Posttranscriptional regulation of mitochondrial DNA in mammalian mitochondria

Mitochondria are the most important organelles for ATP supply in nearly all eukaryotic cells. Besides energy production, mitochondria also play important roles e.g. in calcium homeostasis, apoptosis or fatty acid ß-oxidation. They originated from a proto-bacterium and therefore contain their own genome encoding for a subset of mitochondrial OXPHOS components as well as tRNAs and rRNAs necessary for the translation machinery. Regulation of mtDNA expression is indispensable for normal OXPHOS function and defective mitochondrial function can cause neurodegenerative diseases but is also linked to aging, cancer and diabetes. Mitochondrial transcription factor 1 (MTERF1) has been reported to regulate H-strand transcription of the two ribosomal RNA genes through simultaneous binding of the heavy strand promoter and its termination site based on extensive in vitro studies during the last decades. However, evidence for its function in vivo is still missing. In this work, analysis of the first Mterf1 knockout mouse model reveals that lack of MTERF1 has no effect on ribosomal RNA levels, but instead causes increased RNA levels on the antisense region of mitochondrial rRNAs. At the same time transcription initiation events are decreased at the light-strand promoter suggesting that MTERF1 has a role in transcription termination on the L-strand to prevent transcriptional interference at the light-strand promoter. Studies in mice lacking the mitochondrial transcription termination factor 2 (MTERF2) show apparently healthy and fertile animals with normal lifespan. However, mice challenged with a ketogenic diet have been reported to develop a muscle-specific phenotype including decreased transcription and OXPHOS deficiency. A second Mterf2 knockout mouse model, created in our lab, however, does not confirm the reported phenotype. The viral trap, a genetic tool used to interrupt Mterf2 gene expression in one of the mouse models, could explain the observed differences since it contains a very strong promoter, which can influence the expression of other genes closely located to the target gene. A gene encoding cryptochrome 1 (CRY1) is situated 1,6 kb downstream of Mterf2 and could be influenced by a viral trap targeting the Mterf2 gene. In order to test this hypothesis, we simultaneously analyzed the two Mterf2 knockout mouse models, a Cry1 knockout mouse and controls and found that all mice were healthy and fertile with a normal lifespan. MtDNA levels, mitochondrial transcription as well as steady state levels of OXPHOS protein components are unaffected in mice lacking Mterf2 or Cry1, contradicting a role of MTERF2 in mitochondrial transcription. However, Cry1 expression is decreased in both Mterf2 knockout mouse models, which suggests a putative influence of Cry1 expression when the Mterf2 gene is targeted. The leucine-rich pentatricopeptide repeat domain containing protein (LRPPRC) is an important factor of posttranscriptional regulation of mtDNA expression. Although data from a Lrpprc knockout mouse model and patient fibroblasts carrying decreased LRPPRC protein levels support a role of LRPPRC in mitochondrial mRNA transcript stability and coordination of translation, its in vivo function is still highly debated in the literature. A recent report demonstrated that LRPPRC is involved in mitochondrial transcription initiation through direct interaction with POLRMT. In order to study this protein in a physiological environment we created bacterial artificial chromosome transgenic mice slightly overexpressing LRPPRC and Lrpprc heterozygous knockout mice with moderately decreased LRPPRC levels. Slightly increased or decreased LRPPRC protein levels did not affect steady state transcript levels as well as de novo transcription suggesting that LRPPRC does not have a role in mitochondrial transcription. In addition, increasing amounts of LRPPRC did not stimulate transcription in a recombinant in vitro system and immunoprecipitation as well as size exclusion chromatography did not detect any interaction between LRPPRC and POLRMT

Biotechnological production of value-added chemicals from cis-aconitate with the help of genetically engineered oleophilic yeasts

