brucei, causative agent of African sleeping sickness.Mol Cell Proteomics 8:1527–1538.27. Blatch GL, Lässle M, Zetter BR, Kundra V (1997) Isolation of a mouse cDNA encoding

mSTI1, a stress-inducible protein containing the TPR motif. Gene 194:277–282.28. Dephoure N, et al. (2008) A quantitative atlas of mitotic phosphorylation. Proc Natl

Acad Sci USA 105:10762–10767.29. Murta SM, Vickers TJ, Scott DA, Beverley SM (2009) Methylene tetrahydrofolate

dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in

Leishmania major. Mol Microbiol 71:1386–1401.30. Sikorski RS, Boeke JD (1991) In vitro mutagenesis and plasmid shuffling: From cloned

gene to mutant yeast. Methods Enzymol 194:302–318.31. Sacks DL, Perkins PV (1984) Identification of an infective stage of Leishmania

promastigotes. Science 223:1417–1419.32. Zakai HA, Chance ML, Bates PA (1998) In vitro stimulation of metacyclogenesis in

Leishmania braziliensis, L. donovani, L. major and L. mexicana. Parasitology 116:

305–309.33. Kanei-Ishii C, Tanikawa J, Nakai A, Morimoto RI, Ishii S (1997) Activation of heat shock

transcription factor 3 by c-Myb in the absence of cellular stress. Science 277:246–248.34. Ali A, Bharadwaj S, O’Carroll R, Ovsenek N (1998) HSP90 interacts with and regulates

the activity of heat shock factor 1 in Xenopus oocytes. Mol Cell Biol 18:4949–4960.35. Morales MA, Renaud O, Faigle W, Shorte SL, Späth GF (2007) Over-expression of

Leishmania major MAP kinases reveals stage-specific induction of phosphotransferase

activity. Int J Parasitol 37:1187–1199.36. Wiesgigl M, Clos J (2001) Heat shock protein 90 homeostasis controls stage differen-

tiation in Leishmania donovani. Mol Biol Cell 12:3307–3316.37. Saar Y, et al. (1998) Characterization of developmentally-regulated activities in axenic

amastigotes of Leishmania donovani. Mol Biochem Parasitol 95:9–20.38. Imanishi SY, et al. (2007) Reference-facilitated phosphoproteomics: Fast and reliable

phosphopeptide validation by microLC-ESI-Q-TOF MS/MS. Mol Cell Proteomics 6:

1380–1391.39. Hem S, Rofidal V, Sommerer N, Rossignol M (2007) Novel subsets of the Arabidopsis

plasmalemma phosphoproteome identify phosphorylation sites in secondary active

transporters. Biochem Biophys Res Commun 363:375–380.40. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein

database search programs. Nucleic Acids Res 25:3389–3402.

8386 | www.pnas.org/cgi/doi/10.1073/pnas.0914768107 Morales et al.

Supporting InformationMorales et al. 10.1073/pnas.0914768107SI Materials and Methods2D Gel Electrophoresis (2DE). A total of 90 μg of protein samplecontaining 30 μg of Cy3- and Cy5-labeled samples was pooledtogether with 30 μg of Cy2-labeled control and adjusted with 150μL Destreak rehydration buffer (GE Healthcare) containing0.5% IPG buffer 4-7 and 1.0% DTT. Samples were simulta-neously separated in the first dimension by iso-electric focusing(IEF) overnight at 20 °C using the gel-based IPGphor isoelectricfocusing system (GE Healthcare) and 11-cm DryStrip (pH 4–7)immobiline strips. Strips were rehydrated overnight at roomtemperature in Destreak rehydratation solution containing 0.5%IPG Buffer 4-7 (GE Healthcare). Samples were applied to theacidic end of the strip using a sample cup. The maximun currentsetting was 50 μA/strip and the IEF run was carried out using thefollowing conditions: 100-V gradient step for 5 h, 300-V gradientstep for 5 h, 1,000-V gradient step for 2 h, 6,000-V gradient stepfor 8 h, and 6,000 V for 5 h (60,550 Vh).Following IEF, strips were sequentially incubated for 15 min in

6M urea, 75mMTris/HCl (pH 8.8), 29.3% glycerol, 4% SDS, and0.002%bromophenol blue supplemented with either 65mMDTTor 13.5 mM iodoacetamide. The strips were transferred to SDSpolyacrylamide gels and sealed with 0.5% agarose in 25 mMTris-base, 0.19 M glycine, 0.2% SDS, 0.01% bromophenol blue.Electrophoresis was carried out in an Ettan DALT six electro-phoresis system (GE Healthcare) using 12.5% SDS/PAGE gelsand two-step runs (1 W/gel for 1 h and 13 W/gel for 6 h).In some experiments, phosphoprotein extracts were subjected

to dephosphorylation before 2DE analysis. Samples were incu-bated for 30 min at 30 °C with 4,000 units of lambda proteinphosphatase (New England BioLabs) in reaction buffer con-taining 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT,0.01% Brij, and 35.2 mM MnCl2.

