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Current Research

Research

Our 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  as our model system.Our interests follow two major lines:

a) genetically, biochemically and biophysically dissect structure-function relationships underlying transporter mechanism of transport, and address how specificity is determined or transporter function evolves at the molecular level

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

In addition, we develop serval other parallel projects via collaborations (see Current research)

Structure-function relationships in nutrient transporters

  • We  use classical and reverse genetics, direct biochemical transport assays, in vivo fluorescent microscopy, Molecular Dynamics, crystallography, Hydrogen-Deuterium Exchange (HDX) and recently cryo-EM,  to understand how eukaryotic  transporters fold and how they work.
  • We study many transporters, but 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).
  • In 2016 we published  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, one of the first eukaryotic transporters structures determined, has 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.
  • In 2025 we published (preprint, Under revision in February 2025) very high-resolution new structures of UapA (2.05-3.5 Å) via cryo-EM through a collaboration with C. Gatsogiannis lab in Munster. The structures reveal in an unprecedented level of detail the role of water molecules and lipids in substrate binding, specificity, dimerization, and activity, rationalizing accumulated functional data. Combined with mutational and functional studies and MD, our work points out how N-tail interactions couple proper subcellular trafficking and transport activity by wrapping UapA in a conformation necessary for ER-exit but also critical for elevator-type conformational changes associated with substrate translocation once UapA has integrated into the plasma membrane. Our study also provides detailed insights into important aspects of the elevator-type transport mechanism and opens novel issues on how the evolution of extended cytosolic tails in eukaryotic transporters, apparently needed for subcellular trafficking, might have been integrated into the transport mechanism. [High-resolution structures of the UapA purine transporter reveal unprecedented aspects of the elevator-type transport mechanism. [Broutzakis G, et al., bioRxiv 2024 Aug 24:2024.08.23.609436. doi: 10.1101/2024.08.23.609436.]

Membrane trafficking and endocytosis of transporters
 
The sum of complex processes underlying membrane protein biogenesis  is called membrane protein or cargo trafficking. The mechanism controlling membrane protein trafficking are essentially conserved from fungi to mammals, and in the current consensus trafficking is centrally via Golgi maturations, post-Golgi vesicular secretion and controlled endocytosis and protein turnover.The primary contributions of our lab in this direction are:

  • Identification of two distinct mechanisms controlling transporter down-regulation by endocytic internalization. The first occurs in response to a shift from poor to rich nitrogen media (ammonium ions) and the second in response to substrate excess (Pantazopoulou et al. 2007; Gournas et al. 2010, Karachaliou et al., 2013). Interestingly, substrate-induced endocytosis, unlike ammonium-induced internalization, takes place only for active transporters. The use of specific functional mutations of the UapA transporter has shown that conformational movements associated with the transport process constitute the primary signal for substrate-induced endocytosis.
  • 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).
  • Identification of multiple mechanisms underlying 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 ubiquitin ligase and promoting autophagy (Evangelinos et al., 2016).
  • Showing that the sorting of neosynthesized transporters to the plasma membrane (PM) bypasses the Golgi and does not necessitate key Rab GTPases, AP adaptors, microtubules or endosomes. Instead, transporter PM localization is found to depend on functional COPII vesicles, actin polymerization, clathrin heavy chain and the PM t-SNARE SsoA. Our findings break current dogmas on membrane trafficking  suggesting that specific membrane cargoes drive the formation of distinct early secretory carrierts that bypass the Golgi to be sorted non-polarly to the PM, and thus serving house-keeping cell functions [Sagia GM, et al., Elife. 2024 Oct 21;13:e103355. doi: 10.7554/eLife.103355.; Dimou S, et al. Front Cell Dev Biol. 2022 Apr 7;10:852028. doi: 10.3389/fcell.2022.852028.; Dimou S, et al. EMBO Rep. 2020 Jul 3;21(7):e49929. doi:10.15252/embr.201949929]

 Current Aims

  • Among our primer goals 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 gating elements synergize with the major substrate binding site, and how ions drive solute symport. For this we use cryo-EM and other biochemical and biophysical approaches to get novel UapA structures and understand UapA transport dynamics. This line also includes an effort to understand the role of membrane lipids in transporter biogenesis, traffic, function and turnover.
  • A second major goal of the lab is to fully dissect the molecular and mechanistic details of transporter Golgi-independent trafficking. For this we try to identify partners of transporters during their dynamic trafficking using rational approaches and unbiased genetic screen, biochemical reconstitution of selected cargoes in proteoliposomes, and use the most advanced microscopic modalities (Lattice Light Sheet Microscopy,  Lattice-SIM2 (Zeiss Elyra 7, Spinning Disc Confocal and Cryo-CLEM tomography), to follow the dynamics and establish their structural details of Golgi-bypassing cargo carriers.
  • We also try to genetically manipulate trafficking for enabling the functional expression of mammalian transporters in Aspergillus
  • Additionally, we are also interested in identifying, via a semi-rational approach, based on transporter structure-function relationships, novel antifungal drugs.
To achieve the aforementioned goals, we collaborate with top experts in biochemical and biophysical approaches, namely Christos Gatsogiannis and Seraphine Wegner (Munster), Bernadete Byrne (Imperial College) and Argyris Politis (University of Manchester) and Emmanuel Mikros (NKUA, Athens).

 

Lastly, we have several ongoing parallel projects concerning:

  • the identification and characterization of mechanosensitive channels in fungi (collaboration with Christos Pliotas at University of Manchester and Mihailo Rabasovic at Belgrade University)
  • the functional evolution of neurotransmitter homologues in fungi (collaboration with Alex Pittis at IMBB, Crete)
  • the mechanisms of macrophage-fungal interactions (collaboration what George Chamillos at IMMB Crete)
  • Functionally characterize carboxylic acid transporters in pathogenic and other yeasts (collaboration with Isabel João Soares Silva and Sandra Paiva, University of Minho).