Published by the International Society of Protistologists
The Journal of
Eukaryotic Microbiology
Journal of Eukaryotic Microbiology ISSN 1066-5234
SHORT COMMUNICATION
Tetrahymena Expresses More than a Hundred Proteins with Lipid-binding MORN Motifs that can Differ in their Subcellular Localisations €rn Habichta, Christian Woehleb & Sven B. Goulda Jo €sseldorf, Germany a Institute for Molecular Evolution, Heinrich-Heine-University, 40225 Du b Institute for Microbiology, Christian-Albrecht-University, 24118 Kiel, Germany
Keywords Ciliates; membrane targeting; membrane tethering; MORN. Correspondence S.B. Gould, Institute for Molecular Evolution, €sselHeinrich-Heine-University, 40225 Du dorf, Germany Telephone number: +49 211 81 13983; FAX number: +49 211 81 13554; e-mail: [email protected]
ABSTRACT Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein-encoding genes, some of which are specifically up-regulated during conjugation. A lipid-binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.
Received: 6 November 2014; accepted January 20, 2015. doi:10.1111/jeu.12216
THE membrane occupation and recognition nexus (MORN) motif was first described as part of the mammalian junctophilin protein 1 (JP-1), which mediates the tethering of the sarcoplasmic reticulum to the plasma membrane (Takeshima et al. 2000). The motif consists of a consensus sequence that stretches across 14 amino acids (Fig. 1A) and the number of MORN motifs within animal, plant, and protozoan MORN proteins that have been studied so far varies from 2 to 17 individual motifs (Choi et al. 2010; Shimada et al. 2004). The motifs can cluster predominantly at the N-terminus (Bhattacharya et al. 2012; Ma et al. 2006; Shetty et al. 2007; Takeshima et al. 2000) or the C-terminus of the proteins (Choi et al. 2010; Kunita et al. 2007; Lee et al. 2009; Shimada et al. 2004), but are sometimes also found along the entire protein sequence and in conjunction with other domains of known function (Gubbels 2006; Takeshima et al. 2000). Since the initial description of the MORN motif, several MORN motif-harbouring proteins have been analysed, which include the junctophilin family JP-1 to JP-4 (Ito et al. 2001; Nishi et al. 2000, 2003; Takeshima et al. 2000), MORN4 of Drosophila (Bhattacharya et al. 2012), the amyotrophic lateral sclerosis protein ALS2 (Yang et al. 2001), and the radial spoke protein 44 (RSP44; also referred to as
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meichroacidin, MCA) (Shetty et al. 2007; Tokuhiro et al. 2008). Characterised plant proteins that harbour MORN motifs are phosphatidylinositol monophosphate kinases (Im et al. 2007; Ma et al. 2006; Mikami et al. 2010), the stromal accumulation and replication of chloroplasts 3 (ARC3) protein (Shimada et al. 2004), as well as the Brassica rapa MORN motif protein (BrMORN), which in Arabidopsis promotes an increase in the cell size, vegetative organs, and seed production (Lee et al. 2009). In apicomplexan parasites, MORN1 is the best characterised. Within this group, it acts as an early endodyogeny marker during the asexual and sexual development and has established itself as a reliable marker for the alveoli, too, in Apicomplexa more often referred to as the inner membrane complex (Ferguson et al. 2008; Gould et al. 2008; Gubbels 2006; Lorestani et al. 2010). Knockout mutants of TgMORN1 show a disorganisation of the posterior end and basal complex of the parasite, and fail to complete daughter cell budding (Heaslip et al. 2010; Lorestani et al. 2010). The use of the MORN motif in proteins with a variety of different duties underpins its function as a more universal domain. Here, we took a look at the phylogenetic distribution of this motif and used the ciliate Tetrahymena thermophila as a model to shed light on this complex protein
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 694–700
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Figure 1 The MORN motif is highly conserved across eukaryotes, present in 129 expressed proteins of Tetrahymena and is able to bind to isolated lipids. A. A sequence logo generated from 630 individual MORN motifs stemming from 100 proteins of 10 different organisms shows the conserved nature of the 14-amino acid long motif. B. Four exemplary MORN proteins as representatives for the three main classes we defined: (i) the MORN motifs are found at the N-terminus (N; light green), or (ii) at the C-terminus (C; dark green), or (iii) otherwise and/or in combination with other known domains such as the IQ motif (O; yellow). C. Expression profiles of 129 Tetrahymena thermophila MORN protein-encoding genes (x-axis) during the exponential growth phase (L), a 24 h long starvation phase (S) and an 18 h long conjugation phase (C). The number of MORN motifs in each protein is indicated by a yellow to red gradient. Genes are sorted according to their co-expression correlation values among the three different conditions and expression values are shown in arbitrary units (AU; y-axis). For individual expression values please refer to Table S1. Association of the MORN domains (again N, C, or O) with other protein domains are indicated additionally by coloured squares: SK = serine/ threonine protein kinases; PK = phosphokinase; IQ = IQ motif; ZF = zinc finger domain; TM = transmembrane domain; LRR = leucine-rich repeats; WD40 = tryptophan-aspartic acid repeat; SAM = sterile alpha motif. D. The 14 MORN motifs of MRNO51 mediate the association of glutatione S-transferase (GST) with lipids when fused to the C-terminus of GST (GST::MRNO51). This fusion protein binds predominantly to cardiolipin and to 1,2-diacylglycerol at a concentration of 1 lg/ml. At a higher concentration of 100 lg/ml, GST::MRNO51 furthermore associates with phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylserine. Purified GST alone served as control. On the right a schematic representation of the blot. PtdIns, Phosphatidylinositol; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate.