Staining Procedures and Image Analysis. After electrophoresis, gelswere scanned on a Typhoon 9410 Variable Mode Imager (GEHealthcare), using 488/520 nm for Cy2, 532/580 nm for Cy3, 633/670 nm for Cy5, and 100 μm as pixel size. Gel images werenormalized by adjusting voltage to obtain appropriate pixel valuewithout any saturation. Images were analyzed with DeCyder 6.5software (GE Healthcare). Spot detection and quantification foreach gel were carried out in a DeCyder Differential In-gelAnalysis (DIA) module. The automatic spot detection settingswere 1,500. After spot detection, spots located outside of thearea of interest or nonproteinaceous spots corresponding to dustparticles were excluded. Gels were matched in the DeCyderBiological Variation Analysis (BVA) module. A 1.5-fold differ-ence in abundance, with a confidence of P values <0.05, wasconsidered significant.For MS analysis, gels were fixed in 50% methanol and 7%

acetic acid, restained with SYPRO Ruby Staining (Invitrogen)overnight, and visualized on the Typhoon at 532/610 nm and 100μm pixel size.

Protein Identification by Mass Spectrometry. Spots of interest wereexcised from gels using the ProPic Investigator robotic system(Genomic Solutions) and collected in 96-well plates. Destaining,

reduction, alkylation, and trypsin digestion of the proteins fol-lowed by peptide extraction were carried out with the ProGestInvestigator (Genomic Solutions).After a desalting step using C18-μZipTip (Millipore), peptides

were eluted directly using the ProMS Investigator (GenomicSolutions) onto a 96-well stainless steel MALDI target plate(Applied Biosystems/MDS SCIEX) with 0.5 μl of CHCA matrix(10 mg/mL in 70% ACN/30% H2O/0.1% TFA).MS and MS/MS raw data for protein identification were

obtained on the 4800 Proteomics Analyzer (Applied Biosystems)and analyzed by GPS Explorer 2.0 software (Applied Biosystems/MDS SCIEX). For positive-ion reflector mode spectra 3,000laser shots were averaged. For MS calibration, autolysis peaks oftrypsin ([M + H]+ = 842.5100 and 2,211.1046) were used as in-ternal calibrates. Monoisotopic peak masses were automaticallydetermined within the mass range 800–4,000 Da with a signal-to-noise ratio minimum set to 30. Up to 12 of the most intense ionsignals were selected as precursors for MS/MS acquisition, ex-cluding common trypsin autolysis peaks and matrix ion signals. InMS/MS positive ion mode, 4,000 spectra were averaged, collisionenergy was 2 kV, collision gas was air, and default calibration wasset using the Glu1-Fibrino-peptide B ([M + H]+ = 1,570.6696)spotted onto 14 positions of the MALDI target. Combined pep-tide mass fingerprint and MS/MS queries were performed usingthe MASCOT search engine 2.1 (Matrix Science) embedded intoGPS-Explorer Software 3.5 (Applied Biosystems/MDS SCIEX)on the Leishmania major and Leishmania infantum databases(Gene DB) with the following parameter settings: 50 ppm massaccuracy, trypsin cleavage, one missed cleavage allowed, carba-midomethylation set as fixed modification, oxidation of methio-nines allowed as variable modification, and MS/MS fragmenttolerance set to 0.3 Da. Protein hits with MASCOT proteinscore ≥50 and a GPS Explorer protein confidence index ≥95%were used for further manual validation.

Phosphopeptide Identification. Protein digests, obtained as de-scribed above, were diluted in loading buffer (80% ACN, 5%TFA) (1) and loaded on TiO2 microcolumns as described pre-viously (2). After two washing steps in 10 μL loading buffer and60 μL buffer 2 (80% ACN, 1% TFA), phosphopeptides wereeluted using 2 μL NH4OH (pH 12) onto the MALDI target. Forcrystallization, 0.8 μL of 20 g/L DHB dissolved in ACN, water,and phosphoric acid (50/44/6, vol/vol/vol) was added. All MS/MSraw spectra were externally calibrated and annotated as forclassical protein identification. The L. major and L. infantumdatabases (Gene DB) were searched using MASCOT softwarewith the following parameters: trypsin cleavage, two missedcleavage sites allowed, carbamidomethylation set as fixed mod-ification, 110 ppm mass tolerance for MS, and 0.25 Da for MS/MS fragment ions. Phosphorylation (STY) and Deamidation(NQ) were allowed as variable modifications. All phosphorylatedpeptides were first checked for the presence of the major frag-ment ion [MH-H3PO4]

+ = MH − 98 Da corresponding to theloss of the phosphate moiety and identified positively by MAS-COT. In addition, all MS/MS spectra were carefully checkedmanually for assignment of phosphorylation sites.

