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 relationships 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 Diallinas 2016, Diallinas & Martzoukou 2019, Mikros & Diallians 2019)
- 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 the last 22 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 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 come from direct structural studies on several transporters, but mostly by 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)
- 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.
- Understand the role of membrane lipids in transporter biogenesis, traffic, function and turnover (see Pyle et al., 2018, Kourkoulou et al., 2019)
- Identify, via a semi-rational approch, based on transporter structure-function relationships, novel antifungal drugs
Membrane trafficking and endocytosis of transporters
- Eukaryotic polytopic membrane proteins 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 biogenesis 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. Interestingly, 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. The mechanisms controlling membrane protein trafficking are essentially conserved from fungi to mammals.
- Studies on the intracellular trafficking of yeast and A. nidulans (from our group) have contributed to knowledge concenring the mechanisms of transporter endocytosis and regulated turnover. The primary contributions of our lab in this direction are:
b) Identification of functional transporter dimerization. UapA was shown, with several 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.
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).
e) 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 (see Figure below) break current dogmas on membrane trafficking suggesting that specific membrane cargoes drive the formation of distinct COPII subpopulations that bypass the Golgi to be sorted non-polarly to the PM, and thus serving house-keeping cell functions (Dimou et al, EMBO R, 2020, in press)
Current aims and specific questions
- Dissect the molecular and mechanistic details of transporter Golgi-independent trafficking
- Identify partners of transporters during their dynamic trafficking
- Genetically manipulate trafficking for enabling the functional expression of mammalain transporters in Aspergillus