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 694–700
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family a bit more in detail. Proteins with MORN motifs can reach more than a hundred in some ciliates and we show that the MORN motifs allow an otherwise soluble protein to bind to a variety of different lipids. The exemplary localisation of only a few candidate proteins demonstrates that the subcellular localisation can be multifaceted, even when the MORN motifs and their distribution among the individual proteins are rather similar. MATERIAL AND METHODS Bioinformatics The InterPro database (Hunter et al. 2012) served as the initial source to screen for proteins of the MORN family. The distribution of MORN motifs among individual proteins was determined using SMART (Letunic et al. 2012) and RADAR (Heger and Holm 2000). For the sequence logo, MORN motifs were first aligned with Clustal Omega (Sievers et al. 2011) and then plotted using WebLogo (Crooks et al. 2004). All T. thermophila gene expression values were extracted from the Tetrahymena Functional Genomics Database (TetraFGD) (Xiong et al. 2013) and the data visualised using Matlab (The MathWorks GmbH, Ismaning, Germany).
and TAG—encoding glutamines in ciliates, but stop codons in Escherichia coli—were substituted with CAA and CAG, respectively. The synthesised sequence was cloned into pGEX-4T-2_G029 that provided an N-terminal glutathione S-transferase (GST) tag. E. coli BL21 cells were transfected and cells were allowed to grow to an optical density of 0.6, and fusion protein expression then induced with isopropyl-b-D-thiogalactopyranosid overnight at 20 °C and continuous shaking. Cells were harvested, resuspended in PBS (pH 7.4) and homogenised using a cell disrupter system (Constant Systems Ltd., Daventry, UK). GST-tagged protein was purified via fast protein liquid chromatography using a GSTrap FF column (GE Healthcare, Berlin, Germany) with the recommended buffer. The buffer of the purified protein was changed to TBS using Vivaspinâ 15R Centrifugal Concentrator (Sartorius Stedim Biotech, €ttingen, Germany) and three washing steps. Purified Go GST::TTHERM_00584930 was used at a concentration of 1 and 100 lg/ml (both diluted in TBST) in the lipid overlay assays that were performed on Membrane Lipid StripsTM (Echelon Biosciences Inc., Salt Lake City, UT) according to the manufacturer’s recommendations. Bound fusion protein was detected through standard Western blots using a horse radish peroxidase conjugated anti-GST antibody (Sigma-Aldrich) at a dilution of 1:1,000.
Tetrahymena culturing and cloning The T. thermophila strain CU522 was cultured in SPP medium (Gorovsky et al. 1975) at 15 °C and transferred to 30 °C to obtain high-density cultures overnight. For C-terminal green fluorescent protein (GFP)-tagging, the genes of interest were cloned via the restriction sites SphI and MluI into pTtag (accession no. FJ789658), which has a cadmium-inducible metallothionein (MTT1) promoter. Tetrahymena thermophila CU522 cells were transfected with linearised GFP constructs using a PDS-1000 Gene Gun (Bio-Rad Laboratories GmbH, Munich, Germany) as previously described (Cassidy-Hanley et al. 1997) and then selected for 10 d in SPP medium supplemented with 30 lg/ml paclitaxel (LC Laboratories, Woburn, MA). The correct molecular weight was verified by Western blotting (Fig. S2) and cells fixed after 45 min of induction to localise the proteins as previously described (El-Haddad et al. 2013). The primary antibodies (a-GFP, Abcam, Cambridge, MA; a-alpha-tubulin, DSHB) were used at a concentration of 1:100, diluted in PBS containing 0.1% BSA and 0.1% Triton X-100. Secondary Alexa Fluor 488 and TRITC antibodies Invitrogen / Life Technologies GmbH, Darmstadt, Germany; Sigma-Aldrich Chemie Gmbh, Munich, Germany were used at 1:1,000. Fixed and labelled cells were transferred onto Superfrost slides (Menzel, Braunschweig, Germany) and mounted with Vectashield medium (Vector Laboratories, Peterborough, UK) that included 40 ,6-diamidino-2-phenylindole (DAPI) to stain the DNA. Recombinant overexpression and lipid-binding assay We synthesised a variant of TTHERM_00584930 (GENEART AG, Regensburg, Germany), in which the two codons TAA
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RESULTS AND DISCUSSION MORN motifs occur in conjunction with a variety of other protein domains The MORN motifs are found among all domains of life and some viruses. However, at the InterPro database (Hunter et al. 2012) thousands of MORN proteins are listed each for bacteria and eukaryotes, but only two for Archaea. The isolated occurrence of two almost identical MORN proteins (they differ only in one single amino acid at the very C-terminus) suggests a recent acquisition of these genes in the two Euryarchaeota trough lateral gene transfer, which occurs fivefold more frequently from bacteria to Archaea than vice versa (Nelson-Sathi et al. 2014). In any case, humans encode the largest variety of MORN proteins among mammals, but ciliates the largest variety among all eukaryotes. For the ciliate T. thermophila 132 individual genes are listed at InterPro that encode proteins that harbour at least one MORN motif (129 at ciliate.org, for which expression data are available and that we all annotated). We sorted all Tetrahymena MORN proteins into three classes: (i) those in which the MORN motifs localise predominantly towards the N-terminus (MRNN, 17 proteins), (ii) those in which the MORN motifs localise predominantly towards the C-terminus (MRNC, 60 proteins), and (iii) those cases, in which the MORN motifs are distributed throughout the entire protein and/or are associated with other domains of known function (MRNO, 55 proteins) (Fig. 1B, C and Table S1). Furthermore, ciliate MORN proteins can contain other domains of known function, including calmodulin-binding domains, zinc fingers, transmembranedomains, and WD-40-repeats (Fig. 1C and Table S1).
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 694–700
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MRNC38
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Figure 2 Localisations of four MORN proteins in Tetrahymena thermophila. All four MORN proteins show a distinct localisation. MRNC36::GFP localises to focal dots at the apical tip of cell (arrowhead), to the contractile vacuole pore (CVP; arrow), and to the very tip of the cilia (*). MRNC38::GFP appears associated with the undulating membrane of the oral apparatus (OA, arrowhead). MRNC39::GFP binds to the entire cell membrane of the ciliate. MRNO51::GFP localises predominantly to the OA (*) and the CVP (arrow) but also weakly resembles the localisation observed for MRNC36, although especially the localisation associated with the OA completely differs. For each protein, a schematic representation of the MORN motif composition is shown below the images. All images were captured 45 min after expression was induced with CdCl2. Scale 10 lm.
