Joachim Tjaden

Molecular Identification and Physiological Characterization of a Novel Monosaccharide Transporter from Arabidopsis Involved in Vacuolar Sugar Transport

The tonoplast monosaccharide transporter (TMT) family comprises three isoforms in Arabidopsis thaliana, and TMT–green fluorescent protein fusion proteins are targeted to the vacuolar membrane. TMT promoter–β-glucuronidase plants revealed that the TONOPLAST MONOSACCHARIDE TRANSPORTER1 (TMT1) and TMT2 genes exhibit a tissue- and cell type–specific expression pattern, whereas TMT3 is only weakly expressed. TMT1 and TMT2 expression is induced by drought, salt, and cold treatments and by sugar. During cold adaptation, tmt knockout lines accumulated less glucose and fructose compared with wild-type plants, whereas no differences were observed for sucrose. Cold adaptation of wild-type plants substantially promoted glucose uptake into isolated leaf mesophyll vacuoles. Glucose uptake into isolated vacuoles was inhibited by NH4+, fructose, and phlorizin, indicating that transport is energy-dependent and that both glucose and fructose were taken up by the same carrier. Glucose import into vacuoles from two cold-induced tmt1 knockout lines or from triple knockout plants was substantially lower than into corresponding wild-type vacuoles. Monosaccharide feeding into leaf discs revealed the strongest response to sugar in tmt1 knockout lines compared with wild-type plants, suggesting that TMT1 is required for cytosolic glucose homeostasis. Our results indicate that TMT1 is involved in vacuolar monosaccharide transport and plays a major role during stress responses.

Molecular and biochemical analysis of the plastidic ADP-glucose transporter (ZmBT1) from Zea mays

Physiological studies on the Brittle1 maize mutant have provided circumstantial evidence that ZmBT1 (Zea mays Brittle1 protein) is involved in the ADP-Glc transport into maize endosperm plastids, but up to now, no direct ADP-Glc transport mediated by ZmBT1 has ever been shown. The heterologous synthesis of ZmBT1 in Escherichia coli cells leads to the functional integration of ZmBT1 into the bacterial cytoplasmic membrane. ZmBT1 transports ADP-Glc in counterexchange with ADP with apparent affinities of about 850 and 465 μm, respectively. Recently, a complete ferredoxin/thioredoxin system has been identified in cereal amyloplasts and BT1 has been proposed as a potential Trx target protein (Balmer, Y., Vensel, W. H., Cai, N., Manieri, W., Schurmann, P., Hurkman, W. J., and Buchanan, B. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 2988–2993). Interestingly, we revealed that the transport activity of ZmBT1 is reversibly regulated by redox reagents such as diamide and dithiothreitol. The expression of ZmBT1 is restricted to endosperm tissues during starch synthesis, whereas a recently identified BT1 maize homologue, the ZmBT1–2, exhibits a ubiquitous expression pattern in hetero- and autotrophic tissues indicating different physiological roles for both maize BT1 isoforms. BT1 homologues are present in both mono- and dicotyledonous plants. Phylogenetic analyses classify the BT1 family into two phylogenetically and biochemically distinct groups. The first group comprises BT1 orthologues restricted to cereals where they mediate the ADP-Glc transport into cereal endosperm storage plastids during starch synthesis. The second group occurs in mono- and dicotyledonous plants and is most probably involved in the export of adenine nucleotides synthesized inside plastids.

Characterization of the Arabidopsis Brittle1 transport protein and impact of reduced activity on plant metabolism

The Arabidopsis genome contains a gene (Atbt1) encoding a highly hydrophobic membrane protein of the mitochondrial carrier family, with six predicted transmembrane domains, and showing substantial structural similarity to Brittle1 proteins from maize and potato. We demonstrate that AtBT1 transports AMP, ADP and ATP (but not ADP-glucose), shows a unidirectional mode of transport, and locates to the plastidial membrane and not to the ER as previously proposed. Analysis using an Atbt1 promoter-GUS construct revealed substantial gene expression in rapidly growing root tips and maturating or germinating pollen. Survival of homozygous Atbt1::T-DNA mutants is very limited, and those that do survive produce non-fertile seeds. These observations indicate that no other carrier protein or metabolic mechanism can compensate for the loss of this transporter. Atbt1 RNAi dosage mutants show substantially retarded growth, adenylate levels similar to those of wild-type plants, increased glutamine contents and unchanged starch levels. Interestingly, the growth retardation of Atbt1 RNAi mutant plants was circumvented by adenosine feeding, and was accompanied by increased adenylate levels. Further observations showed the presence of a functional nucleotide salvage pathway in Atbt1 RNAi mutants. In summary, our data indicate that AtBT1 is a plastidial nucleotide uniport carrier protein that is strictly required to export newly synthesized adenylates into the cytosol.