1. Imanishi SY, et al. (2007) Reference-facilitated phosphoproteomics: Fast and reliablephosphopeptide validation by microLC-ESI-Q-TOF MS/MS. Mol Cell Proteomics 6:1380–1391.

2. Hem S, Rofidal V, Sommerer N, Rossignol M (2007) Novel subsets of the Arabidopsisplasmalemma phosphoproteome identify phosphorylation sites in secondary activetransporters. Biochem Biophys Res Commun 363:375–380.

Morales et al. www.pnas.org/cgi/content/short/0914768107 1 of 9

A

270303

309

220,256,248

250

464

494

547520

519

533

529

534

566

574

525

537

553

569

572

539

573

604

675

657

655

628

610

639

586

629

642 644

663

691 704

695 693

782

767

93

743

401

394

342

321

327

713

715

714

708

382

381

388

389

392

475

433

352

374

369

300

295

244

187

159

161183

171

177

172

204

206

207

205

90

89

162

152

154

91

100

65

63

61

75

95

115

66

127

122

141

144

146

155

308

200

182

449

460

468

482

596

615

669

668

672

722

703

763

760

764

619

648

779

783

809

813

436455

462

328

338

305

290

291

302

298

331

343

425403

363

358

339

336

319

380

383

630

361

148169

314

258

360

324

364

750751

768

362

683685

B

-10 -5 -1/1

01

23

45

67

average ratio

Sta

tisitc

alsig

nifican

ce

(n

eg

ative

lo

g1

0 o

fP

valu

e)

5 10

Fig. S1. (A) Identification of stage-specific phosphoproteins in L. donovani. Extracts from promastigotes and host-free amastigotes were differentially labeledwith the spectrally resolvable CyDye fluors Cy3 and Cy5, separated by 2DE on 11-cm (pH 4–7) IPG strips and 12.5% polyacrylamide gels, and sequentially stainedwith SyproRuby. A total of 189 protein spots (yielding 167 identifications), indicated by the numbered circles, were analyzed by MALDI-MS and MS/MS. (B)Global analysis of the stage-specific phosphoproteome. Graphical representation of the ratio of the promastigote/amastigote protein abundance as a functionof the P value. This volcano plot allows for a more global analysis of the stage-specific phosphoproteome and indicates that 4.4% of the promastigote and10.2% of the amastigote phosphoproteomes, respectively, show statistically significant, stage-specific increase of >2-fold (red spots).

Morales et al. www.pnas.org/cgi/content/short/0914768107 2 of 9

0 2.5 5 10 15 24 144

-3

-2

-1

0

1

2

3

ratio

time (h)

HSP100

PFR2C

Fig. S2. Amastigote chaperones are differentially phosphorylated. Expression patterns of HSP proteins during differentiation using a quantitative iTRAQapproach based on the data published in ref. 1 are shown. Log2-transformed expression values of HSPs show a constitutive expression of most of the proteins,with the exception of CLP1 (CLP) (♢). HSP100 (●) and paraflagellar rod protein PFR2C (shaded circle) are shown as representative examples of amastigote- andpromastigote-specific proteins, respectively. ●, PPI; △, HSP putative; ▽, HSP70.4; □, HSP70L; ◆, Cyclophilin-40; ×, STI1; □, HIP.

1. Rosenzweig D, et al. (2008) Retooling Leishmania metabolism: From sand fly gut to human macrophage. FASEB J 22:590–602.

Morales et al. www.pnas.org/cgi/content/short/0914768107 3 of 9

A B

m/z300 600 900 1200

0

10

20

30

74 9. 37 993 .4 0 37 6. 24 620 .3 2 1108 .3 9 491 .2 8 86 4. 38

27 5. 19 y 2

y 4 y 3 y 5 y 9

y 6 y 8 y 7 y 10

* y 10

b 9 b 8

b 7

b 10

[M H- P- 18] +

[MH] +

1191 .4 3

1142 .2 56

1 289 .2 6 89 8. 22

1 027 .2 9

1243 .4 0

1419 .4 8

1 044 .3 1 b 9

*

b 11 *

127 3. 38

y 2 y 4 y 5 y 11 y 6 y 9 y 7 y 10 y 3

EV pTDE DE E D T K K y 8

y 10 *

b 7 b 8 b 9 b 10 b 9

*

b 11 *

m/z600 1300 2000 2600

0

20

40

60

80

100

y 1

y 3

y 5

y 11 y 6

y 9

y 7

y 10

y 19 b 26

*

[M H - P- 18 ] +

[MH] +

304 0. 68

1638 .6 6

1824 .6 8 36 0. 16

1540 .6 9 726 .2 5 2 052 .8 2

85 5. 34

y 17

y 15

1142 .4 0

124 1. 43

1055 .3 5

17 5. 10

62 8. 28 y 6

*

598 .1 2

y 7 *

75 7. 39

y 15 *

1726 .6 9 y 17

*

1955 .0 1 y 19

*

268 1. 53

443 .1 8 y 4

*

y 5 *

500 .2 2

Rel.