On the basis of microarray data we extracted from the TetraFGD, we clustered the expression profiles of 129 MORN encoding genes according to their co-expression throughout logarithmic growth, starvation, and conjugation. Genes encoding MORN proteins show four main patterns of expression: (i) the first set of genes is likely encoding for “housekeeping MORNs”, they show high levels of expression throughout all time points analysed, (ii) the second set of genes is highly expressed during the logarithmic growth phase, starvation, and later phases of conjugation, (iii) the third set of genes is up-regulated specifically during early conjugation, (iv) and the fourth set— the majority of the MORN protein-encoding genes—is generally expressed at low levels, but above background expression that is already subtracted at TGED (Fig. 1C, S1). There is, however, no connection of certain gene expression profiles to the localisation, domain composition, or association with other protein domains. It
suggests that the MORN proteins in Tetrahymena have not evolved like a typical gene family, but that due to the more universal duty of the motif, MORN domains have found residence in a variety of different proteins through exon shuffling or recombination events. Do the MORN motifs alone determine localisation? To first investigate the lipid-binding ability of MORN motifs from a Tetrahymena protein, we performed a lipid overlay assay using the MORN motifs of MRNO51 (TTHERM_00584930) for a case study. The 14 MORN motifs span almost across the entire sequence of MRNO51 that is predicted to be 426 amino acids long. We synthesised a codon-optimised variant of MRNO51 to allow overexpression in E. coli BL21 cells. The protein was fused to the C-terminus of GST for the efficient purification through fast protein liquid chromatography of the recombinant protein.
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 694–700
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At a concentration of 1 lg/ml, GST::MRNO51 bound to cardiolipin (CL) and 1,2-diacylglycerol (DAG), the latter a lipid second messenger that is able to recruit cytosolic proteins to a membrane (van Meer et al. 2008). The increase in the fusion protein concentration to 100 lg/ml revealed additional lipids, such as phosphatidylinositol and phosphatidylserine, with which GST::MRNO51, but not the control (GST alone), interacted (Fig. 1D). The MORN domain of MRNO51 is able to recruit an otherwise soluble protein, GST, to a variety of isolated lipids. Membrane targeting and binding properties are known for the C1 domain of protein kinase C conserved 1, the C2 domain of protein kinase C conserved 2 and the pleckstrin homology (PH) domains. The C1, C2, and PH domains are approximately 50, 130, and 120 amino acids long, respectively (Cho 2001; Yu et al. 2004). In general, they show a much higher sequence variability than the MORN motifs that are highly conserved among protists and eumetazoans (Ferguson et al. 2008; Gubbels 2006; Takeshima et al. 2000). Not the sequence similarity, but rather a common fold of each domain in this case provides the lipid affinity of the C1, C2 (Cho 2001), and PH domains (Lemmon 2007; Lemmon et al. 2002). The general membrane tethering ability of MORN domains is provided through the conserved 14-amino acid repeat (Fig. 1A). It appears the motifs have to be somehow arranged in tandem to mediate reliable membrane association (Ma et al. 2006). However, the exact limit or individual arrangement of the MORN motifs needed to guarantee specific lipid binding has not been experimentally addressed in detail yet. If the purpose of the MORN motif itself is to first and foremost tether a protein to a lipid bilayer, the question arises to what degree the subcellular localisation of MORN proteins differs, when the composition of the MORN motifs is almost identical. We chose to localise four proteins through C-terminal GFP tagging. All four share a high sequence similarity among each other and are homologues of PfMORN1 (PF3D7_1031200) and TgMORN1 (TGME49_310440); well-studied MORN proteins of the apicomplexan parasites Plasmodium and Toxoplasma, respectively (Ferguson et al. 2008; Gubbels 2006; Lorestani et al. 2010). Three of the four proteins have nine MORN motifs arranged in tandem at the C-terminus (MRNC36, TTHERM_01084170; MRNC38, TTHERM_ 00378500; MRNC39, TTHERM_00590130) and one has 14 consecutive MORN motifs that stretch almost across the entire protein (MRNO51; TTHERM_00584930). Studies of truncated rice and rabbit MORN proteins showed that the number of MORN motifs in combination with their orientation influenced the subcellular localisation in comparison to the wild-type protein (Ma et al. 2006; Takeshima et al. 2000). Therefore, one could predict that the same orientation and the exact same number, and hence a similar predicted lipid-binding efficiency of MORN motifs, would lead to an identical localisation. However, the four MORN proteins we analysed—three of which have the same MORN motif amount and composition—all showed distinct subcellular localisation (Fig. 2). This has two major implica-