Hintergrund: Die Synthese von Chemikalien aus fossilen Rohstoffen wird wegen ihrer begrenzten Verfügbarkeit und ihren negativen Auswirkungen auf die Umwelt zunehmend kritisch bewertet. Eine Alternative bietet die „Weiße Biotechnologie“, insbesondere die Fermentation nachwachsender Rohstoffe mithilfe von Hefen. Die oleophilen Hefen Pseudozyma (P.) tsukubaensis und Yarrowia (Y.) lipolytica sind natürliche Säureproduzenten. Ihre Hauptprodukte sind Metabolite des Tricarbonsäurezyklus: Citrat (CA), α-Ketoglutarat und Malat. In kleineren Mengen werden auch andere Stoffe wie Isocitrat (ICA) oder Itaconat (ITA, nur von P. tsukubaensis) sekretiert. Das Interesse an den beiden Letztgenannten hat in den vergangenen Jahrzehnten stetig zugenommen. Bis heute gibt es allerdings keinen etablierten Wirtsorganismus für die ICA-Produktion. ITA hingegen wird mithilfe von Aspergillus terreus synthetisiert. Jedoch stößt die ITA-Produktivität dieses Hyphenpilzes auch mit großem wissenschaftlichem Aufwand an ihre Grenzen. Daher wird ein neuer Wirtsorganismus benötigt. Ergebnisse: In dieser Studie wurden ein vielversprechender P. tsukubaensis-Stamm für die Produktion von ITA und ein Y. lipolytica-Stamm für ICA konstruiert. Zunächst wurde das Genom von P. tsukubaensis sequenziert. Infolgedessen wurde ein Gencluster für die Synthese und den Export von ITA identifiziert, das homolog zu dem von Ustilago maydis ist. Die Überexpression von vier der fünf Clustergene erhöhte die ITA-Sekretion nicht deutlich. Das fünfte Gen kodiert den vermeintlichen Transkriptionsfaktor Ria1p, der vermutlich das Gencluster steuert. Die Überexpression des PtRIA1 Gens führte zu einer signifikant erhöhten ITA-Produktion von bis zu 31,4 g/l in Mikrotiterplatten. Durch die Optimierung der Wachstumsbedingungen wurden im Bioreaktor innerhalb von 7 d 113,6 g/l ITA ohne die Notwendigkeit eines Triggers produziert. Für die ICA-Produktion wurden zwei mutmaßliche mitochondriale Citrat-Transportproteine in Y. lipolytica identifiziert, welche von den Genen YlCTP1 sowie YlYHM2 kodiert werden. Die Funktionsweise der beiden Proteine scheint sich stark voneinander zu unterscheiden. Die Deletion von YlCTP1 führte zu einer leichten Verschiebung des ICA:CA-Verhältnisses, aber die Gesamtmenge beider Säuren nahm stark ab. Durch die Deletion von YlYHM2 stieg die ICA:CA-Produktrate von 12 % auf 95 % im Vergleich zum Wildtyp. Innerhalb von 5 d wurden bis zu 131,9 g/l ICA mit Sonnenblumenöl, bzw. 22,0 g/l ICA mit Glukose als einzige Kohlenstoffquelle in einem Bioreaktor unter kontrollierten Produktionsbedingungen erreicht. Durch die zusätzliche Hemmung des Isocitratlyase-Proteins mit ITA stieg das ICA:CA-Verhältnis bis 98 %. Fazit: Mittels Metabolic Engineering wurden im Rahmen dieser Arbeit die beiden Hefestämme P. tsukubaensis HR12 und Y. lipolytica ΔYHM2 erzeugt. Mit ihrer Hilfe ist es möglich, die hochwertigen Chemikalien ITA oder ICA in hohen Mengen (> 100 g/l) aus nachwachsenden Rohstoffen wie Glukose oder sogar Pflanzenölen herzustellen.Background: The synthesis of chemicals from fossil fuels is being evaluated increasingly critically, mainly due to its expected exhaustion and negative impact on the environment. An alternative offers ‘white biotechnology’, especially the fermentation of renewable resources with the help of yeasts. The oleophilic yeast species Pseudozyma (P.) tsukubaensis and Yarrowia (Y.) lipolytica are both natural organic acid producers. Their main products are metabolites of the tricarboxylic acid cycle, namely citrate, α-ketoglutarate and malate. In smaller amounts, other compounds like isocitrate (ICA) or itaconate (ITA, solely with P. tsukubaensis) are also secreted. The interest for the latter two has been rising steadily during the last decades. However, to this date, there is no established host organism for the ICA production. ITA, on the other hand, is being synthesised with Aspergillus terreus. Even with great scientific effort, the ITA productivity of this hyphal fungus appears to reach its limits. Therefore, a different host organism is needed. Results: In this study, a promising P. tsukubaensis strain has been constructed for the production of ITA and a Y. lipolytica strain for ICA. First, the genome of the ITA producer P. tsukubaensis has been sequenced. As a result, a gene cluster for the synthesis and export of ITA, homologous to that of Ustilago maydis, has been identified. By overexpressing four of the five cluster genes, respectively, none to low increases in ITA secretion were observed. The fifth gene is encoding the putative transcription factor Ria1p which probably controls the gene cluster. The overexpression of the gene PtRIA1 led to a significantly increased ITA production of up to 31.4 g/l in micro-wells. By optimizing the growth conditions 113.6 g/l ITA could be produced within 7 d under controlled conditions in a bioreactor without the need of a trigger like phosphate limitation. For the production of ICA, two putative mitochondrial citric acid transporter proteins were identified in Y. lipolytica. One carrier protein is encoded by the novel gene YlYHM2, the other one by YlCTP1. The mode of function for the two deduced proteins appears to be very distinct from one another. The deletion of YlCTP1 led to a minor shift in the ICA:CA ratio but the total amount of acids decreased greatly. By deleting YlYHM2, the ICA:CA product ratio could be increased from 12 % to 95 % compared to the wild type strain. Within 5 d up to 131.9 g/l ICA with sunflower oil and 22.0 g/l with glucose as the sole carbon source could be achieved under controlled production conditions in a bioreactor. Further inhibition of the isocitrate lyase protein with ITA increased the ICA:CA ratio to 98 %. Conclusion: Within this work, the two yeast strains P. tsukubaensis (HR12) and Y. lipolytica (ΔYHM2) have been created via metabolic engineering. With their help, it is possible to produce the value-added chemicals ITA or ICA on a high scale (> 100 g/l) from renewable resources like glucose or even vegetable oils