Multiple origins of hydrogenosomes : functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp.

A mitochondrial‐type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP – similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial‐type AACs. Hydrogenosome‐bearing anaerobic ciliates possess clearly distinct mitochondrial‐type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial‐type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.

Mitochondria, hydrogenosomes and mitosomes: products of evolutionary tinkering!

One hundred years ago, C. Mereschkowsky, “Privatdozent an der Kaiserlichen Universitat in Kasan (Russia)” published a notoriously ignored landmark paper: “Uber Natur und Ursprung der Chromatophoren im PXanzenreiche.” (“On the nature and origin of the chromatophores in the plant kingdom”; Mereschkowsky 1905). In spite of the fact that this paper was written in German (the lingua franca in biology at the time), its fate was similar to Mendel’s publication, which signiWed, in retrospect, the birth of genetics (Mendel 1865). Both before and after the publication of Mereschkowsky’s article there were many publications dealing with plant “chimera’s” and cytoplasmic inheritance in plants, which should have favoured the interpretation of plastids as “semi-autonomous” symbiotic entities in the cytoplasm of the eukaryotic plant cell (e.g. Braun 1873; Hildebrand 1908; Baur 1909; Renner 1922, 1924, 1934, 1936a, b; Darlington 1929; Stubbe 1959; Tilney-Basset 1963). In addition, millions of people had variegated Pelargonium and other green-and-white spotted plants in their homes, or variegated plants such as Euonymus, Hedera or Ilex aquifolius in their gardens, which could have provided comprehensive evidence for plastid inheritance to the naked eye. Therefore, it is one of the mysteries of the 20th century that an endosymbiotic origin of plastids had not been generally accepted before the 1970s and 1980s, especially after the courageous paper of Lynn Margulis (Sagan 1967) and the unequivocal demonstration of DNA in plastids (Gibor and Izawa 1963). Twenty years after Mereschkowsky’s plea for an endosymbiotic origin of plastids, Wallin (1925, 1927) postulated the “bacterial nature of mitochondria”. The reasons for this postulate were less obvious, since—in contrast to chloroplast mutations—you cannot experience the consequences of mutations in the mitochondrial genome by naked eye—you need at least a good microscope and basic experience in cytochemical staining techniques (reviewed by Ernster and Schatz 1981). Moreover, Wallin’s claim of having cultivated mitochondria in vitro turned out (of course) to be wrong, just as Portier’s fancy speculation that food-associated bacteria could fuse with mitochondria in order to rejuvenate the latter (Portier 1918). Most importantly, however, the genetic evidence for the presence of hereditary factors in mitochondria was diYcult to interpret. The reasons for these peculiarities of mitochondrial genetics are well understood today, but still notoriously ignored in many textbooks. First of all, mitochondrial DNA is usually present in multiple copies in one and the same mitochondrion and, notably, the hundreds to thousands of mitochondria in a single Communicated by R. Bock

Identification of a Novel Adenine Nucleotide Transporter in the Endoplasmic Reticulum of Arabidopsis

Many metabolic reactions in the endoplasmic reticulum (ER) require high levels of energy in the form of ATP, which is important for cell viability. Here, we report on an adenine nucleotide transporter residing in the ER membranes of Arabidopsis thaliana (ER-ANT1). Functional integration of ER-ANT1 in the cytoplasmic membrane of intact Escherichia coli cells reveals a high specificity for an ATP/ADP antiport. Immunodetection in transgenic ER-ANT1-C-MYC-tag Arabidopsis plants and immunogold labeling of wild-type pollen grain tissue using a peptide-specific antiserum reveal the localization of this carrier in ER membranes. Transgenic ER-ANT1-promoter-β-glucuronidase Arabidopsis lines show high expression in ER-active tissues (i.e., pollen, seeds, root tips, apical meristems, or vascular bundles). Two independent ER-ANT1 Arabidopsis knockout lines indicate a high physiological relevance of ER-ANT1 for ATP transport into the plant ER (e.g., disruption of ER-ANT1 results in a drastic retardation of plant growth and impaired root and seed development). In these ER-ANT1 knockout lines, the expression levels of several genes encoding ER proteins that are dependent on a sufficient ATP supply (i.e., BiP [for luminal binding protein] chaperones, calreticulin chaperones, Ca2+-dependent protein kinase, and SEC61) are substantially decreased.