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tions: (i) one cannot use the results obtained for the domain architecture of one MORN protein to predict the localisation of another, and (ii) the fine localisation specificity of MORN proteins must lay either within the less conserved amino acids of the motif itself (Fig. 1A) or is MORN motif independent. The MORN motifs, when arranged in tandem, function as a universal membrane-binding domain in a variety of different proteins, similar to the function of the C1, C2, and pleckstrin homology domains. The ubiquitous presence of the MORN motifs among eukaryotic groups reflects the importance of this domain in general, and particular in ciliates, in which the MORN protein family has been massively expanded. For ciliates it has been suggested that expanded gene families—that are also specifically selected to persist after genome duplication events —are of special biological importance for the protists (Chalker and Stover 2007; Eisen et al. 2006). The overall amount of MORN proteins in ciliates might at first stun, but especially the MORN proteins of Tetrahymena that are up-regulated during conjugation for example, have the potential to identify new crucial players with regard to how the two complementary cells communicate and fuse during the sexual reproduction stage. ACKNOWLEDGMENTS Funding through the Deutsche Forschungsgemeinschaft to SBG (GO1825/3-1) is gratefully acknowledged. LITERATURE CITED Bhattacharya, M. R. C., Gerdts, J., Naylor, S. A., Royse, E. X., Ebstein, S. Y., Sasaki, Y., Milbrandt, J. & DiAntonio, A. 2012. A model of toxic neuropathy in Drosophila reveals a role for MORN4 in promoting axonal degeneration. J. Neurosci., 32:5054–5061. Cassidy-Hanley, D., Bowen, J., Lee, J. H., Cole, E., VerPlank, L. A., Gaertig, J., Gorovsky, M. A. & Bruns, P. J. 1997. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics, 146:135–147. Chalker, D. L. & Stover, N. A. 2007. Genome evolution: a double take for Paramecium. Curr. Biol., 17:R97–R99. Cho, W. 2001. Membrane targeting by C1 and C2 domains. J. Biol. Chem., 276:32407–32410. Choi, Y.-J., Hwang, K.-C., Park, J.-Y., Park, K.-K., Kim, J.-H., Park, S.-B., Hwang, S., Park, H., Park, C. & Kim, J.-H. 2010. Identification and characterization of a novel mouse and human MOPT gene containing MORN-motif protein in testis. Theriogenology, 73:273–281. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. 2004. WebLogo: a sequence logo generator. Genome Res., 14:1188– 1190. Eisen, J. A., Coyne, R. S., Wu, M., Wu, D., Thiagarajan, M., Wortman, J. R., Badger, J. H., Ren, Q., Amedeo, P., Jones, K. M., Tallon, L. J., Delcher, A. L., Salzberg, S. L., Silva, J. C., Haas, B. J., Majoros, W. H., Farzad, M., Carlton, J. M., Smith Jr, R. K., Garg, J., Pearlman, R. E., Karrer, K. M., Sun, L., Manning, G., Elde, N. C., Turkewitz, A. P., Asai, D. J., Wilkes, D. E., Wang, Y., Cai, H., Collins, K., Stewart, B. A., Lee, S. R., Wilamowska, K., Weinberg, Z., Ruzzo, W. L., Wloga, D., Gaertig,
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Letunic, I., Doerks, T. & Bork, P. 2012. SMART 7: recent updates to the protein domain annotation resource. Nucleic Acids Res., 40:D302–D305. Lorestani, A., Sheiner, L., Yang, K., Robertson, S. D., Sahoo, N., Brooks, C. F., Ferguson, D. J. P., Striepen, B. & Gubbels, M. J. 2010. A Toxoplasma MORN1 null mutant undergoes repeated divisions but is defective in basal assembly, apicoplast division and cytokinesis. PLoS ONE, 5:e12302. Ma, H., Lou, Y., Lin, W. H. & Xue, H. W. 2006. MORN motifs in plant PIPKs are involved in the regulation of subcellular localization and phospholipid binding. Cell Res., 16:466–478. van Meer, G., Voelker, D. R. & Feigenson, G. W. 2008. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol., 9:112–124. Mikami, K., Saavedra, L. & Sommarin, M. 2010. Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases? Plant Signal Behav., 5:1241–1244. Nelson-Sathi, S., Sousa, F. L., Roettger, M., Lozada-Chavez, N., Thiergart, T., Janssen, A., Bryant, D., Landan, G., Schonheit, P., Siebers, B., McInerney, J. O. & Martin, W. F. 2014. Origins of major archaeal clades correspond to gene acquisitions from bacteria. Nature, 517:77–80. Nishi, M., Mizushima, A., Nakagawara, K. & Takeshima, H. 2000. Characterization of human junctophilin subtype genes. Biochem. Biophys. Res. Commun., 273:920–927. Nishi, M., Sakagami, H., Komazaki, S., Kondo, H. & Takeshima, H. 2003. Coexpression of junctophilin type 3 and type 4 in brain. Brain Res. Mol. Brain Res., 118:102–110. Shetty, J., Klotz, K. L., Wolkowicz, M. J., Flickinger, C. J. & Herr, J. C. 2007. Radial spoke protein 44 (human meichroacidin) is an axonemal alloantigen of sperm and cilia. Gene, 396:93–107. Shimada, H., Koizumi, M., Kuroki, K., Mochizuki, M., Fujimoto, H., Ohta, H., Masuda, T. & Takamiya, K-I. 2004. ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase. Plant Cell Physiol., 45:960–967. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Soding, J., Thompson, J. D. & Higgins, D. G. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol., 7:539. Takeshima, H., Komazaki, S., Nishi, M., Iino, M. & Kangawa, K. 2000. Junctophilins: a novel family of junctional membrane complex proteins. Mol. Cell, 6:11–22. Tokuhiro, K., Hirose, M., Miyagawa, Y., Tsujimura, A., Irie, S., Isotani, A., Okabe, M., Toyama, Y., Ito, C., Toshimori, K., Takeda, K., Oshio, S., Tainaka, H., Tsuchida, J., Okuyama, A., Nishimune, Y. & Tanaka, H. 2008. Meichroacidin containing the membrane occupation and recognition nexus motif is essential for spermatozoa Morphogenesis. J. Biol. Chem., 283:19039– 19048. Xiong, J., Lu, Y., Feng, J., Yuan, D., Tian, M., Chang, Y., Fu, C., Wang, G., Zeng, H. & Miao, W. 2013. Tetrahymena functional genomics database (TetraFGD): an integrated resource for Tetrahymena functional genomics. Database (Oxford), 2013: bat008. Yang, Y., Hentati, A., Deng, H. X., Dabbagh, O., Sasaki, T., Hirano, M., Hung, W. Y., Ouahchi, K., Yan, J., Azim, A. C., Cole, N., Gascon, G., Yagmour, A., Ben-Hamida, M., Pericak-Vance, M., Hentati, F. & Siddique, T. 2001. The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat. Genet., 29:160–165.