DNA Interstrand Crosslink Repair in Trypanosoma brucei

PhDGenomes are constantly challenged by agents that promote DNA damage, with interstrand crosslinks (ICLs) representing a particularly dangerous lesion. Ongoing work in the Wilkinson laboratory aimed at identifying novel agents that target Trypanosoma brucei, the causative agent of African trypanosomiasis, identified several prodrugs that once activated form ICLs in this protozoan parasite. To understand the complexity of ICL repair systems that T. brucei employs to resolve such damage, a variety of null mutant lines were generated that lack activities postulated to fix such lesions. Phenotypic screens using various DNA damaging agents revealed that TbMRE11, TbEXO1, TbCSB, TbCHL1, TbFAN1, TbBRCA2 and TbRAD51 all help to resolve ICLs, implicating components of the homologous recombination, nucleotide excision repair and mismatch repair pathways in resolving this form of damage: This approach demonstrated that components of the translesion synthesis pathway (TbREV2 and TbREV3) do not play a significant role in ICL repair. In many organisms, nucleases belonging to the SNM1/PSO2 family play a key and specific role in the repair of ICLs with this property extending to the T. brucei homologue, TbSNM1. To assess whether there is a functional linkage between the DNA repair factors noted above and TbSNM1, a series of double null mutants were constructed and the susceptibility of these lines to ICL inducing agents determined. Identification of their epistatic/non-epistatic interactions revealed that T. brucei expresses at least two ICL repair systems with one pathway involving the concerted activities of TbSNM1/TbCSB/TbEXO1, that we postulate functions to repair ICLs encountered by the transcriptional machinery, while the other is centred upon TbMRE11/TbFAN1/TbEXO1 that may help resolve lesions which cause stalling of DNA replication forks. By unravelling how T. brucei repairs ICLs, specific inhibitors against key components of these pathways could be developed and used in combination with DNA damaging agents to target trypanosomal infections.Queen Mary University of Londo

Understanding the role of unusual dynamin- related proteins in the lizard pathogen Entamoeba invadens

