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Multifunctional class I transcription in Trypanosoma brucei depends on a novel protein complex

2007; Springer Nature; Volume: 26; Issue: 23 Linguagem: Inglês

10.1038/sj.emboj.7601905

1460-2075

Jens Brandenburg, Bernd Schimanski, Everson Nogoceke, Tu N. Nguyen, Júlio C. Padovan, Brian T. Chait, George Cross, Arthur Günzl,

Biochemical and Molecular Research

Article1 November 2007free access Multifunctional class I transcription in Trypanosoma brucei depends on a novel protein complex Jens Brandenburg Jens Brandenburg Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Bernd Schimanski Bernd Schimanski Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Everson Nogoceke Everson Nogoceke Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY, USAPresent address: Pharmaceuticals Research Division, Roche, Basel CH-4052, Switzerland Search for more papers by this author Tu N Nguyen Tu N Nguyen Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Júlio C Padovan Júlio C Padovan Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author Brian T Chait Brian T Chait Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author George AM Cross George AM Cross Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY, USA Search for more papers by this author Arthur Günzl Corresponding Author Arthur Günzl Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Jens Brandenburg Jens Brandenburg Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Bernd Schimanski Bernd Schimanski Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Everson Nogoceke Everson Nogoceke Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY, USAPresent address: Pharmaceuticals Research Division, Roche, Basel CH-4052, Switzerland Search for more papers by this author Tu N Nguyen Tu N Nguyen Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Júlio C Padovan Júlio C Padovan Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author Brian T Chait Brian T Chait Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA Search for more papers by this author George AM Cross George AM Cross Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY, USA Search for more papers by this author Arthur Günzl Corresponding Author Arthur Günzl Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Author Information Jens Brandenburg1,‡, Bernd Schimanski1,‡, Everson Nogoceke2, Tu N Nguyen1, Júlio C Padovan3, Brian T Chait3, George AM Cross2 and Arthur Günzl 1,4 1Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA 2Laboratory of Molecular Parasitology, The Rockefeller University, New York, NY, USA 3Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY, USA 4Department of Molecular, Microbial and Structural Biology, University of Connecticut Health Center, Farmington, CT, USA ‡These authors contributed equally to this study *Corresponding author. Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3710, USA. Tel.: +1 860 679 8878; Fax: +1 860 679 8130; E-mail: [email protected] The EMBO Journal (2007)26:4856-4866https://doi.org/10.1038/sj.emboj.7601905 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The vector-borne, protistan parasite Trypanosoma brucei is the only known eukaryote with a multifunctional RNA polymerase I that, in addition to ribosomal genes, transcribes genes encoding the parasite's major cell-surface proteins—the variant surface glycoprotein (VSG) and procyclin. In the mammalian bloodstream, antigenic variation of the VSG coat is the parasite's means to evade the immune response, while procyclin is necessary for effective establishment of trypanosome infection in the fly. Moreover, the exceptionally high efficiency of mono-allelic VSG expression is essential to bloodstream trypanosomes since its silencing caused rapid cell-cycle arrest in vitro and clearance of parasites from infected mice. Here we describe a novel protein complex that recognizes class I promoters and is indispensable for class I transcription; it consists of a dynein light chain and six polypeptides that are conserved only among trypanosomatid parasites. In accordance with an essential transcriptional function of the complex, silencing the expression of a key subunit was lethal to bloodstream trypanosomes and specifically affected the abundance of rRNA and VSG mRNA. The complex was dubbed class I transcription factor A. Introduction The protistan parasite Trypanosoma brucei is transmitted by Glossina spp. and lives freely in the bloodstream and extracellular tissue spaces of its mammalian host, causing the lethal disease known as African Sleeping Sickness in humans. Key factors for successful T. brucei infection of mammalian and insect hosts are the parasite's variant surface glycoprotein (VSG) and procyclin, respectively. In the mammalian bloodstream, antigenic variation of the dense VSG coat is the parasite's means to evade the immune system (reviewed by Pays et al, 2007), whereas procyclins are important to establish an infection in the fly midgut (Ruepp et al, 1997). VSG and procyclin genes are expressed with an extremely high efficiency in bloodstream and procyclic stages, respectively. The cell coat of a bloodstream trypanosome consists of 107 identical VSG molecules, which are derived from a single gene drawn from a large VSG repertoire. Accordingly, in vivo RNA labeling experiments indicated that VSG mRNA synthesis is approximately 100 times higher than that of a single β-tubulin gene (Ehlers et al, 1987). This level of VSG expression is essential for parasite growth and persistence of infection: RNAi-mediated VSG silencing caused rapid cell-cycle arrest in vitro and efficient clearance of trypanosomes from the bloodstream of infected mice (Sheader et al, 2005). The requirement for this extraordinarily high expression is probably the reason why T. brucei has evolved the unique capability of utilizing the high efficiency of RNA polymerase (pol) I-mediated transcription for VSG and procyclin genes (Kooter and Borst, 1984; Rudenko et al, 1989; Clayton et al, 1990; Günzl et al, 2003). In other eukaryotes, RNA pol I exclusively transcribes ribosomal RNA genes (RRNA), as is also the case in trypanosomes, whereas mRNA is invariably synthesized by RNA pol II. This clear division of function is most likely due to the specific association of the mRNA capping enzyme with RNA pol II and the co-transcriptional mode of mRNA capping (McCracken et al, 1997; Yue et al, 1997). In trypanosomes, however, protein-coding genes are arranged in long tandem arrays that are transcribed in a polycistronic manner. Individual mRNAs are resolved from their precursors by trans splicing a 39-nt 5′ leader sequence, derived from a capped RNA pol II-synthesized spliced leader donor RNA (SL RNA), and by polyadenylation. This post-transcriptional capping process uncouples capping from mRNA synthesis and enables T. brucei to efficiently express protein-coding genes by RNA pol I (Rudenko et al, 1991; Zomerdijk et al, 1991) or even by bacteriophage RNA polymerases (Wirtz et al, 1994). The active VSG gene is always located in a telomeric expression site (ES), whereas there are two chromosome-internal loci of procyclin genes, termed GPEET and EP1. While the VSG promoter is very short with two distinct sequence boxes upstream of the transcription initiation site (TIS; Vanhamme et al, 1995; Pham et al, 1996), the RRNA, GPEET, and EP1 promoters are larger and consist of four distinct domains (Sherman et al, 1991; Brown et al, 1992; Janz and Clayton, 1994; Laufer and Günzl, 2001). Although there are no obvious sequence homologies between these promoters, an in vitro transcription competition study indicated that they bind a common trans-activating factor (Laufer and Günzl, 2001). In addition to the structural variation among T. brucei class I promoters, the nuclear distribution of RNA pol I makes class I transcription in this parasite even more complex. While in other eukaryotes, RNA pol I transcribes RRNA genes exclusively in the nucleolus, in bloodstream T. brucei RNA pol I is sequestered in the nucleolus and in a novel DNase I-resistant compartment dubbed the ES body (Navarro and Gull, 2001). It was hypothesized that this compartment accommodates only a single ES, ensuring mono-allelic VSG expression. Despite the importance and multifaceted nature of class I transcription in T. brucei, a class I transcription factor has not been identified and our knowledge of the transcription machinery has been restricted to conserved subunits of RNA pol I (Walgraffe et al, 2005; Nguyen et al, 2006). Only very recently, a novel RNA pol I subunit with an essential transcriptional function was characterized (Nguyen et al, 2007). Our knowledge about class I transcription factors stems from research on mammals and the budding yeast Saccharomyces cerevisiae (recently reviewed in Grummt, 2003; Russell and Zomerdijk, 2005). In mammals, the basal RRNA promoter-binding factor is termed selectivity factor 1 (SL-1) in humans and TIF-IB in the mouse. It consists of the TATA-binding protein (TBP) and four TBP-associated proteins (TAFs). The interaction between RNA pol I and SL1 is mediated by a single polypeptide-dubbed RRN3 in humans or TIF-IA in the mouse, and activated RRNA transcription requires a dimer of the 94 kDa-large upstream binding factor UBF. In yeast, RRN3 is conserved and TBP stimulates RNA pol I transcription in the absence of TAFs, whereas the three subunits of the core factor, which