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Data S1. Materials and methods. Table S1. Tetrahymena thermophila MORN proteins are sorted by the MORN protein annotation ID of the Tetrahymena genome database (ciliate.org), not available values are abbreviated with na (not applicable). The MORN nomenclature MRNN, MRNC, and MRNO was assigned according to the composition of the MORN domains throughout the protein and/or the association with other domains. Numbers of predicted MORN motifs are shown in column three. Predicted associations with further domains are indicated by the following abbreviations in column four: SK = serine/threonine protein kinases; PK = phosphokinase; IQ = IQ motif, EF-hand binding site; ZF = zinc finger domain; TM = transmembrane domain; LRR = leucine-rich repeats; WD40 = tryptophan-aspartic acid repeat; SAM = sterile alpha motif; na, not applicable. Columns 5–20 show the expression values without decimal places during logarithmic growth (L), starvation (S), and
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conjugation (C) of each gene encoding MORN motifs. The numbers behind the letters in columns 5–20 indicate the hours for each condition analysed (see TGED for details). Figure S1. Pearson correlations of the co-expression of genes encoding MORN proteins. Genes are sorted in correspondence to those of Fig. 1C. All expression values were normalised and plotted from 1 to 1. Three distinct clusters, highlighted by triangles, reflect the three main expression patterns observed for highly expressed MORN proteins as seen in Fig. 1C. Figure S2. GSTtrap FF column FPLC purification of GST:: MRNO51. A. FPLC curve showing the elution of GST:: MRNO51 (peak at about 2,250 mAU; fractions A, B, and C). B. SDS-Gel electrophoresis of fractions A, B, and C. Lanes 1, 2, 3: whole lysate after cell-disruption, before centrifugation and after centrifugation, respectively; combined lane 4: purified GST:MRNO51 corresponding to fraction A and B; lane 5: fraction C. C. Western blot of the four fusion-proteins MRNC36:GFP (lane 1), MRNC38:GFP (lane 2), MRNC39:GFP (lane 3), and MRNO51:GFP (lane 4), which demonstrates that all proteins are expressed fulllength together with the C-terminal GFP tag. Figure S3. Comparison of the endogenous expression of MRNC36, MRNC38, MRNC39, MRNO51 based on microarray data extracted from TGED. MTT1 expression (whose promoter was used for the GFP-fusion protein localisation studies) is always low and only induced through CdCl2.
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 694–700
The Journal of
Eukaryotic Microbiology
Journal of Eukaryotic Microbiology ISSN 1066-5234
SHORT COMMUNICATION
Tetrahymena Expresses More than a Hundred Proteins with Lipid-binding MORN Motifs that can Differ in their Subcellular Localisations €rn Habichta, Christian Woehleb & Sven B. Goulda Jo €sseldorf, Germany a Institute for Molecular Evolution, Heinrich-Heine-University, 40225 Du b Institute for Microbiology, Christian-Albrecht-University, 24118 Kiel, Germany
Keywords Ciliates; membrane targeting; membrane tethering; MORN. Correspondence S.B. Gould, Institute for Molecular Evolution, €sselHeinrich-Heine-University, 40225 Du dorf, Germany Telephone number: +49 211 81 13983; FAX number: +49 211 81 13554; e-mail: [email protected]
ABSTRACT Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein-encoding genes, some of which are specifically up-regulated during conjugation. A lipid-binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.
Received: 6 November 2014; accepted January 20, 2015. doi:10.1111/jeu.12216
THE membrane occupation and recognition nexus (MORN) motif was first described as part of the mammalian junctophilin protein 1 (JP-1), which mediates the tethering of the sarcoplasmic reticulum to the plasma membrane (Takeshima et al. 2000). The motif consists of a consensus sequence that stretches across 14 amino acids (Fig. 1A) and the number of MORN motifs within animal, plant, and protozoan MORN proteins that have been studied so far varies from 2 to 17 individual motifs (Choi et al. 2010; Shimada et al. 2004). The motifs can cluster predominantly at the N-terminus (Bhattacharya et al. 2012; Ma et al. 2006; Shetty et al. 2007; Takeshima et al. 2000) or the C-terminus of the proteins (Choi et al. 2010; Kunita et al. 2007; Lee et al. 2009; Shimada et al. 2004), but are sometimes also found along the entire protein sequence and in conjunction with other domains of known function (Gubbels 2006; Takeshima et al. 2000). Since the initial description of the MORN motif, several MORN motif-harbouring proteins have been analysed, which include the junctophilin family JP-1 to JP-4 (Ito et al. 2001; Nishi et al. 2000, 2003; Takeshima et al. 2000), MORN4 of Drosophila (Bhattacharya et al. 2012), the amyotrophic lateral sclerosis protein ALS2 (Yang et al. 2001), and the radial spoke protein 44 (RSP44; also referred to as
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meichroacidin, MCA) (Shetty et al. 2007; Tokuhiro et al. 2008). Characterised plant proteins that harbour MORN motifs are phosphatidylinositol monophosphate kinases (Im et al. 2007; Ma et al. 2006; Mikami et al. 2010), the stromal accumulation and replication of chloroplasts 3 (ARC3) protein (Shimada et al. 2004), as well as the Brassica rapa MORN motif protein (BrMORN), which in Arabidopsis promotes an increase in the cell size, vegetative organs, and seed production (Lee et al. 2009). In apicomplexan parasites, MORN1 is the best characterised. Within this group, it acts as an early endodyogeny marker during the asexual and sexual development and has established itself as a reliable marker for the alveoli, too, in Apicomplexa more often referred to as the inner membrane complex (Ferguson et al. 2008; Gould et al. 2008; Gubbels 2006; Lorestani et al. 2010). Knockout mutants of TgMORN1 show a disorganisation of the posterior end and basal complex of the parasite, and fail to complete daughter cell budding (Heaslip et al. 2010; Lorestani et al. 2010). The use of the MORN motif in proteins with a variety of different duties underpins its function as a more universal domain. Here, we took a look at the phylogenetic distribution of this motif and used the ciliate Tetrahymena thermophila as a model to shed light on this complex protein
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Figure 1 The MORN motif is highly conserved across eukaryotes, present in 129 expressed proteins of Tetrahymena and is able to bind to isolated lipids. A. A sequence logo generated from 630 individual MORN motifs stemming from 100 proteins of 10 different organisms shows the conserved nature of the 14-amino acid long motif. B. Four exemplary MORN proteins as representatives for the three main classes we defined: (i) the MORN motifs are found at the N-terminus (N; light green), or (ii) at the C-terminus (C; dark green), or (iii) otherwise and/or in combination with other known domains such as the IQ motif (O; yellow). C. Expression profiles of 129 Tetrahymena thermophila MORN protein-encoding genes (x-axis) during the exponential growth phase (L), a 24 h long starvation phase (S) and an 18 h long conjugation phase (C). The number of MORN motifs in each protein is indicated by a yellow to red gradient. Genes are sorted according to their co-expression correlation values among the three different conditions and expression values are shown in arbitrary units (AU; y-axis). For individual expression values please refer to Table S1. Association of the MORN domains (again N, C, or O) with other protein domains are indicated additionally by coloured squares: SK = serine/ threonine protein kinases; PK = phosphokinase; IQ = IQ motif; ZF = zinc finger domain; TM = transmembrane domain; LRR = leucine-rich repeats; WD40 = tryptophan-aspartic acid repeat; SAM = sterile alpha motif. D. The 14 MORN motifs of MRNO51 mediate the association of glutatione S-transferase (GST) with lipids when fused to the C-terminus of GST (GST::MRNO51). This fusion protein binds predominantly to cardiolipin and to 1,2-diacylglycerol at a concentration of 1 lg/ml. At a higher concentration of 100 lg/ml, GST::MRNO51 furthermore associates with phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylserine. Purified GST alone served as control. On the right a schematic representation of the blot. PtdIns, Phosphatidylinositol; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PtdIns(3,4,5)P3, phosphatidylinositol (3,4,5)-trisphosphate.