ab

un

dan

ce

(%

)

GP QI EV TF DL DAnG IL NVS AEE KG pTGK R y 1 y 3 y 5 y 11 y 6 y 9 y 7 y 10 y 19

b 26 *

y 17 y 15

y 6 *y 7

*y 15 *y 17

*y 19 *

y 4 *

y 5 *

GP QI EV TF DL DAnG IL NVS AEE KG pTGK R y 1 y 3 y 5 y 11 y 6 y 9 y 7 y 10 y 19

b 26 *

y 17 y 15

y 6 *y 7

*y 15 *y 17

*y 19 *

y 4 *

y 5 *

Rel.

ab

un

dan

ce

(%

)

00

00

*

*

A AA AG

26

37

49

64

82

115

C

Fig. S3. (A) MALDI-TOF/TOF spectra. [MH]+, precursor ion; [MH-P-18]+, precursor ion with a loss of one phosphoric acid (−98 Da); n, N-deamination; pT,phosphorylated threonine; *, fragment ions arising from loss of phosphoric acid (−98 Da). (A) Peptide 214EVTDEDEEDTKK225 (m/z 1516.71) of HSP 83-1 (Upper)contains y9, y10, and y10* ions, which allows us to identify Thr223 as the phosphorylated residue. Peptide 473GVPQIEVTFDLDANGILNVSAEEKGTGKR501 (m/z3137.76) of HSP70 (Lower) contains y3 and y4* ions, which allows us to identify Thr498 as the phosphorylated residue. (B) Multiple alignment analysis ofconserved sequence elements comprising phosphorylation sites of HSP90 (Upper) and HSP70 (Lower) in trypanosomatids, humans, and yeast. The phos-phoresidues are marked by the asterisks. (C) Coimmunoprecipitation. Amastigotes were lysed and incubated with anti-STI1 polyclonal antibody and protein AMicroBeads (Miltenyi Biotec). Eluates were separated by denaturing SDS/PAGE and gels stained with SyproRuby. Amastigote lysates were loaded onto the gelsafter incubation with STI1 antibody and protein A microbeads (lane A), protein A microbeads alone (lane AA), and control IgG and protein A microbeads (laneAG). MW of marker proteins in kilodaltons is shown. The gel is representative of two independent experiments.

Morales et al. www.pnas.org/cgi/content/short/0914768107 4 of 9

STI1

STI1 flank rev

STI1 flank fwd

ST

I1

ST

I1

ST

I1

ST

I1

ST

I1

ST

I1

ST

I1

WT STI1 locus

rec. STI1 locus

STI1 5’UTR

STI1 5’UTR STI1 3’UTR

STI1 3’UTRPAC

BLEO

Fig. S4. Creation of sti1−/− null mutants. (A and B) Schematic representations of the Δsti1::PAC and Δsti1::BLEO gene replacement constructs. Relevant re-striction sites are shown, as are length rulers (base paris) referring to the absolute position in the plasmids. UTR, untranslated regions. (C) Schematic repre-sentation of the L. infantum STI1 gene locus (solid box), with a neighboring coding region (open box), the annealing sites for primers STI1 flank fwd and STI1flank rev, and a size ruler (base pairs). (D) Verification of gene replacement by PCR. Genomic DNA purified from L. donovani wild type (STI1+/+, lane 1), theΔsti1::PAC mutant (sti1+/−, lane 2), and four putative Δsti1::PAC/Δsti1::BLEO/[pXNG-STI1] mutants (sti1−/−[+], lanes 3–6) is shown. The STI1 gene locus was am-plified with primers STI1 flank fwd and STI1 flank rev. The wild-type allele yields 3,795-bp amplification products, whereas the size of the products afterhomologous recombination with PAC and BLEO is predicted as 2,761 and 2,530 bp, respectively. An unspecific product appears in all samples. Note the ap-pearance of the 3,795-bp product with sti1−/−[+], clone 2, indicating that a wild-type allele was retained.

Morales et al. www.pnas.org/cgi/content/short/0914768107 5 of 9

Table S1. Leishmania IDs identified by GO analysis

Yeast ID Yeast name Leishmania ID Leishmania annotation BLAST E-value BLAST Score

GO-ID 42026 (protein refolding)NP_013911 HSC82 LinJ33_V3.0350 Heat-shock protein 83-1 0NP_009728 SSE2 LinJ18_V3.1350 Heat-shock protein, putative 2.00E-78 288.0NP_014670 STI1 LinJ27_V3.2340 TPR-repeat protein, putative 2.00E-08 55.8NP_012579 SSC1 LinJ30_V3.2540 Heat-shock 70-rel. protein 1, putative 0 671.0