is the functional equivalent of SL1, and the six subunits of the upstream activation factor, share no sequence similarity with the mammalian proteins. Here, we have purified and characterized a protein that recognizes the VSG ES promoter with sequence specificity. We show by independent criteria that this protein is indispensable for class I transcription in vitro and for VSG and RRNA expression in vivo. Tandem affinity purification of the protein revealed a complex of seven subunits that contained five additional parasite-specific polypeptides and, surprisingly, a dynein light chain. We demonstrated that the protein complex is the functional entity that binds to the VSG ES promoter and conclude that we have characterized a novel multi-subunit transcription factor which we named class I transcription factor A (CITFA). Results Identification of two VSG ES promoter-binding proteins In a previous study, an electrophoretic mobility shift assay (EMSA) was used to convincingly demonstrate specific protein binding to the VSG ES promoter in crude trypanosome extracts (Pham et al, 1997). These extracts were prepared from procyclic trypanosomes because, in contrast to bloodstream forms, procyclics can easily be grown in large numbers in vitro and because the VSG ES promoter can direct accurate transcription in procyclic cells (Lee and Van der Ploeg, 1997, and references therein) and in procyclic extracts (Laufer et al, 1999). To identify factors that specifically interact with the VSG ES promoter, we grew 30 l of procyclic forms, prepared a crude extract and, by testing individual fractions with EMSA, purified the VSG ES promoter-binding activity by a combination of ion exchange, heparin affinity, and DNA affinity chromatography (data not shown). Mass spectrometric analysis of the promoter-binding activity revealed 17 proteins with calculated masses ranging from 41.3 to 181.5 kDa (Figure 1A). While some of these proteins probably co-purified due to nonspecific DNA–protein interactions, a protein with an apparent size of ∼50 kDa could be specifically UV-crosslinked to VSG ES promoter DNA (Figure 1B). For further analysis, we therefore chose four proteins (Figure 1A arrowheads) with a predicted size in the 50 kDa range that had been annotated as 'conserved hypothetical proteins' in the T. brucei genome database (http://www.genedb.org/genedb/tryp/index.jsp). The four proteins were C-terminally tagged in individual cell lines either with the HA epitope or the 19-kDa PTP tag (Schimanski et al, 2005b), which is suitable for tandem affinity purification. The DNA-binding activity of these proteins was tested by promoter pull-down and immunoblot experiments, using a VSG ES promoter containing the wild-type sequence or a few point mutations in either or both of the two essential sequence elements (Figure 1C), and a nonspecific control DNA. In addition, DNAs comprising the complete SLRNA, RRNA and GPEET promoters were co-analyzed. The proteins encoded by Tb927.2.3800 and Tb927.4.1310 bound to DNA in a nonspecific manner (data not shown), whereas the wild-type VSG ES promoter bound the Tb09.211.3440 protein very efficiently, and the nonspecific control and the class II SLRNA promoter DNA did not (Figure 1D: compare lane 3 with lanes 2 and 7). Binding of this protein to the VSG ES promoter required the integrity of both promoter elements, because mutation of either element strongly diminished the binding activity, and mutation of both elements abolished it completely (lanes 4–6). Albeit to a lesser extent, Tb09.211.3440 reproducibly bound to the other two class I promoters, which raised the possibility that this protein is a general class I transcription factor in T. brucei (compare lane 3 with lanes 8 and 9). The pull-down results with protein Tb11.47.0008 were similar but the binding specificity was not as clear (data not shown). We therefore continued our analysis with Tb09.211.3440, which from now on we refer to as CITFA-2 (as will become apparent below, CITFA-2 is the second largest subunit of the complex). Figure 1.Identification of VSG ES promoter-binding proteins. (A) List of identified proteins with their GeneDB gene accession numbers, calculated molecular weights, and current GeneDB annotations. Arrowheads indicate proteins that were further analyzed. (B) Autoradiograph of proteins from S-Sepharose or Resource Q (Res Q) fractions that were UV crosslinked to radio-labeled VSG ES promoter, DNAse-digested, and separated by SDS–PAGE. –pa, no polyamines added. (C) Sequence of the VSG ES 118 promoter from position −67 to +1 relative to the TIS. The important residues of boxes 1 and 2 are underlined and the point mutations are indicated below the wild-type sequence. (D) Immunoblot