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family a bit more in detail. Proteins with MORN motifs can reach more than a hundred in some ciliates and we show that the MORN motifs allow an otherwise soluble protein to bind to a variety of different lipids. The exemplary localisation of only a few candidate proteins demonstrates that the subcellular localisation can be multifaceted, even when the MORN motifs and their distribution among the individual proteins are rather similar. MATERIAL AND METHODS Bioinformatics The InterPro database (Hunter et al. 2012) served as the initial source to screen for proteins of the MORN family. The distribution of MORN motifs among individual proteins was determined using SMART (Letunic et al. 2012) and RADAR (Heger and Holm 2000). For the sequence logo, MORN motifs were first aligned with Clustal Omega (Sievers et al. 2011) and then plotted using WebLogo (Crooks et al. 2004). All T. thermophila gene expression values were extracted from the Tetrahymena Functional Genomics Database (TetraFGD) (Xiong et al. 2013) and the data visualised using Matlab (The MathWorks GmbH, Ismaning, Germany).
and TAG—encoding glutamines in ciliates, but stop codons in Escherichia coli—were substituted with CAA and CAG, respectively. The synthesised sequence was cloned into pGEX-4T-2_G029 that provided an N-terminal glutathione S-transferase (GST) tag. E. coli BL21 cells were transfected and cells were allowed to grow to an optical density of 0.6, and fusion protein expression then induced with isopropyl-b-D-thiogalactopyranosid overnight at 20 °C and continuous shaking. Cells were harvested, resuspended in PBS (pH 7.4) and homogenised using a cell disrupter system (Constant Systems Ltd., Daventry, UK). GST-tagged protein was purified via fast protein liquid chromatography using a GSTrap FF column (GE Healthcare, Berlin, Germany) with the recommended buffer. The buffer of the purified protein was changed to TBS using Vivaspinâ 15R Centrifugal Concentrator (Sartorius Stedim Biotech, €ttingen, Germany) and three washing steps. Purified Go GST::TTHERM_00584930 was used at a concentration of 1 and 100 lg/ml (both diluted in TBST) in the lipid overlay assays that were performed on Membrane Lipid StripsTM (Echelon Biosciences Inc., Salt Lake City, UT) according to the manufacturer’s recommendations. Bound fusion protein was detected through standard Western blots using a horse radish peroxidase conjugated anti-GST antibody (Sigma-Aldrich) at a dilution of 1:1,000.
Tetrahymena culturing and cloning The T. thermophila strain CU522 was cultured in SPP medium (Gorovsky et al. 1975) at 15 °C and transferred to 30 °C to obtain high-density cultures overnight. For C-terminal green fluorescent protein (GFP)-tagging, the genes of interest were cloned via the restriction sites SphI and MluI into pTtag (accession no. FJ789658), which has a cadmium-inducible metallothionein (MTT1) promoter. Tetrahymena thermophila CU522 cells were transfected with linearised GFP constructs using a PDS-1000 Gene Gun (Bio-Rad Laboratories GmbH, Munich, Germany) as previously described (Cassidy-Hanley et al. 1997) and then selected for 10 d in SPP medium supplemented with 30 lg/ml paclitaxel (LC Laboratories, Woburn, MA). The correct molecular weight was verified by Western blotting (Fig. S2) and cells fixed after 45 min of induction to localise the proteins as previously described (El-Haddad et al. 2013). The primary antibodies (a-GFP, Abcam, Cambridge, MA; a-alpha-tubulin, DSHB) were used at a concentration of 1:100, diluted in PBS containing 0.1% BSA and 0.1% Triton X-100. Secondary Alexa Fluor 488 and TRITC antibodies Invitrogen / Life Technologies GmbH, Darmstadt, Germany; Sigma-Aldrich Chemie Gmbh, Munich, Germany were used at 1:1,000. Fixed and labelled cells were transferred onto Superfrost slides (Menzel, Braunschweig, Germany) and mounted with Vectashield medium (Vector Laboratories, Peterborough, UK) that included 40 ,6-diamidino-2-phenylindole (DAPI) to stain the DNA. Recombinant overexpression and lipid-binding assay We synthesised a variant of TTHERM_00584930 (GENEART AG, Regensburg, Germany), in which the two codons TAA
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RESULTS AND DISCUSSION MORN motifs occur in conjunction with a variety of other protein domains The MORN motifs are found among all domains of life and some viruses. However, at the InterPro database (Hunter et al. 2012) thousands of MORN proteins are listed each for bacteria and eukaryotes, but only two for Archaea. The isolated occurrence of two almost identical MORN proteins (they differ only in one single amino acid at the very C-terminus) suggests a recent acquisition of these genes in the two Euryarchaeota trough lateral gene transfer, which occurs fivefold more frequently from bacteria to Archaea than vice versa (Nelson-Sathi et al. 2014). In any case, humans encode the largest variety of MORN proteins among mammals, but ciliates the largest variety among all eukaryotes. For the ciliate T. thermophila 132 individual genes are listed at InterPro that encode proteins that harbour at least one MORN motif (129 at ciliate.org, for which expression data are available and that we all annotated). We sorted all Tetrahymena MORN proteins into three classes: (i) those in which the MORN motifs localise predominantly towards the N-terminus (MRNN, 17 proteins), (ii) those in which the MORN motifs localise predominantly towards the C-terminus (MRNC, 60 proteins), and (iii) those cases, in which the MORN motifs are distributed throughout the entire protein and/or are associated with other domains of known function (MRNO, 55 proteins) (Fig. 1B, C and Table S1). Furthermore, ciliate MORN proteins can contain other domains of known function, including calmodulin-binding domains, zinc fingers, transmembranedomains, and WD-40-repeats (Fig. 1C and Table S1).