GO-ID 6454 (translational initiation)NP_013866 TIF34 LinJ36_V3.4070 eIF 3 subunit, putative 3.00E-28 121.0NP_012540 SUI2 LinJ03_V3.0960 eIF2 α-subunit, putative 1.00E-39 159.0NP_015206 DBP1 LinJ35_V3.3150 ATP-dependent RNA helicase, putative 2.00E-106 381.0NP_012581 ANB1 LinJ25_V3.0750 Eukaryotic initiation factor 5a, putative 4.00E-40 160.0NP_012397 TIF2 LinJ01_V3.0790 Eukaryotic initiation factor 4a, putative 8.00E-111 395.0

GO-ID 6511 (ubiquitin-dependent protein catabolic process)NP_012709 DOA1 LinJ24_V3.1970 Hypothetical protein, conserved 3.00E-37 151.0NP_014760 RPT5 LinJ22_V3.0490 Proteasome regulatory ATPase subunit 5,

putative4.00E-130 460.0

NP_116710 RPN12 LinJ32_V3.1260 Proteasome regulatory non-ATPase subunit,putative

3.00E-19 92.0

NP_012777 RPT1 LinJ22_V3.0440 Proteasome regulatory ATPase subunit 1,putative

0 515.0

NP_014800 PUP1 LinJ36_V3.4180 hs1vu complex proteolytic subunit-like 0.003 38.9NP_013064 PRP19 LinJ27_V3.2430 Hypothetical protein, conserved 7.00E-14 73.9NP_010134 RPN5 LinJ21_V3.0840 Proteasome regulatory non-ATPase subunit 5,

putative2.00E-45 178.0

GO-ID 7001 (chromosome organization and biogenesis)NP_011059 GLC7 LinJ05_V3.0100 Phosphoprotein phosphatase, putative 9.00E-38 153.0NP_010993 RNR1 LinJ28_V3.0980 Ribonucleoside diphosphate reductase,

putative0 974.0

NP_009645 POL30 LinJ15_V3.1500 Proliferative cell nuclear antigen (PCNA),putative

3.00E-40 161.0

NP_010199 SUB2 LinJ21_V3.1820 RNA helicase, putative 8.00E-112 399.0NP_013834 ASC1 LinJ28_V3.2940 Activated protein kinase c receptor (LACK) 4.00E-65 244.0NP_015219 SSE1 LinJ29_V3.1330 Hypothetical protein, unknown function 3.00E-17 85.1NP_013387 NNT1 LinJ15_V3.0870 Hypothetical protein, conserved 4.00E-18 88.2NP_015089 RVB2 LinJ34_V3.2440 ATP-dependent DNA helicase, putative 2.00E-154 540.0NP_014264 FPR1 LinJ19_V3.1560 Peptidylprolyl isomerase-like protein 1.00E-21 99.8NP_116616 TUB2 LinJ21_V3.2240 β-Tubulin 0 631.0NP_012980 DYN1 LinJ26_V3.1960 Hypothetical protein, conserved 3.00E-04 42.0NP_116614 ACT1 LinJ04_V3.1250 Actin 6.00E-165 575.0NP_015467 KAR3 LinJ19_V3.0250 C-terminal motor kinesin, putative 4.00E-56 214.0NP_013911 HSC82 LinJ33_V3.0350 Heat-shock protein 83-1 0NP_009916 GBP2 LinJ35_V3.2240 RNA-binding protein, putative 1.00E-09 60.1

GO-ID 48248 (organellar fusion)NP_015467 KAR3 LinJ19_V3.0250 C-terminal motor kinesin, putative 4.00E-56 214.0NP_116616 TUB2 LinJ21_V3.2240 β-Tubulin 0 631.0NP_010643 SPC110 LinJ14_V3.0990 Immunodominant antigen, putative 5.00E-09 57.8

GO-ID 6519 (amino acid and derivative metabolic process)NP_010322 KRS1 LinJ15_V3.0270 Lysyl-tRNA synthetase, putative 0 518.0NP_012737 SPE1 LinJ12.0100 Ornithine decarboxylase, putative 5.00E-63 237.0NP_015360 GLN1 LinJ06_V3.0370 Glutamine synthetase, putative 4.00E-88 320.0NP_014264 FPR1 LinJ19_V3.1560 Peptidylprolyl isomerase-like protein 1.00E-21 99.8NP_009679 GRS1 LinJ36_V3.4030 Glycyl tRNA synthetase, putative 2.00E-139 491.0NP_010868 GLY1 LinJ01_V3.0500 Hypothetical protein, conserved 3.00E-10 62.0NP_014980 ALA1 LinJ22_V3.1390 Alanyl-tRNA synthetase, putative 8.00E-05 43.9NP_010790 SAM2 LinJ30_V3.3560 S-adenosylmethionine synthetase 0 457.0

