Our current research is centered on the regulation of cellular expression, structure, function and evolution of eukaryotic transporters.
For this, we principally use the genetically tractable fungi Aspergillus nidulans and Saccharomyces cerevisiae as our model systems.
Our interests follow three major lines:
a) genetically, biochemically and biophysically dissect structure-function relationships underlying transporter function, specificity and molecular evolution
b) identify the pathways and molecular mechanisms involved in the membrane trafficking, exocytosis, endocytosis and turnover of specific transporters and other membrane cargoes in response to fungal growth and to various physiological or stress signals
c) study the role of transporters in fungal pathogenicity and use in silico modeling of specific purine transporters for rational antifungal drug design.
Structure-function relatiosniops in purine and pyrimidine transporters
- We use classical and reverse genetics, direct biochemical transport assays, in vivo fluorescent microscopy, Molecular Dynamics and crystallography, to understand how specific purine or pyrimidine of eukaryotic transporters work (for reviews see Pantazopoulou and Diallinas, 2007; 2008; Gournas et al, 2008; Diallinas & Gournas 2008, Krypotou et al., 2012; 2014, 2015; Diallinas 2014; 2016; 2017; Sioupouli et al., 2017, see publication list next pages).
- Our favorite molecule is the UapA uric acid-xanthine/H+ symporter, which is the prototype and founding member of an important and ubiquitous transporter family, called Nucleobase Ascorbate Transporters (NAT). Interestingly, mammalian NATs transport either L-ascorbic acid (SVCT1 and SVCT2) or nucleobases (rSNBT17), and thus how NAT specificity has shifted, in the course of evolution, from nucleobases to L-ascorbic acid is a major actual research issue of the lab.
- In the last 18 years, hundreds of UapA mutations, obtained by classical or reverse genetic approaches, as well as chimeric constructs, have been analyzed at the molecular, cellular and functional level, giving rise to unprecedented knowledge of the molecular elements underlying the function of this eukaryotic carrier. Based on our results, we have proposed models on how the UapA recognizes and translocates its substrates, and provided data on which amino residues are involved in substrate selection, binding and transport, and on which amino acid residues are key elements for protein stability and trafficking to the plasma membrane. In fact, it is through our studies that gating has been proposed to exist and be critical in determining UapA transporter specificity. The existence of distinct gating elements or molecular filters is an entirely novel concept that breaks the dogmatic distinction of transporters and channels. Supporting evidence for this idea has recently come from direct structural studies on several transporters, but mostly by the recent determination of the structure of UapA at 3.5 A (collaboration with Dr. B. Byrne and A. Cameron, Imperial College & Warwick University). [Alguel et al, Nat Com, April 2016] The crystal structure of UapA has also confirmed parallel genetic and molecular data that suggested that UapA functions as a dimer and that dimerization is critical for specificity. Functional dimerization is an entirely novel aspect in the field of solute transporters.
Upper panel: Crystal structure of UapA dimer. Middle panel: Functional analysis of UapA mutants supporting functional dimerization.
Lower panel: MD analysis showing the substrate (xanthine) translocation pathway and supporting the functional role of dimerization
(for details see Alguel et al., 2016)
- At present, we try to understand in more detail how the binding and release of substrates leads to the opening and closing of the substrate translocation trajectory, how the gates synergize with the major substrate binding site, how our findings are related to the generally accepted model of outward- and inward-facing alternating transporter conformers, and finally, how ions drive solute symport.
- Our current efforts include more sophisticated Molecular Dynamics (MD) and determination of X-ray structures of several distinct conformations of UapA. For these approaches, we actively collaborate with the groups of Emmanuel Mikros (University of Athens, Pharmacy Department) and Bernadette Byrne (Imperial College, London, UK). In particular, with the group at Imperial College, we are implementing a high-throughput platform for expression, screening and purification of wild-type or stable mutant forms of UapA and other NAT homologues.
- Similar approaches of those used to study UapA, are currently followed to study other transporters (AzgA and NCS1 transporter families; see Krypotou et al., 2012; 2014, 2015;Sioupouli et al., 2017, Papadaki et al, in press).