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Figure 2 Localisations of four MORN proteins in Tetrahymena thermophila. All four MORN proteins show a distinct localisation. MRNC36::GFP localises to focal dots at the apical tip of cell (arrowhead), to the contractile vacuole pore (CVP; arrow), and to the very tip of the cilia (*). MRNC38::GFP appears associated with the undulating membrane of the oral apparatus (OA, arrowhead). MRNC39::GFP binds to the entire cell membrane of the ciliate. MRNO51::GFP localises predominantly to the OA (*) and the CVP (arrow) but also weakly resembles the localisation observed for MRNC36, although especially the localisation associated with the OA completely differs. For each protein, a schematic representation of the MORN motif composition is shown below the images. All images were captured 45 min after expression was induced with CdCl2. Scale 10 lm.
On the basis of microarray data we extracted from the TetraFGD, we clustered the expression profiles of 129 MORN encoding genes according to their co-expression throughout logarithmic growth, starvation, and conjugation. Genes encoding MORN proteins show four main patterns of expression: (i) the first set of genes is likely encoding for “housekeeping MORNs”, they show high levels of expression throughout all time points analysed, (ii) the second set of genes is highly expressed during the logarithmic growth phase, starvation, and later phases of conjugation, (iii) the third set of genes is up-regulated specifically during early conjugation, (iv) and the fourth set— the majority of the MORN protein-encoding genes—is generally expressed at low levels, but above background expression that is already subtracted at TGED (Fig. 1C, S1). There is, however, no connection of certain gene expression profiles to the localisation, domain composition, or association with other protein domains. It
suggests that the MORN proteins in Tetrahymena have not evolved like a typical gene family, but that due to the more universal duty of the motif, MORN domains have found residence in a variety of different proteins through exon shuffling or recombination events. Do the MORN motifs alone determine localisation? To first investigate the lipid-binding ability of MORN motifs from a Tetrahymena protein, we performed a lipid overlay assay using the MORN motifs of MRNO51 (TTHERM_00584930) for a case study. The 14 MORN motifs span almost across the entire sequence of MRNO51 that is predicted to be 426 amino acids long. We synthesised a codon-optimised variant of MRNO51 to allow overexpression in E. coli BL21 cells. The protein was fused to the C-terminus of GST for the efficient purification through fast protein liquid chromatography of the recombinant protein.
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At a concentration of 1 lg/ml, GST::MRNO51 bound to cardiolipin (CL) and 1,2-diacylglycerol (DAG), the latter a lipid second messenger that is able to recruit cytosolic proteins to a membrane (van Meer et al. 2008). The increase in the fusion protein concentration to 100 lg/ml revealed additional lipids, such as phosphatidylinositol and phosphatidylserine, with which GST::MRNO51, but not the control (GST alone), interacted (Fig. 1D). The MORN domain of MRNO51 is able to recruit an otherwise soluble protein, GST, to a variety of isolated lipids. Membrane targeting and binding properties are known for the C1 domain of protein kinase C conserved 1, the C2 domain of protein kinase C conserved 2 and the pleckstrin homology (PH) domains. The C1, C2, and PH domains are approximately 50, 130, and 120 amino acids long, respectively (Cho 2001; Yu et al. 2004). In general, they show a much higher sequence variability than the MORN motifs that are highly conserved among protists and eumetazoans (Ferguson et al. 2008; Gubbels 2006; Takeshima et al. 2000). Not the sequence similarity, but rather a common fold of each domain in this case provides the lipid affinity of the C1, C2 (Cho 2001), and PH domains (Lemmon 2007; Lemmon et al. 2002). The general membrane tethering ability of MORN domains is provided through the conserved 14-amino acid repeat (Fig. 1A). It appears the motifs have to be somehow arranged in tandem to mediate reliable membrane association (Ma et al. 2006). However, the exact limit or individual arrangement of the MORN motifs needed to guarantee specific lipid binding has not been experimentally addressed in detail yet. If the purpose of the MORN motif itself is to first and foremost tether a protein to a lipid bilayer, the question arises to what degree the subcellular localisation of MORN proteins differs, when the composition of the MORN motifs is almost identical. We chose to localise four proteins through C-terminal GFP tagging. All four share a high sequence similarity among each other and are homologues of PfMORN1 (PF3D7_1031200) and TgMORN1 (TGME49_310440); well-studied MORN proteins of the apicomplexan parasites Plasmodium and Toxoplasma, respectively (Ferguson et al. 2008; Gubbels 2006; Lorestani et al. 2010). Three of the four proteins have nine MORN motifs arranged in tandem at the C-terminus (MRNC36, TTHERM_01084170; MRNC38, TTHERM_ 00378500; MRNC39, TTHERM_00590130) and one has 14 consecutive MORN motifs that stretch almost across the entire protein (MRNO51; TTHERM_00584930). Studies of truncated rice and rabbit MORN proteins showed that the number of MORN motifs in combination with their orientation influenced the subcellular localisation in comparison to the wild-type protein (Ma et al. 2006; Takeshima et al. 2000). Therefore, one could predict that the same orientation and the exact same number, and hence a similar predicted lipid-binding efficiency of MORN motifs, would lead to an identical localisation. However, the four MORN proteins we analysed—three of which have the same MORN motif amount and composition—all showed distinct subcellular localisation (Fig. 2). This has two major implica-