of PTP-CITFA-2 subsequent to promoter pull-downs carried out with a nonspecific DNA, with VSG ES promoter DNAs as indicated above, and with wild-type SLRNA, RRNA, and GPEET promoter DNAs. The numbers indicate the end positions of the DNA fragments relative to the TIS. Download figure Download PowerPoint Silencing of CITFA-2 expression is lethal We silenced the expression of CITFA-2 in procyclic and bloodstream T. brucei by using an inducible RNA interference system that is based on stable transfection of cells expressing both the tetracycline repressor and T7 RNA polymerase (Wirtz et al, 1999). We first cloned 530 bp of the CITFA-2-coding region into the inducible construct pZJM, which harbors T7 but no class I promoters (Wang et al, 2000). While this strategy led to inducible procyclic cell lines, it failed to generate corresponding bloodstream forms. We therefore modified the available and more tightly regulated stem-loop vector for dsRNA synthesis (Shi et al, 2000) by replacing the inducible class I promoter with an inducible T7 promoter and by inserting two T7 terminators downstream of the dsRNA cassette before cloning the CITFA-2 sequence in opposite orientations into this T7-stl vector (Supplementary Figure S1). While T. brucei growth is not affected by the doxycycline concentrations used (Luu et al (2006) and data not shown), procyclic and bloodstream cell lines that were stably transfected with these plasmids rapidly ceased growth when CITFA-2 dsRNA expression was induced (Figure 2A and data not shown). Typically, cell growth was inhibited after 24 h and cell cultures died out after day 5, indicating that CITFA-2 is essential for both life-cycle stages. Semiquantitative RT–PCR revealed that CITFA-2 mRNA abundance was specifically reduced in induced cells within the first 24 h (data not shown). To evaluate the CITFA-2 protein levels in these cells, we raised a rat polyclonal antiserum against the N-terminal half of CITFA-2, which recognizes the endogenous protein with high specificity (Supplementary Figure S2). On immunoblots, CITFA-2 levels were seen to be strongly reduced, 24 and 48 h after RNAi induction, which confirmed effective silencing of CITFA-2 expression in these cells (Figure 2B). Analysis of total RNA prepared from noninduced and induced bloodstream cells showed that the abundance of RNA pol I-synthesized 18S, 28Sα and 28Sβ rRNAs and of VSG 221 mRNA was clearly reduced 42 and 48 h after induction (Figure 2C). Concomitant with the decrease of these major RNAs, the RNA pol II transcripts smD1 mRNA and SL RNA and the RNA pol III transcript U2 snRNA increased in these samples of equal amounts. When standardized against smD1 signals, rRNA/VSG mRNA decreased to 56/44% and 30/29% 42 and 48 h after induction, respectively. The latter numbers are in close accordance with the 73% reduction of rRNA found upon silencing an essential RNA pol I subunit in procyclic trypanosomes (Nguyen et al, 2007). We therefore concluded that silencing of CITFA-2 specifically affected RNA pol I transcripts. Together with our finding that CITFA-2 specifically bound to class I promoters, these results suggested that this protein is involved in class I transcription. Figure 2.CITFA-2 is an essential class I transcription factor. (A) Growth curve of a bloodstream-form RNAi cell line in the presence (circles, gray) or absence (diamonds, black) of doxycycline, which induces the expression of CITFA-2 dsRNA. (B) Immunoblot of whole-cell lysates prepared from bloodstream RNAi cells before and 24 and 48 h after induction of CITFA-2 dsRNA synthesis. Detection of the nuclear protein U2-40K served as a loading control. (C) Analysis of total RNA prepared from noninduced cells and from cells 24, 42, and 48 h after induction. The rRNAs were visualized by ethidium bromide staining, VSG 221 and smD1 mRNAs by hybridization with appropriate probes, and the small SL and U2 RNAs by a primer extension assay. (D) In vitro transcription of the class I templates VSG-trm, GPEET-trm, and Rib-trm and the class II template SLins19 in the absence of rat serum (no IS) or in the presence of anti-CITFA-2 pre-immune serum (pre-IS), TFIIB antiserum (α-TFIIB), or CITFA-2 antiserum (α-CITFA-2). The indicated transcription signals were obtained by primer extension assays and extension products were separated by denaturing PAGE and visualized by autoradiography. The asterisk designates an aberrantly initiated SLins19 transcript caused by the TFIIB antiserum. Marker, pBR322-MspI. (E) Co-transcription of GPEET-trm and SLins19 in extracts of procyclic 29-13 cells in which CITFA-2 silencing was not induced (−RNAi) or induced for 36 h (+RNAi). Download figure Download PowerPoint CITFA-2 is essential for class