Biochemistry and Physiological Impact of Selected Transport Proteins
Our lab is highly interested in the biochemical properties and cellular functions of membrane located transport proteins. Since the foundation of our laboratory, we identified several so far hidden transporters on the molecular level, studied their biochemical properties in recombinant systems or after reconstitution in artificial vesicles, and revealed their cellular impact on corresponding mutants.
Nucleoside and nucleotide transporters involved in energy provision and cell signaling
Nucleosides and nucleotides represent essential primary metabolites in all living cells and cross bio-membranes via various types of transporters.
In chloroplasts, we discovered several nucleotide transporter (NTT) isoforms able to import ATP from the cytosol in counter exchange to stromal ADP + Pi (Winkler and Neuhaus, 1999). It is highly likely, that vascular plants received first NTT proteins via horizontal gene transfer from bacteria during early evolution (Haferkamp et al., 2004). In the plasma membrane from higher plants an ATP exporter (PM-ANT1) exports ATP for signaling processes required for proper pollen development (Rieder and Neuhaus, 2011), while a ATP/ADP exchanger located to the Endoplasmic Reticulum is surprisingly involved in homeostasis of photorespiration (Hoffmann et al., 2013).
Equilibrative nucleoside transporters (ENT) are present in a wide range of eukaryotes mediating exchange of nucleosides. In Arabidopsis, ENTs locate to the plasma-membrane and the tonoplast, supplying the cytosol with nucleotide precursors for recycling (Traub et al., 2007). Proton-dependent nucleoside transport by plant ENTs were identified after heterologous expression in bakers-yeast or Xenopus oocytes. Besides supplying the cytosol with nucleosides ENT3 seems to be involved in a signaling function of extracellular nucleosides or nucleotides (Daumann et al., 2015).
The plastidic nucleobase transporter PLUTO is a member of the NCS1 (nucleobase-cation symporter 1) protein family and mediates the proton driven nucleobase (predominantly uracil) import into plastids for recycling (salvage) or catabolism. By this, the cytosolic pyrimidine metabolism is linked to that of the plastid (Witz et al., 2012). Homology modeling and mutational analysis allowed to unravel the structure-function relationship of PLUTO mediated nucleobase transport (Witz et al., 2014).
Transport across the vacuolar membrane
The vacuole can occupy up to 90% of the cell volume rendering this compartment the prime storage location for a wide number of metabolites. We identified the sugar transporter SWEET17 as a highly specific fructose transporter determining leaf sugar composition (Chardon et al., 2013). In addition we were able to identify the vacuolar malate exchanger tDT, which is critical for cellular malate accumulation and exhibited, after expression in Xenopus oocytes and reconstitution in artificial vesicles, an unexpected malate/citrate counter exchange (Emmerlich et al., 2003).
Figure: Transport processes across cellular membranes. Over the past years several nucleoside and nucleotide transporters, involved in energy provision and cellular signaling, were identified in the plastid (NTT and PLUTO), the plasma membrane (ENT) and the endoplasmic reticulum (ER-ANT1). In the tonoplast, a fructose carrier (SWEET17) and a malate/citrate antiporter (tDT) could be described. Created with BioRender.com.
Chardon F, Bedu M, Calenge F, Klemens PA, Spinner L, Clement G, Chietera G, Leran S, Ferrand M, Lacombe B, Loudet O, Dinant S, Bellini C, Neuhaus HE, Daniel-Vedele F, Krapp A (2013) Leaf fructose content is controlled by the vacuolar transporter SWEET17 in Arabidopsis. Curr.Biol. 23, 697-702 doi: 10.1016/j.cub.2013.03.021
Daumann M, Fischer M, Niopek-Witz S, Girke C, Möhlmann T (2015) Apoplastic nucleoside accumulation in Arabidopsis leads to reduced photosynthetic performance and increased susceptibility against Botrytis cinerea. Front Plant Sci. 6, 1158 doi: 10.3389/fpls.2015.01158
Emmerlich V, Linka N, Reinhold T, Hurth MA, Traub M, Martinoia E, Neuhaus HE (2003) The plant homolog to the human sodium/dicarboxylic cotransporter is the vacuolar malate carrier. Proc.Natl.Acad.Sci.USA. 100, 11122-11126 doi: 10.1073/pnas.1832002100
Haferkamp I, Schmitz-Esser S, Linka N, Urbany C, Collingro A, Wagner M, Horn M, Neuhaus HE (2004) A candidate NAD+ transporter in an intracellular bacterial symbiont related to Chlamydiae. Nature 432, 622-625 doi: 10.1038/nature03131
Hoffmann C, Plocharski B, Haferkamp I, Leroch M, Ewald R, Bauwe H, Riemer J, Herrmann JM, Neuhaus HE (2013) From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter Endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration. Plant Cell 25, 2647-2660 doi: 10.1105/tpc.113.113605
Rieder B, Neuhaus HE (2011) Identification of an Arabidopsis plasma membrane located ATP transporter important for anther development. Plant Cell 23, 1932-1944 doi: 10.1105/tpc.111.084574
Traub M, Flörchinger M, Piecuch J, Kunz HH, Weise‐Steinmetz A, Deitmer JW, Möhlmann, T (2007). The fluorouridine insensitive 1 (fur1) mutant is defective in equilibrative nucleoside transporter 3 (ENT3), and thus represents an important pyrimidine nucleoside uptake system in Arabidopsis thaliana. Plant J. 49, 855-864 doi: 10.1111/j.1365-313X.2006.02998.x
Winkler HH, Neuhaus HE (1999) Non-mitochondrial ATP transport. Trends Biochem.Sci. 24, 64-68 doi: 10.1016/S0968-0004(98)01334-6
Witz S, Jung B, Fürst S, Möhlmann T (2012) De novo pyrimidine nucleotide synthesis mainly occurs outside of plastids, but a previously undiscovered nucleobase importer provides substrates for the essential salvage pathway in Arabidopsis. Plant Cell 24, 1549-1559 doi: 10.1105/tpc.112.096743
Witz S, Panwar P, Schober M, Deppe J, Pasha FA, Lemieux MJ, Möhlmann T (2014). Structure-function relationship of a plant NCS1 member–homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis. PLoS One 9, e91343. doi: 10.1371/journal.pone.0091343