Morales et al. www.pnas.org/cgi/content/short/0914768107 6 of 9

Table S2. Stage-specific phosphoproteins and phosphoprotein isoforms

Accession no. Ratio T test Protein ID

LinJ35_V3.4830 6.07 0.000001 Cyclophilin-40* 574LinJ15_V3.0040 5.32 8.3E-05 Hypothetical protein, nd 685LinJ31_V3.2400 5.11 0.00003 3,2-transenoyl-CoA isom. mito.† 630LinJ29_V3.1330 4.72 0.00003 Hypothetical protein, rel. HSP70* 172LinJ18_V3.1350 4.44 0.00001 Heat-shock protein* 144LinJ29_V3.1330 4.35 0.00003 Hypothetical protein, rel. HSP70* 177LinJ32_V3.2410 4.27 0.002 Hypothetical protein, rel. HSP20* 767LinJ33_V3.0350 4.19 0.00002 Heat-shock protein 83-1* 258LinJ08_V3.1020 4.06 0.00005 Stress-induced protein STI1* 361LinJ18_V3.1350 3.96 0.00001 Heat-shock protein* 141LinJ35_V3.4830 3.89 0.000003 Cyclophilin-40* 572LinJ29_V3.0120 3.38 0.0001 Proteasome regulatory non-ATPase SU* 169LinJ04_V3.0230 3.35 0.01136 Hypothetical protein† 683LinJ18_V3.1350 3.19 1.37E-07 Heat-shock protein* 146LinJ33_V3.0350 2.85 0.0003 Heat-shock protein 83-1* 248LinJ19_V3.1560 2.83 0.000002 Peptidylprolyl isomerase-like protein* 468LinJ28_V3.2960 2.74 0.0008 Hsp70, nd 321LinJ33_V3.0350 2.6 0.00003 Heat-shock protein 83-1* 256LinJ29_V3.0330 2.55 0.00001 Hypothetical protein, rel. HIP* 520LinJ28_V3.2960 2.55 0.0008 Hsp70, nd 328LinJ33_V3.0350 2.32 0.007 Heat-shock protein 83-1* 220LinJ19_V3.1560 2.25 0.0002 Peptidylprolyl isomerase-like protein* 482LinJ11_V3.0210 2.21 0.004 Acidocalcisomal pyrophosphatase* 494LinJ26_V3.1220 2.12 0.002 HSP70.4 hsp70 related* 331LinJ28_V3.2960 2.07 0.004 Hsp70, nd 314LinJ19_V3.0250 2.03 0.002 C-terminal motor kinesin† 159LinJ31_V3.1100 2.02 0.026 Hypothetical protein, nd 547LinJ35_V3.3390 2.00 0.00067 6-phosphoglucon. dehydrogen.* 537LinJ26_V3.1960 2.00 0.002 Hypothetical protein† 360LinJ12_V3.0100 1.88 0.008 Ornithine decarboxylase, nd 205LinJ28_V3.1310 1.86 0.00001 Glucose-regulated protein 78† 298LinJ28_V3.2960 1.83 0.0009 Hsp70, nd 327LinJ21_V3.2240 1.81 0.0004 β-Tubulin† 449LinJ29_V3.1330 1.81 0.0004 Hypothetical protein* 171LinJ21_V3.2240 1.79 0.000004 β-Tubulin† 460LinJ28_V3.1310 1.70 0.000007 Glucose-regulated protein 78† 302LinJ08_V3.1020 1.68 0.00006 Stress-induced protein STI1* 352LinJ32_V3.1000 1.67 0.01 Hypothetical protein* 122LinJ12_V3.0100 1.65 0.0002 Ornithine decarboxylase, nd 207LinJ34_V3.3890 1.63 0.003 Ubiquitin hydrolase, nd 127LinJ34_V3.0080 1.60 0.0001 G6PD, glucose-6-phosphate* 392LinJ28_V3.2960 1.58 0.002 Hsp70, nd 321LinJ04_V3.1250 1.47 0.00001 Actin* 586LinJ34_V3.0080 1.45 0.0001 G6PD, glucose-6-phosphate* 389LinJ30_V3.3130 1.45 0.0006 Hypothetical protein* 610LinJ21_V3.1820 1.43 0.04 RNA helicase* 566LinJ30_V3.3130 1.42 0.002 Hypothetical protein* 604LinJ02_V3.0680 1.41 0.003 ATP-dep. Clp protease subunit† 300LinJ35_V3.3110 1.36 0.02 Ubiquitine act. enzyme E1, nd 91LinJ26_V3.1220 1.35 0.01 HSP70.4, hsp70 related, nd 324LinJ12_V3.0100 1.22 0.03 Ornithine decarboxylase, nd 206LinJ22_V3.1390 1.19 0.004 Alanyl-tRNA synthetase, nd 152LinJ20_V3.1210 −1.11 0.00002 Calpain-like cysteine peptidase 115LinJ34_V3.3460 −1.15 0.02 Vacuolar ATP synthase subunit a 305LinJ06_V3.0370 −1.24 0.03 Glutamine synthetase 628LinJ30_V3.2540 −1.24 0.02 HSP70-related protein 1, mito. 309LinJ36_V3.7240 −1.26 0.005 Chaperonin, T-complex prot. 1 403LinJ36_V3.4030 −1.27 0.005 Glycyl tRNA synthetase 295LinJ20_V3.1210 −1.27 0.01 Calpain-like cysteine peptidase 95LinJ12_V3.0620 −1.27 0.02 Cytochrome c oxidase subunit iv 642LinJ30_V3.0250 −1.28 0.01 Ubiquitin hydrolase, cysteine pept. 66LinJ15_V3.0870 −1.29 0.03 Hypothetical protein 100LinJ29_V3.1330 −1.30 0.02 Hypothetical protein 182