I transcription in vitro To directly evaluate the role of CITFA-2 in class I transcription, we employed a homologous in vitro transcription system. This system, which is based on a crude mix of cytoplasmic and extracted nuclear components of procyclic trypanosomes, is active for both class I and class II SLRNA transcription (Laufer et al, 1999). The template plasmids VSG-trm, GPEET-trm and Rib-trm, which contain a VSG ES, the GPEET procyclin and an RRNA promoter, respectively, were co-transcribed with the SLRNA template SLins19. These templates contain insertions of unrelated oligonucleotide sequences downstream of the transcription initiation site, which allow specific detection of transcripts by primer extension assays. In a first analysis, we tested whether the addition of CITFA-2 antiserum affected RNA pol I-mediated transcription in vitro. In these reactions, pre-immune serum did not significantly affect transcription of either the class I templates or of SLins19 (Figure 2D, compare lanes 2, 6, and 10 with lanes 1, 5, and 9, respectively). When we added, as another control, a similarly derived antiserum directed against the general class II transcription factor TFIIB of T. brucei (Schimanski et al, 2006), correctly initiated SLins19 transcription was abolished whereas class I transcription was only mildly affected, most likely due to a nonspecific effect from this particular serum (lanes 3, 7, and 11; Nguyen et al, 2007). In contrast, anti-CITFA-2 serum specifically interfered with class I transcription. While VSG-trm transcription was strongly reduced (lane 4), GPEET-trm and Rib-trm transcription was abolished (lanes 8 and 12). This differential and reproducible effect may be a consequence of higher affinity binding of CITFA-2 to the VSG ES promoter (see Figure 1D). To confirm this observation, we prepared transcription extracts from procyclic cells in which CITFA-2 expression was silenced for 36 h, and from non-RNAi-induced cells. CITFA-2 silencing was less effective in procyclic than in bloodstream RNAi cells and reduced the CITFA-2 protein level by only 64% (Supplementary Figure S3). Nevertheless, when GPEET-trm, which drives the most efficient class I transcription in this in vitro system (Laufer et al, 1999), was co-transcribed with SLins19, GPEET-trm transcription was decreased by ∼73% when CITFA-2 was silenced, whereas the same extract supported SLins19 transcription nearly as well (∼13% reduction) as the extract prepared from noninduced cells (Figure 2E). Moreover, as a third criterion, depletion of CITFA-2 from the extract by IgG affinity chromatography specifically abolished class I transcription (see below, Figure 6). We therefore concluded that CITFA-2 is a general class I transcription factor in T. brucei. Six proteins specifically co-purify with CITFA-2 While a single CITFA-2 ortholog is encoded in the genomes of the trypanosomatids Trypanosoma vivax, Trypanosoma cruzi, Leishmania major and Leishmania infantum (for a sequence alignment, see Supplementary Figure S4), we were unable to identify homologous sequences outside of this taxonomic family or to detect a characteristic sequence motif. Hence, by primary structure, CITFA-2 and its orthologs appear to be trypanosomatid-specific proteins. In the next step, we therefore asked whether this protein is associated with more conserved proteins resembling known transcription factors. We generated the procyclic cell line TbT2 which exclusively expressed CITFA-2 with an N-terminal PTP tag at a level corresponding to wild-type CITFA-2 expression (PTP-CITFA-2; Figure 3A and B). Since we had shown that CITFA-2 is encoded by an essential gene and TbT2 cells grew normally, we concluded that the tag did not interfere with protein function. The PTP tag is a modified tandem affinity purification tag consisting of a protein A tandem domain and a protein C (ProtC) epitope separated by a tobacco etch virus (TEV) protease cleavage site. PTP fusion proteins can be sequentially purified by IgG chromatography, TEV protease elution, anti-ProtC immunoaffinity chromatography, and elution with an EGTA-containing buffer (Schimanski et al, 2005b). According to the anti-ProtC immunoblot analysis, both PTP-CITFA-2 purification steps were highly efficient and resulted in the near depletion of the protein from input material (Figure 3C). In the final eluate, 13.7% (360 ng) of the CITFA-2 protein was recovered from the input material. Since approximately 81% of CITFA-2 was extracted from cells (data not shown), we estimate that a single trypanosome harbors ∼750 CITFA-2 molecules. Coomassie staining of the final eluate detected several distinct protein bands (Figure 3D), whose excision and analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS) led to the identification of P-CITFA-2 and six co-purified proteins (identified peptides are listed in Supplementary Table S1). The mix of bands around 55 kDa contained tagged CITFA-2 and proteins encoded by genes Tb11.47.0008 and Tb11.47.0010. While Tb11.47.0008 had been purified by our initial chromatography approach, Tb11.47.0010 was a new identification. Similarly, the bands with apparent sizes of 43 and 23 kDa were the products of genes Tb11.01.0240 and Tb927.5.970, respectively, and were not identified before. The band with an apparent size of 28 kDa reproducibly appeared to be fuzzy, suggesting that it contained more than one protein or differently modified versions of the same protein. Interestingly, the identified protein is encoded by three genes, which are part of a tandem repeat of five genes on chromosome 8. Tb927.8.4030 and Tb927.8.4080 encode the same protein, whereas the product of gene Tb927.8.4130 has a different C-terminal region. According to four diagnostic peptide identifications (Supplementary Table S1), both protein versions co-purified with P-CITFA-2. Figure 3.Six proteins co-purify with CITFA-2. (A) Schematic depiction (not to scale) of the CITFA-2 gene locus in cell line TbT2. In one allele, the CITFA-2-coding region was replaced by a hygromycin-resistance gene (HYG-R) and in the second allele the PTP sequence was fused to the 5′ end of the coding region by targeted insertion of pPURO-PTP-CITFA-2. Coding regions are represented by open boxes, the PTP tag by a black box, and introduced gene flanks by small gray boxes. (B) Immunoblot analysis of PTP-CITFA-2 in extracts of wild-type (WT) and TbT2 cells. The tagged protein was detected with the protein A-specific PAP reagent (top panel) or with the polyclonal anti-CITFA-2 serum (middle panel). Protein loading was controlled by reprobing the same blot with an antibody against T. brucei TFIIB. (C) Immunoblot monitoring of PTP-CITFA-2 purification. Aliquots of the input material (INP), the flow-through of the IgG affinity chromatography (FT-IgG), the TEV protease elution (Elu TEV), the flow-through of the anti-ProtC affinity chromatography (FT-ProtC), and the final EGTA eluate (Elu) were separated on a 10% SDS/polyacrylamide gel, blotted, and probed with anti-ProtC antibody. The relative amount of each sample to the input material is specified. It should be noted that the size of the tagged protein (PTP-CITFA-2) was reduced by ∼15 kDa after protease cleavage (P-CITFA-2). (D) Coomassie staining of purified proteins. The total eluate of a standard PTP-CITFA-2 purification was separated on a 15% SDS/polyacrylamide gel and stained with Coomassie. For comparison, 0.003% of the input material (Inp) and 5% of the TEV protease eluate (TEV) were loaded. On the right, proteins identified by mass spectrometry are specified by their GeneDB accession numbers or protein name. The asterisk marks a minor IgG kappa light chain contamination of the anti-ProtC matrix. (E) Immunoblot of whole-cell lysates derived from cell lines that express the proteins of the indicated genes as C-terminal PTP fusions. In a control cell line, the spliceosomal smD1 protein was PTP-tagged. (F) Co-precipitation of CITFA-2 with PTP-tagged proteins. Proteins were eluted from IgG beads either with glycine (Tb11.47.0008 and Tb11.47.0010) or by TEV protease digest (smD1, Tb11.01.0240, Tb927.8.4130, Tb927.5.970) that reduced the protein sizes. In the precipitates (P), the tagged proteins were detected with the anti-ProtC antibody, and CITFA-2 and TFIIB with polyclonal antisera. For comparison, 20% of input material (INP) was co-analyzed. Download figure Download PowerPoint These six identified proteins are conserved among trypanosomatids (Supplementary Figures S4–S9), but they exhibit no similarity to proteins of other organisms. Nor could we identify a conserved sequence motif using the InterPro (http://www.ebi.ac.uk/interpro), Motif scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan), SMART (http://smart.embl-heidelberg.de/), and Minimotif miner (http://sms.engr.uconn.edu/servlet/SMS SearchServlet) search engines. Hence, based on primary structure, these six proteins appear to be specific to trypanosomatid parasites. In contrast, the smallest co-purified protein with an apparent size of 9 kDa was identified by a single peptide as the motor protein subunit dynein light chain DYNLL, which is also known as LC8 (Pfister et al, 2006). In T. brucei, this protein is encoded by the genes Tb11.50.0007 and Tb11.0845. Among several T. brucei DYNLL paralogs, this dynein light chain is most closely related to human DYNLL1 and 2 (data not shown). Accordingly, we propose to name the identified protein TbDYNLL1.