A divergent ADP/ATP carrier in the hydrogenosomes of Trichomonas gallinae argues for an independent origin of these organelles

The evolution of mitochondrial ADP and ATP exchanging proteins (AACs) highlights a key event in the evolution of the eukaryotic cell, as ATP exporting carriers were indispensable in establishing the role of mitochondria as ATP‐generating cellular organelles. Hydrogenosomes, i.e. ATP‐ and hydrogen‐generating organelles of certain anaerobic unicellular eukaryotes, are believed to have evolved from the same ancestral endosymbiont that gave rise to present day mitochondria. Notably, the hydrogenosomes of the parasitic anaerobic flagellate Trichomonas seemed to be deficient in mitochondrial‐type AACs. Instead, HMP 31, a different member of the mitochondrial carrier family (MCF) with a hitherto unknown function, is abundant in the hydrogenosomal membranes of Trichomonas vaginalis. Here we show that the homologous HMP 31 of closely related Trichomonas gallinae specifically transports ADP and ATP with high efficiency, as do genuine mitochondrial AACs. However, phylogenetic analysis and its resistance against bongkrekic acid (BKA, an efficient inhibitor of mitochondrial‐type AACs) identify HMP 31 as a member of the mitochondrial carrier family that is distinct from all mitochondrial and hydrogenosomal AACs studied so far. Thus, our data support the hypothesis that the various hydrogenosomes evolved repeatedly and independently.

Characterization and Developmentally Regulated Localization of the Mitochondrial Carrier Protein Homologue MCP6 from Trypanosoma brucei

ABSTRACT Proteins of the mitochondrial carrier family (MCF) are located mainly in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates. The genome of Trypanosoma brucei harbors 29 genes encoding different MCF proteins. We describe here the characterization of MCP6, a novel T. brucei MCF protein. Sequence comparison and phylogenetic reconstruction revealed that MCP6 is closely related to different mitochondrial ADP/ATP and calcium-dependent solute carriers, including the ATP-Mg/Pi carrier of Homo sapiens. However, MCP6 lacks essential amino acids and sequence motifs conserved in these metabolite transporters, and functional reconstitution and transport assays with E. coli suggested that this protein indeed does not function as an ADP/ATP or ATP-Mg/Pi carrier. The subcellular localization of MCP6 is developmentally regulated: in bloodstream-form trypanosomes, the protein is predominantly glycosomal, whereas in the procyclic form, it is found mainly in the mitochondria. Depletion of MCP6 in procyclic trypanosomes resulted in growth inhibition, an increased cell size, aberrant numbers of nuclei and kinetoplasts, and abnormal kinetoplast morphology, suggesting that depletion of MCP6 inhibits division of the kinetoplast.

Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp.: Hydrogenosomal ADP/ATP carriers

A mitochondrial‐type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP – similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial‐type AACs. Hydrogenosome‐bearing anaerobic ciliates possess clearly distinct mitochondrial‐type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial‐type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.

Multiple origins of hydrogenosomes

A mitochondrial‐type ADP/ATP carrier (AAC) has been identified in the hydrogenosomes of the anaerobic chytridiomycete fungus Neocallimastix sp. L2. Biochemical and immunocytochemical studies revealed that this ADP/ATP carrier is an integral component of hydrogenosomal membranes. Expression of the corresponding cDNA in Escherichia coli confers the ability on the bacterial host to incorporate ADP at significantly higher rates than ATP – similar to isolated mitochondria of yeast and animals. Phylogenetic analysis of this AAC gene (hdgaac) confirmed with high statistical support that the hydrogenosomal ADP/ATP carrier of Neocallimastix sp. L2 belongs to the family of veritable mitochondrial‐type AACs. Hydrogenosome‐bearing anaerobic ciliates possess clearly distinct mitochondrial‐type AACs, whereas the potential hydrogenosomal carrier Hmp31 of the anaerobic flagellate Trichomonas vaginalis and its homologue from Trichomonas gallinae do not belong to this family of proteins. Also, phylogenetic analysis of genes encoding mitochondrial‐type chaperonin 60 proteins (HSP 60) supports the conclusion that the hydrogenosomes of anaerobic chytrids and anaerobic ciliates had independent origins, although both of them arose from mitochondria.