- We finally also ask whether we can identify, via a semi-rational approch, based on transporter structure-function relationships, novel antifungal drugs [see Lougiakis et al., 2016].
Membrane trafficking and endocytosis of transporters
- Eukaryotic polytopic membrane proteins, such as transporters and channels, or receptors, are co-translationally inserted into the ER membrane through the action of the so-called translocase complex. Once a membrane protein is properly folded within the ER membrane, an important check-point by itself, it then follows a vesicular or tubular trafficking pathway, initially towards the Golgi, and subsequently towards the endosomal pathway, the vacuole or the plasma membrane. The sum of complex processes underlying membrane protein exocytosis, but also endocytosis in response to specific signals, is called membrane protein or cargo trafficking. In this process, both cis-acting elements on the cargo proteins and trans-acting factors need to be orchestrated in a sequential and flexible manner to achieve proper trafficking. Addtitionally, the lipid composition of membranes also plays a pivotal role in protein trafficking. The dynamic control of the trafficking of membrane proteins constitutes an essential mechanism for cell homeostasis and for the communication of cells with their environment.
- Strikingly, the mechanisms controlling transporter trafficking are essentially conserved from fungi to mammals. In fact, studies on the intracellular trafficking of yeast permeases have contributed to revealing the central role played by the small protein ubiquitin (Ub), a sorting signal of eukaryotic membrane proteins. In yeasts and A. nidulans the covalent attachment of Ub on cargoes depends on Rsp5/HulA, a Ub ligase of the Nedd4 HECT family. Recent studies suggested a general model in which different Rsp5/HulA adaptor proteins recognize different transporters, or the same transporter in response to different stimuli. Finally, lipid rafts, formed by the lateral association of sphingolipids and cholesterol (mammals) or ergosterol (fungi) in the external membrane leaflet have been implicated in transporter traffic and cell signaling in mammalian cells and yeast.
- Unique genetic and molecular tools for specific sub-cellular organelles/compartments have been developed for A. nidulans and several transporters of purines, pyrimidines and amino acids, belonging to evolutionary discrete families, have been used as protein cargoes to study endocytosis in response to a shift in nitrogen source or excess substrate (our lab).As a consequence, important aspects of trafficking mechanisms have been revealed in this model fungus.
- The primary contributions of our lab in this direction are:
b) Identification of functional transporter dimerization. UapA was shown, with four different assays, to dimerize in vivo and that dimerization is critical for ER-exit, membrane traffic, turnover and transport activity (Martzoukou et al., 2015; Alguel et al, 2016a; 2016b). Subsequent crystallization of UapA confirmed the role of UapA functional dimeization (see above)
c) Identification of multiple mechanisms undelying turnover of misfolded transporters. Partially misfolded UapA versions trapped in the ER are down-regulated by ERAD and endocytosis, but also via selective autophagy. A major factor in the latter process is an ER transmembrane adaptor,
called BsdA, recruiting HulA ubiqutin ligase and promoting autophagy (Evangelinos et al., 2016).
d) Identification that the AP-2 adaptor complex, which in mammals is a major partner of clathrin-mediated endocytosis, has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi. The role of AP-2 in the maintenance of proper apical membrane lipid and cell wall composition was supported by its functional interaction with sphingolipid biosynthesis, apical sterol-rich membrane domains and its essentiality in polar deposition of chitin. These findings supported that the AP-2 complex of fungi has acquired, in the course of evolution, a specialized clathrin-independent function necessary for polar growth Martzoukou et al., 2016). The pharmacological importance of these findings in antifungal research are apparent.
Role of the AP-2 adaptor complex in specialized clathrin-independent apical endocytosis and polar growth in fungi.
Current aims and specific question
- What is thew role of the AP-1 and AP-3 adaptor complexes in membrane traffic, sorting and turnover of protein cargoes and how this is related to fungal growth and pathogenicity?
- What is the role of lipid composition in transporter trafficking, endocytosis, degradation or function?
- Most of our initial studies employ as a model cargo the uric acid transporter UapA.Subsequent studies will investigate the trafficking of several other purine, pyrimidine and nucleoside transporters (UapC, FurD, AzgA, FcyB, CntA) readily available in our lab.