Amoebiasis is the third most common cause of death due to parasitic diseases in the world after malaria and schistosomiasis. In developing countries, it infects more than 50 million individuals annually causing 50,000 -100,000 deaths. Entamoeba. histolytica, an intestinal protozoan parasite, is the causative agent of amoebiasis in humans. The cyst is responsible for the transmission of the disease, in which the host gets infected through the ingestion of cyst-contaminated foods and water. About 90% of infections are asymptomatic cyst carriers and spreaders, in which each individual shed up to 45 million cysts per day. The process of conversion from motile trophozoite to dormant cyst is called encystation. However, little known about the molecular and cellular events that trigger this encystation. Numerous studies have attempted to investigate the molecules and genes involved in encystation. Dynamin, which is a conserved family of large GTPase, were suggested to play a role during encystation in Entamoeba. The unusual dynamin-related proteins Drp3 and Drp4 were revealed to be upregulated during stage transition. Despite the fact that all eukaryotes contain at least one dynamin protein, no homologues of Drp3 and Drp4 were identified in mammals. In this study, I attempted to study potential role of Drp3 and Drp4 during encystation in the laboratory model Entamoeba invadens. Furthermore, study the effect of the overexpression of these dynamins in mammalian cells. Using molecular biology techniques, immunofluorescence microscopy, transmission electron microscopy, and immunoelectron microscopy. In this study, we conducted stage conversion experiments to understand the importance of dynamins during cyst formation. Localization experiments showed that Drp3 and Drp4 have dual localization to both nucleus and cytoplasm. Drp3 existed as punctate structures localized to the cytoplasm and the nucleus in trophozoites and elongated structures localized to the nucleus in the cyst. The localization of the mutated form of Drp3 lacking the key residues in the GTPase domain (Drp3-mutant) was mainly nuclear and to a lesser extent cytoplasmic. The nuclear localization revealed the presence of Drp3 on the nucleus, the nuclear vesicles, and the nuclear envelope. Interestingly, cells overexpressing Drp3-mutant exhibited cytokinesis failure and multinucleation, suggesting a possible role in cytokinesis. In the mammalian cells, both Drp3 and Drp4 displayed the negative dominant effect on COS-7 cells. Drp3 localized to the nucleus and Drp4 localized to the mitochondria. However, both dynamins induced membranous tubulation in COS-7 cells. The work presented in this study demonstrated that Drp3 and Drp4 have nuclear and cytoplasmic functions. Drp3 is associated with the nucleus in mammalian cells and with the nucleus and the cytoplasm in E. invadens. The effect of Drp3 mutant on E. invadens cells indicates a potential role in cytokinesis and nuclear division. Drp4 is associated with Mitochondria in mammalian cells and some cytoplasmic compartments in E. invadens might involve the mitosomes

Understanding the pathogenesis of myotonic dystrophy type 1

To identify the full range of targets and the pathogenic consequences, we sought to mimic the pathogenesis of myotonic dystrophy type 1 with temporal and spatial control: temporal to reproduce the developmental pathogenesis of the congenital form, and spatial to isolate tissue specific pathology. To do this, we attempted to use the Cre-lox system for the conditional expression of an EGFP reporter-linked expanded CUG repeat RNA in the mouse. Expression of the transgene was controlled by Cre excision of a transcriptional stop, placed upstream of the EGFP-expanded repeat open reading frame. The transgenes were constructed and tested successfully, and a normal length repeat transgenic line was established. Unfortunately generation of the expanded repeat line was not successful. The constructs were used to generate cell-culture models of DM1, in both human and murine cells, which mimicked the nuclear foci formation and MBNL1 co-localisation seen in patient cells. Expression of exogenous MBNL1/GFP fusion protein in this model resulted in an increase in the size of foci, indicating that MBNL1 protein is limiting within the cell, and may possibly play a protective role. The murine DM1 cell-culture model was used to investigate the effects of expanded CUG repeat expression on splicing within the transcriptome. The differential effect between 5 and 250 repeat RNA expression using Affymetrix whole transcript and exon arrays was compared. Using whole genome arrays, 6 genes were down-regulated and 128 upregulated. With exon arrays, 58 genes showed alternative exon usage. Six genes were selected for further bioinformatics analysis: MtmR4, which has possible neuromuscular involvement; Kcnk4, Narg1, Ttyh1 and Bptf, potentially related to brain development; and Cacna1c, a promising candidate for heart conductance defects and sudden death

Lipoic acid protein ligases in Plasmodium spp.