Morales et al. www.pnas.org/cgi/content/short/0914768107 7 of 9

Table S2. Cont.

Accession no. Ratio T test Protein ID

LinJ23_V3.0090 −1.35 0.03 Hypothetical protein 425LinJ29_V3.0330 −1.38 0.004 Hypothetical protein 519LinJ15_V3.0270 −1.39 0.00008 Lysyl-tRNA synthetase 374LinJ11_V3.0210 −1.40 0.03 Acidocalcisomal pyrophosphat. 464LinJ35_V3.0920 −1.41 0.009 Hypothetical protein 691LinJ35_V3.3150 −1.43 0.004 ATP-dep. RNA helicase 90LinJ03_V3.0960 −1.43 0.0003 eIF2 α-subunit 553LinJ27_V3.2430 −1.44 0.0005 Hypothetical protein 381LinJ28_V3.2940 −1.49 0.003 Activated protein kinase c receptor 675LinJ34_V3.2530 −1.49 0.007 Hypothetical protein 693LinJ27_V3.1300 −1.63 0.01 60S acidic rib. subunit protein 669LinJ34_V3.2530 −1.64 0.003 Hypothetical protein 695LinJ35_V3.3150 −1.68 0.005 ATP-dep. RNA helicase 93LinJ07_V3.0710 −1.69 0.003 Hypothetical protein 629LinJ25_V3.1670 −1.71 0.0005 Hypothetical protein 619LinJ20_V3.0790 −1.72 0.04 Hypothetical protein 250LinJ33_V3.0600 −1.72 0.0004 Hypothetical protein 303LinJ30_V3.3560 −1.73 0.0004 S-adenosylmethionine synth. 534LinJ05_V3.0100 −1.76 0.0002 Phosphoprotein phosphatase 401LinJ36_V3.4070 −1.77 0.00005 eIF3 subunit 615LinJ11_V3.0820 −1.85 0.02 Hypothetical protein 364LinJ27_V3.1300 −1.88 0.0006 60s acidic rib. subunit prot. 668LinJ33_V3.0350 −1.96 0.002 Heat-shock protein 83-1 290LinJ27_V3.2480 −2.04 0.002 60s acidic rib. subunit prot. 672LinJ26_V3.1960 −2.05 0.04 Hypothetical protein 342LinJ29_V3.0120 −2.25 0.01 Proteasome reg. non-ATPase subunit 148LinJ11_V3.0820 −2.39 0.001 Hypothetical protein 383LinJ31_V3.1930 −2.51 0.0005 Ubiquitin-fusion protein 75LinJ35_V3.2240 −2.83 0.0003 RNA-binding protein 708LinJ05_V3.0100 −2.91 0.0006 Phosphoprotein phosphatase 394LinJ35_V3.2240 −3.11 0.0003 RNA-binding protein 714LinJ03_V3.0910 −3.43 0.004 Hypothetical protein 270LinJ35_V3.2240 −3.51 0.00003 RNA-binding protein 715LinJ30_V3.0580 −3.97 0.0007 Hypothetical protein 779LinJ34_V3.3030 −4.01 8.62E-07 Pyruvate carboxylase 382LinJ20_V3.1320 −5.89 0.007 Calpain-like cysteine peptidase 813LinJ20_V3.1320 −10.3 0.001 Calpain-like cysteine peptidase 809

nd, protein expression not determined by ref. 1.*According to ref. 1, protein expression is equal or decreased in amastigotes compared to promastigotes.†According to ref. 1, protein expression is increased in amastigotes compared to promastigotes.

1. Rosenzweig D, et al. (2008) Retooling Leishmania metabolism: From sand fly gut to human macrophage. FASEB J 22:590–602.