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tions: (i) one cannot use the results obtained for the domain architecture of one MORN protein to predict the localisation of another, and (ii) the fine localisation specificity of MORN proteins must lay either within the less conserved amino acids of the motif itself (Fig. 1A) or is MORN motif independent. The MORN motifs, when arranged in tandem, function as a universal membrane-binding domain in a variety of different proteins, similar to the function of the C1, C2, and pleckstrin homology domains. The ubiquitous presence of the MORN motifs among eukaryotic groups reflects the importance of this domain in general, and particular in ciliates, in which the MORN protein family has been massively expanded. For ciliates it has been suggested that expanded gene families—that are also specifically selected to persist after genome duplication events —are of special biological importance for the protists (Chalker and Stover 2007; Eisen et al. 2006). The overall amount of MORN proteins in ciliates might at first stun, but especially the MORN proteins of Tetrahymena that are up-regulated during conjugation for example, have the potential to identify new crucial players with regard to how the two complementary cells communicate and fuse during the sexual reproduction stage. ACKNOWLEDGMENTS Funding through the Deutsche Forschungsgemeinschaft to SBG (GO1825/3-1) is gratefully acknowledged. LITERATURE CITED Bhattacharya, M. R. C., Gerdts, J., Naylor, S. A., Royse, E. X., Ebstein, S. Y., Sasaki, Y., Milbrandt, J. & DiAntonio, A. 2012. A model of toxic neuropathy in Drosophila reveals a role for MORN4 in promoting axonal degeneration. J. Neurosci., 32:5054–5061. Cassidy-Hanley, D., Bowen, J., Lee, J. H., Cole, E., VerPlank, L. A., Gaertig, J., Gorovsky, M. A. & Bruns, P. J. 1997. Germline and somatic transformation of mating Tetrahymena thermophila by particle bombardment. Genetics, 146:135–147. Chalker, D. L. & Stover, N. A. 2007. Genome evolution: a double take for Paramecium. Curr. Biol., 17:R97–R99. Cho, W. 2001. Membrane targeting by C1 and C2 domains. J. Biol. Chem., 276:32407–32410. Choi, Y.-J., Hwang, K.-C., Park, J.-Y., Park, K.-K., Kim, J.-H., Park, S.-B., Hwang, S., Park, H., Park, C. & Kim, J.-H. 2010. Identification and characterization of a novel mouse and human MOPT gene containing MORN-motif protein in testis. Theriogenology, 73:273–281. Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. 2004. WebLogo: a sequence logo generator. Genome Res., 14:1188– 1190. Eisen, J. A., Coyne, R. S., Wu, M., Wu, D., Thiagarajan, M., Wortman, J. R., Badger, J. H., Ren, Q., Amedeo, P., Jones, K. M., Tallon, L. J., Delcher, A. L., Salzberg, S. L., Silva, J. C., Haas, B. J., Majoros, W. H., Farzad, M., Carlton, J. M., Smith Jr, R. K., Garg, J., Pearlman, R. E., Karrer, K. M., Sun, L., Manning, G., Elde, N. C., Turkewitz, A. P., Asai, D. J., Wilkes, D. E., Wang, Y., Cai, H., Collins, K., Stewart, B. A., Lee, S. R., Wilamowska, K., Weinberg, Z., Ruzzo, W. L., Wloga, D., Gaertig,
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SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Data S1. Materials and methods. Table S1. Tetrahymena thermophila MORN proteins are sorted by the MORN protein annotation ID of the Tetrahymena genome database (ciliate.org), not available values are abbreviated with na (not applicable). The MORN nomenclature MRNN, MRNC, and MRNO was assigned according to the composition of the MORN domains throughout the protein and/or the association with other domains. Numbers of predicted MORN motifs are shown in column three. Predicted associations with further domains are indicated by the following abbreviations in column four: SK = serine/threonine protein kinases; PK = phosphokinase; IQ = IQ motif, EF-hand binding site; ZF = zinc finger domain; TM = transmembrane domain; LRR = leucine-rich repeats; WD40 = tryptophan-aspartic acid repeat; SAM = sterile alpha motif; na, not applicable. Columns 5–20 show the expression values without decimal places during logarithmic growth (L), starvation (S), and
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conjugation (C) of each gene encoding MORN motifs. The numbers behind the letters in columns 5–20 indicate the hours for each condition analysed (see TGED for details). Figure S1. Pearson correlations of the co-expression of genes encoding MORN proteins. Genes are sorted in correspondence to those of Fig. 1C. All expression values were normalised and plotted from 1 to 1. Three distinct clusters, highlighted by triangles, reflect the three main expression patterns observed for highly expressed MORN proteins as seen in Fig. 1C. Figure S2. GSTtrap FF column FPLC purification of GST:: MRNO51. A. FPLC curve showing the elution of GST:: MRNO51 (peak at about 2,250 mAU; fractions A, B, and C). B. SDS-Gel electrophoresis of fractions A, B, and C. Lanes 1, 2, 3: whole lysate after cell-disruption, before centrifugation and after centrifugation, respectively; combined lane 4: purified GST:MRNO51 corresponding to fraction A and B; lane 5: fraction C. C. Western blot of the four fusion-proteins MRNC36:GFP (lane 1), MRNC38:GFP (lane 2), MRNC39:GFP (lane 3), and MRNO51:GFP (lane 4), which demonstrates that all proteins are expressed fulllength together with the C-terminal GFP tag. Figure S3. Comparison of the endogenous expression of MRNC36, MRNC38, MRNC39, MRNO51 based on microarray data extracted from TGED. MTT1 expression (whose promoter was used for the GFP-fusion protein localisation studies) is always low and only induced through CdCl2.
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 694–700
