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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 Aims)

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  [Structure of eukaryotic purine/H(+) symporter UapA suggests a role for homodimerization in transport activity.Alguel Y, Amillis S, Leung J, Lambrinidis G, Capaldi S, Scull NJ, Craven G, Iwata S, Armstrong A, Mikros E, Diallinas G, Cameron AD, Byrne B.Nat Commun. 2016 Apr 18;7:11336. doi: 10.1038/ncomms11336.]. 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 2026 we published 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. [Cryo-EM of the eukaryotic purine transporter UapA demonstrates intramolecular and lipid regulation of transport. Broutzakis G, Pyrris Y, Akrani I, Neuhaus A, Mikros E, Diallinas G, Gatsogiannis C.Proc Natl Acad Sci U S A. 2026 Jun 30;123(26):e2513585123. doi: 10.1073/pnas.2513585123].

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 maturation, 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 carriers 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

    • Understanding the molecular mechanisms of substrate and ion transport. One of our primary goals is to understand in greater detail how substrate binding and release drive the opening and closing of the substrate translocation pathway, how gating elements synergize with the major substrate-binding site, and how ions drive solute symport. To address these questions, we combine cryo-EM with complementary biochemical and biophysical approaches to determine novel structures of UapA and elucidate its transport dynamics. This research also encompasses efforts to understand the role of membrane lipids in transporter biogenesis, trafficking, function, and turnover.
    • Dissecting the molecular mechanisms of Golgi-independent transporter trafficking. A second major goal of the lab is to elucidate the molecular and mechanistic basis of transporter trafficking through Golgi-independent pathways. We aim to identify transporter-interacting partners during dynamic trafficking using both rational approaches and unbiased genetic screens. Selected cargoes are further investigated through biochemical reconstitution in proteoliposomes, while advanced imaging modalities—including Lattice Light-Sheet Microscopy, Lattice-SIM2 (Zeiss Elyra 7), spinning-disk confocal microscopy, and cryo-CLEM tomography—are used to monitor trafficking dynamics and define the structural organization of Golgi-bypassing cargo carriers.
    • Engineering trafficking pathways for heterologous transporter expression. We also seek to genetically manipulate cellular trafficking pathways to enable the functional expression of mammalian transporters in Aspergillus.
    • Discovery of novel antifungal compounds. In addition, we are interested in identifying novel antifungal drugs through a semi-rational approach guided by transporter structure–function relationships.

  • To achieve these goals, we collaborate with leading experts in complementary biochemical and biophysical approaches, including Christos Gatsogiannis and Seraphine Wegner (Münster), Bernadette Byrne (Imperial College London), Argyris Politis (University of Manchester), and Emmanuel Mikros (National and Kapodistrian University of Athens, NKUA).


Other ongoing collaborative projects concern:

  • The identification and characterization of mechanosensitive channels in fungi (collaboration with Christos Pliotas at University of Manchester and Mihailo Rabasovic at Belgrade University)
  • 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).