Protozoan parasites of the genus Plasmodium are the causative agent of malaria. The four human pathogenic species infect more than 500 million people each year, causing the death of at least 1 million people. The most severe form of human malaria is caused by P. falciparum, which is responsible for 90% of the malaria deaths. A major problem in the treatment of this disease is resistance of the parasites against most of the existing chemotherapies. Therefore, there is an urgent need to identify, validate and assess potential new drug targets. The prerequisite of a potential drug target is that it should not be of significance for the human host or it should be sufficiently different from the human counterpart, so that parasite-specific inhibition is feasible. Lipoic acid metabolism in Plasmodium differs from that of mammals in some ways and therefore it might be a promising target for the development of new antimalarials. This study investigated the importance of lipoic acid ligation in P. falciparum using reverse genetic approaches, to assess whether this pathway has potential for drug design. In addition, a spectrophotometric assay system was developed that allowed the biochemical characterisation of lipoic acid ligases and can be adapted to high-throughput screening approaches of inhibitors for these enzymes. Lipoic acid, also known as 6,8-thioctic acid, is an essential cofactor of alpha-keto acid dehydrogenase complexes (KADH) and the glycine cleavage system (GCV). The KADH include the pyruvate dehydrogenase (PDH), branched chain alpha-keto acid dehydrogenase (BCDH) and alpha-ketoglutarate dehydrogenase (KGDH), which are an integral part for any cell's metabolism. In Plasmodium spp. the lipoic acid dependent enzyme complexes are found in the apicoplast, a plastid related organelle, and in the mitochondrion and thus two organelle specific lipoylation pathways are present in these parasites. Biosynthesis of the cofactor occurs in the apicoplast. Octanoyl-[acyl carrier protein]: protein N-octanoyltransferase (LipB) catalyses the attachment of octanoyl-acyl carrier protein (octanoyl-ACP) to the PDH and lipoic acid synthase (LipA) then catalyses the insertion of two sulfurs into the octanoyl-chain to form lipoamide. In the mitochondrion, scavenged lipoic acid is ligated to the enzyme complexes by the action of lipoic acid protein ligase A (LplA1), in an ATP-dependent reaction. However, a second lipoate protein ligase A (LplA2) was identified in the genome of P. falciparum, but its subcellular localisation could not be predicted using the available prediction programs. To further analyse its localisation, parasites were generated expressing full length LplA2 in frame with green fluorescent protein (GFP). In addition, immunofluorescence analyses on wild-type parasites using LplA2 specific antibodies were performed. These studies showed that LplA2 is dually targeted to the apicoplast as well as to the mitochondrion, raising the question about potential redundancy between the ligases present in the parasites. To further analyse this possibility, knock-out studies of lplA1 and lplA2 were performed in the human and rodent malaria parasites P. falciparum and P. berghei, respectively. Knock-out studies showed that LplA1 and LplA2 are non-redundant and strongly suggested that LplA1 is crucial for intraerythrocytic development, whereas LplA2 is essential for sexual development in the mosquito. According to these results it appears that (1) a key regulator of lipoic acid metabolism in Plasmodium spp. is stage specific expression of the relevant proteins and (2) both ligases are potential drug targets as knock-out of lplA1 appeared impossible in the blood stages and knock-out of lplA2 resulted in the interruption of parasite sexual development in the mosquito, and thus transmission of the parasites would be blocked if LplA2 was inhibited. To further analyse the biochemical properties of P. falciparum LplA1 and LplA2, a spectrophotometric assay system was developed, which is also suitable for the development of a high-throughput assay system. The spectrophotometric assay monitors the first part of the LplA reaction - the activation of lipoic acid by ATP. The released pyrophosphate is converted to phosphate which is detected by acidic ammonium molybdate. Using the Escherichia coli LplA protein as a positive control, kinetic parameters for the bacterial protein were determined that are in reasonable agreement with the published data. The results validate the assay and suggest that it might be suitable for inhibitor screening in the future

Towards functional assignment of Plasmodium membrane transport proteins: an experimental genetics study on four diverse proteins

Membrane transport proteins (MTPs) transfer nutrients, metabolic products and inorganic ions across membranes. Many MTPs are essential for Plasmodium blood infection and gain importance as candidate drug targets in malaria therapy, whereas the physiological functions of many MTPs still remain enigmatic. In this thesis, we applied experimental genetics to determine key characteristics of four selected Plasmodium MTPs, including spatio-temporal expression and phenotypical analysis of knockout mutants. We employed the murine malaria model parasite Plasmodium berghei and in vitro blood cultures of the human malaria parasite Plasmodium falciparum. For this study, we selected one conserved MTP called FT2, which was previously shown to transport folate, a P-type ATPase that is specific for P. falciparum as well as two essential MTPs, CRT and ATP4, with important roles in anti-malarial therapy. These targets exemplify the range of druggable candidates and illustrate the potential and limitations of reverse genetics to decipher their physiological roles for Plasmodium life cycle progression. A combination of transgenic and knockout strategies was applied to the P. berghei folate transporter 2 (FT2). We show that endogenously tagged FT2 localises to the apicoplast membranes, and is broadly expressed throughout the parasite’s life cycle. Strikingly, in two stages, schizonts and sporozoites, FT2 displays a dual localisation. Analysis of FT2-deficient parasites revealed a severe sporulation defect in the vector; the vast majority of ft2– oocysts form large intracellular vesicles which displace the cytoplasm. Accordingly, very few sporozoites are generated and these are non-infectious to the mammalian host, resulting in a complete arrest of Plasmodium transmission. A candidate aminophospholipid P-type ATPase, that is encoded by human malaria parasites, but not P. berghei and related Vinckeia parasites, was assessed by a CRISPR/Cas9-mediated gene disruption. Compared to many vital P-type ATPases this gene is dispensable for asexual blood replication. Two MTPs, ATP4 and CRT are prime targets for antimalarial therapies. A comprehensive spatio-temporal expression analysis of transgenic parasites expressing mCherry tagged proteins revealed expression beyond blood infection, indicative of functions in additional parasite stages. Together, the findings of this study contribute towards a better understanding of the roles of the four MTPs based on localisation, expression and functional deletion

