Structure-function relationships in transmembrane transporters

Doctoral Dissertation uoadl:2940718 15 Read counter

Department of Biology
Library of the School of Science
Deposit date:
Kourkoulou Athanasia-Vasiliki
Dissertation committee:
Γεώργιος Διαλλινάς, Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Bernadette Byrne, Καθηγήτρια, Department of Life Sciences, Imperial College
Ευστάθιος Φριλίγγος, Καθηγητής, Τμήμα Ιατρικής, Πανεπιστήμιο Ιωαννίνων
Εμμανουήλ Μικρός, Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Δημήτριος Στραβοπόδης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Κοσμάς Χαραλαμπίδης, Αναπληρωτής Καθηγητής, Τμήμα Βιολογίας, ΕΚΠΑ
Χρήστος Γουρνάς, Ερευνητής Γ, ΕΚΕΦΕ Δημόκριτος
Original Title:
Structure-function relationships in transmembrane transporters
Translated title:
Structure-function relationships in transmembrane transporters
Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Members of the ubiquitous Nucleobase Ascorbate Transporter (NAT) family are H+ or Na+ symporters specific for the cellular uptake of either purines and pyrimidines or L-ascorbic acid [1]. Despite the fact that several members have been extensively characterized at a genetic, biochemical or cellular level, and crystal structures of NAT members from Escherichia coli and Aspergillus nidulans have been determined pointing to a mechanism of transport, the current knowledge cannot explain how substrate selectivity is determined [2,3]. Functionally characterized NATs from bacteria, fungi and plants are specific for nucleobases, but rather surprisingly, mammals and other vertebrates possess NAT homologues that are specific for L-ascorbate transport (SVCT1/2) in addition to nucleobase specific members (i.e. rSNBT1) [4,5]. Vertebrates also include a third distinct paralogue of unknown function called SVCT3 [5].
High-resolution crystal structures from two NAT members have been obtained [2,3,6]. These are the UraA uracil transporter of E. coli and the UapA uric acid-xanthine transporter of A. nidulans. Both proteins are composed of 14 transmembrane segments characterized by a 7 helix inverted repeat (7+7) forming a core and a dimerization domain. Additionally, these proteins exist as dimers, the formation of which is essential for transport activity. UapA is considered the prototypic eukaryotic member of this family as it is one of the most extensively studied eukaryotic transporters in respect to structure-function relationships, substrate specificity, regulation of expression and subcellular trafficking.
All NATs include a highly conserved motif in TMS10 historically referred as the NAT signature motif which includes residues critical for substrate binding and specificity or transport catalysis [1,8]. Previous studies on UapA reported that most specificity substitutions in UapA, selected by direct genetic screens, map outside the major substrate binding site and the NAT signature motif [9,10,13]. The most prominent specificity substitutions concerned residues Arg481, Thr526 or Phe528, which are located along the proposed sliding trajectory of the core domain in the UapA dimer.
Moreover, it has been shown recently that specific interactions with plasma membrane phospholipids at the dimer interface of UapA are essential for the formation and/or stability of functional dimers [12]. More specifically, UapA co-purifies with lipid and delipidation results in dissociation into monomers. Addition of PIs or PEs resulted in the re-formation of the UapA dimer. MDs predicted a specific lipid binding site at the dimer interface that is formed by three arginine residues Arg287, Arg478 and Arg479. Replacement of these arginines by alanine residues led to total loss of UapA function and both native MS and bifluorescence complementation (BiFC) assays indicated that a major fraction of UapA cannot dimerize.
This study is divided in three distinct chapters. In the first one the molecular aspects of NAT substrate specificity and the evolution of ascorbate transporters were investigated by making at first an extensive phylogenetic analysis and then a mutational analysis on the NAT signature motif of UapA. This mutational analysis was coupled with a rational combination of substitutions while new substitutions were also isolated by novel genetic screens. Overall, the results revealed cryptic context-dependent roles of partially conserved residues in the NAT signature motif in determining the specificity of the UapA transporter. Additionally we provided novel findings concerning how Phe528, a residue outside the substrate binding site, might function as a key amino acid in determining UapA specificity.
The second part of the study was focused on the role of lipid interactions in UapA function, stability and trafficking. In particular, the role of UapA-lipid interactions at the dimer interface was further examined and the possible role of other predicted, by MDs, interactions at the membrane facing regions of the core domain of the UapA dimer was identified. We found that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Additionally, through genetic screens, we identified substitutions that restore defects in dimer formation and/or trafficking.
Finally, in the third part of the present study, using knowledge acquired from this work we achieved for the first time the functional expression of a mammalian NAT homologue in A. nidulans.
Main subject category:
UapA, transporters, NAT family, Aspergillus nidulans, genetics, nucleobase, L-ascorbate, lipid interactions, rSNBT1
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