Morales et al. www.pnas.org/cgi/content/short/0914768107 8 of 9

Table S3. Input set of 43 yeast IDs used for network analysis

Yeast ID Yeast name Leishmania ID Leishmania annotation Blast score BlastE-value

NP_010544.1 HSP78 LinJ02_V3.0680 ATP-dep. Clp protease subunit 692 0NP_012540.1 SUI2 LinJ03_V3.0960 eIF2 α-subunit 159 1.00E-39NP_116614.1 ACT1 LinJ04_V3.1250 Actin 575 6.00E-165NP_011059.1 GLC7 LinJ05_V3.0100 Phosphoprotein phosphatase 153 9.00E-38NP_015360.1 GLN1 LinJ06_V3.0370 Glutamine synthetase 320 4.00E-88NP_014670.1 STI1 LinJ08_V3.1020 Stress-induced protein sti1 318 2.00E-87NP_009565.1 IPP1 LinJ11_V3.0210 Acidocalc. pyrophosphatase 162 2.00E-40NP_012737.1 SPE1 LinJ12.0100 Ornithine decarboxylase 237 5.00E-63NP_010322.1 KRS1 LinJ15_V3.0270 Lysyl-tRNA synthetase 518 1.00E-147NP_013387.1 NNT1 LinJ15_V3.0870 Hypothetical protein 88.2 4.00E-18NP_009728.1 SSE2 LinJ18_V3.1350 Heat-shock protein 288 2.00E-78NP_015467.1 KAR3 LinJ19_V3.0250 C-terminal motor kinesin 214 4.00E-56NP_014264.1 FPR1 LinJ19_V3.1560 Peptidylprol. isom.-like protein 99.8 1.00E-21NP_013207.1 MDN1 LinJ20_V3.1210 Calpain-like cysteine peptidase 62.4 2.00E-10NP_010199.1 SUB2 LinJ21_V3.1820 RNA helicase 399 8.00E-112NP_116616.1 TUB2 LinJ21_V3.2240 β-tubulin 631 0NP_014980.1 ALA1 LinJ22_V3.1390 Alanyl-tRNA synthetase 43.9 8.00E-05NP_011029.1 SSA4 LinJ26_V3.1220 HSP70.4 hsp70-related 795 0NP_013064.1 PRP19 LinJ27_V3.2430 Hypothetical protein 73.9 7.00E-14NP_013444.1 RPP0 LinJ27_V3.2480 60s acidic rib. subunit prot. 202 2.00E-52NP_009396.2 SSA1 LinJ28_V3.1310 Glucose-reg. protein 78 693 0NP_013834.1 ASC1 LinJ28_V3.2940 Act. protein kinase c receptor 244 4.00E-65NP_011029.1 SSA4 LinJ28_V3.2960 hsp70 833 0NP_011892.1 RPN1 LinJ29_V3.0120 Proteas. regul. nonATPase SU 276 1.00E-74NP_011639.1 PPT1 LinJ29_V3.0330 Hypothetical protein, rel. HIP 47.8 6.00E-06NP_015219.1 SSE1 LinJ29_V3.1330 Hypoth. protein, rel. HSP70 85.1 3.00E-17NP_011421.1 PAN2 LinJ30_V3.0250 Ubiquitin hydrolase, cyst. pept. 161 3.00E-40NP_012579.1 SSC1 LinJ30_V3.2540 HSP70-rel. protein 1, mitoch. 671 0NP_010790.1 SAM2 LinJ30_V3.3560 S-adenosylmethionine synth. 457 2.00E-129NP_012118.1 RPL40A LinJ31_V3.1930 Ubiquitin-fusion protein 219 1.00E-57NP_013911.1 HSC82 LinJ33_V3.0350 Heat-shock protein 83-1 732 0NP_014158.1 ZWF1 LinJ34_V3.0080 G6PD, glucose-6-phosphate 437 2.00E-123NP_013235.1 PDC5 LinJ34_V3.3030 Pyruvate carboxylase 306 5.00E-84NP_010096.1 TFP1 LinJ34_V3.3460 Vac. ATP synthase subunit a 369 8.00E-103NP_014033.1 UBP15 LinJ34_V3.3890 Ubiquitin hydrolase 178 3.00E-45NP_009916.1 GBP2 LinJ35_V3.2240 RNA-binding protein 60.1 1.00E-09NP_012712.1 UBA1 LinJ35_V3.3110 Ubiquitine act. enzyme E1 426 4.00E-120NP_015206.1 DBP1 LinJ35_V3.3150 ATP-dep. RNA helicase 381 2.00E-106NP_012053.1 GND1 LinJ35_V3.3390 6-phosphoglucon. dehydrogen. 313 7.00E-86NP_013317.1 CPR6 LinJ35_V3.4830 Cyclophilin-40 231 2.00E-61NP_009679.1 GRS1 LinJ36_V3.4030 Glycyl tRNA synthetase 491 2.00E-139NP_013866.1 TIF34 LinJ36_V3.4070 eIF3 subunit 121 3.00E-28NP_012526.1 CCT8 LinJ36_V3.7240 Chaperonin, T-complex prot. 1 418 2.00E-117

The IDs that are part of the network shown in Fig. 3A are indicated in boldface–italic type.

Other Supporting Information Files

Dataset S1 (XLS)Dataset S2 (XLS)

Morales et al. www.pnas.org/cgi/content/short/0914768107 9 of 9