Molecular Characterization and Expression of Two New Members of the SLC10 Transporter Family: SLC10A4 and SLC10A5

Over 10 years the solute carrier family SLC10 comprised two well established sodium-dependent bile acid transporters, i.e. the Na+/taurocholate cotransporting polypeptide NTCP (SLC10A1) and the apical sodium-dependent bile acid transporter ASBT (SLC10A2). These carriers are essentially involved in the maintenance of the enterohepatic circulation of bile acids mediating the first step of active bile acid transport through the membrane barriers in the liver (NTCP) and the intestine (ASBT). Recently, two further members of this transporter family were identified in our group; they are referred to as sodium-dependent organic anion transporter SOAT (SLC10A6) and SLC10A7. While SOAT transports sulfoconjugated steroid hormones and sulfoconjugated bile acids, SLC10A7 remains an orphan carrier with yet unknown substrates. In the present study, two novel members were discovered and referred to as Slc10a4 and SLC10A5 (GenBank accession nos. AY825924, AY825925, and DQ074435). This work describes molecular cloning, expression and molecular analysis as well as functional testing for these novel putative membrane transporters. Sequence lengths of the SLC10A4 and SLC10A5 proteins are between 434-438 amino acids in man, rat, and mouse which is much longer compared with other members this carrier family (i.e. 340-377 amino acids). However, sequence identity/similarity of SLC10A4 is quite high to NTCP, ASBT, and SOAT, being 29%/54%. In contrast, identity/similarity values of SLC10A5 are highest to SLC10A3 which is an orphan and hitherto uncharacterized member of the SLC10 family. Slc10a4 exhibits a seven transmembrane domain topology with Nexo/Ccyt trans-orientation of the N- and C-terminal ends, whereas SLC10A5/Slc10a5 shows nine putative TMDs. Based on real time quantitative PCR and northern blot analyses SLC10A4 expression is predominant in the brain, whereas SLC10A5 is highest expressed in the liver and kidney. A polyclonal rabbit antibody was generated against the rat Slc10a4 protein and used for western blot and immunohistochemical analyses. Slc10a4-immunoreactivity was detected in cholinergic regions throughout the rat central nervous system. Co-localization studies with the cholinergic markers VAChT, ChAT, and CHT1 confirmed the presence of Slc10a4 in cholinergic neurons. Furthermore, the Slc10a4 protein is expressed in two dopaminergic regions: the substantia nigra and ventral tegmental area. Here, this protein was co-localized with ChAT and the catecholaminergic marker tyrosine hydroxylase. Slc10a5 expression was localized by in situ hybridization to hepatocytes and renal proximal tubules in rat liver and kidney sections, respectively. For the functional characterization of Slc10a4 and SLC10A5/Slc10a5, these proteins were expressed in X. laevis oocytes and HEK293 cells. Thereby, the C-terminal end of the proteins was tagged by the FLAG epitope and plasma membrane expression was confirmed by immunofluorescence microscopy. However, up to now transport studies failed to show transport activity of Slc10a4 and SLC10A5/Slc10a5 for bile acids and steroid sulfates, which are the typical substrates for NTCP, ASBT, and SOAT. Regarding SLC10A5/Slc10a5, because similar expression patterns to NTCP and ASBT and as these bile acid carriers are the most related carriers to SLC10A5 though, it's strongly supposed that SLC10A5 also has carrier function. Regarding Slc10a4, this carrier did not transport choline neither, which is a substrate of CHT1. Thus, in this study, the functional properties of Slc10a4 could not be completely elucidated, but Slc10a4 is assumed to be a new marker protein for cholinergic neurons. This unexpected finding for a novel member of the sodium/bile acid cotransporter family SLC10 sets Slc10a4 apart and needs further clarification