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Diallinas G, Martzoukou O. 201. Transporter membrane traffic and function: lessons from a mould. FEBS J. 2019 Dec;286(24):4861-4875. doi: 10.1111/febs.15078. Epub 2019 Oct 17. Review. PubMed PMID: 31583839
Abstract: Transporters are essential transmembrane proteins that mediate the selective translocation of solutes, ions or drugs across biological membranes. Their function is related to cell nutrition, communication, stress resistance and homeostasis. Consequently, their malfunction is associated with genetic or metabolic diseases and drug sensitivity or resistance. A distinctive characteristic of transporters is their cotranslational translocation and folding in a membrane bilayer, this being the endoplasmic reticulum (ER) in eukaryotes or the cell membrane in prokaryotes. In the former case, transporters exit the ER packed in secretory vesicles and traffic via seemingly unconventional, rather than Golgi-dependent, sorting routes to their final destination, the plasma membrane (PM). Proper folding is a prerequisite for ER exit and further trafficking. Misfolded transporters, either due to mutations, high temperature of chemical agents (e.g. DMSO, DTT) are blocked in the ER. The accumulation of ER-retained transporters, in most cases, elicits endoplasmic reticulum-associated degradation, but also ubiquitination-dependent, chaperone-mediated, selective autophagy. The function of PM transporters is finely regulated at the cellular level, in response to physiological or stress signals that promote, via α-arrestin-assisted ubiquitination, their endocytosis and vacuolar/lysosomal degradation, and in some cases recycling to the PM. Importantly, transporter oligomerization and specific interactions with membrane lipids are emerging as important players in transporter expression, function and turnover. This review discusses how paradigmatic work on transporters of a model mould, Aspergillus nidulans, has contributed to novel findings related to transporter functioning in eukaryotes.

Dimou S, Kourkoulou A, Amillis S, Percudani R, Diallinas G. 2019 The peroxisomal SspA protein is redundant for purine utilization but essential for peroxisome localization in septal pores in Aspergillus nidulans. Fungal Genet Biol. 2019 Nov;132:103259. doi: 10.1016/j.fgb.2019.103259. Epub 2019 Aug 5. PubMed PMID: 31394175.

Abstract: In an in silico search for correlated gene loss with fungal peroxisomal uric acid oxidase (UOX), we identified PMP22-like proteins, some of which function as promiscuous channels in organellar membranes. To investigate whether PMP22 channels have a role in peroxisomal uric acid transport and catabolism, we functionally analyzed the closest homologue in Aspergillus nidulans, named SspA. We confirmed that SspA is a peroxisomal membrane protein that co-localizes significantly with PTS1-tagged mRFP, UOX or HexA, the latter considered a protein of Woronin bodies (WB), organelles originating from peroxisomes that dynamically plug septal pores in ascomycetes. Our results suggest that in A. nidulans, unlike some other ascomycetes, there is no strict protein segregation of peroxisomal and WB-specific proteins. Importantly, genetic deletion of sspA, but not of hexA, led to lack of peroxisomal localization at septal pores, suggesting that SspA is a key factor for septal pore functioning. Additionally, ΔsspA resulted in increased sensitivity to oxidative stress, apparently as a consequence of not only the inability to plug septal pores, but also a recorded reduction in peroxisome biogenesis. However, deleting sspA had no effect on uric acid or purine utilization, as we hypothesized, a result also in line with the observation that expression of SspA was not affected by regulatory mutants and conditions known to control purine catabolic enzymes. Our results are discussed within the framework of previous studies of SspA homologues in other fungi, as well as, the observed gene losses of PMP22 and peroxisomal uric acid oxidase.

Kourkoulou A, Grevias P, Lambrinidis G, Pyle E, Dionysopoulou M, Politis A, Mikros E, Byrne B, Diallinas G. 2019 Specific Residues in a Purine Transporter Are Critical for Dimerization, ER Exit, and Function. Genetics. 2019 Dec;213(4):1357-1372. doi: 10.1534/genetics.119.302566. Epub 2019 Oct 14. PubMed  PMID: 31611232; PubMed Central PMCID: PMC6893392.

Abstract: Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Recently, we have shown that specific interactions with plasma membrane phospholipids are essential for the formation and/or stability of functional dimers of the purine transporter UapA, a prototypic eukaryotic member of the ubiquitous nucleobase ascorbate transporter (NAT) family. Here, we provide strong evidence 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. Through genetic screens, we identify mutations that restore defects in dimer formation and/or trafficking. Suppressors of defective dimerization restore ab initio formation of UapA dimers in the ER. Most of these suppressors are located in the movable core domain, but also in the core-dimerization interface and in residues of the dimerization domain exposed to lipids. Molecular dynamics suggest that the majority of suppressors stabilize interhelical interactions in the core domain and thus assist the formation of functional UapA dimers. Among suppressors restoring dimerization, a specific mutation, T401P, was also isolated independently as a suppressor restoring trafficking, suggesting that stabilization of the core domain restores function by sustaining structural defects caused by the abolishment of essential interactions with specific lipids. Importantly, the introduction of mutations topologically equivalent to T401P into a rat homolog of UapA, namely rSNBT1, permitted the functional expression of a mammalian NAT in Aspergillus nidulans. Thus, our results provide a potential route for the functional expression and manipulation of mammalian transporters in the model Aspergillus system.

Ch. Michelaki, N.M. Fyllas, A. Galanidis, M. Aloupi, E. Evangelou, M. Arianoutsou, P.G. Dimitrakopoulos. 2019. An integrated phenotypic trait-network in thermo-Mediterranean vegetation describing alternative, coexisting resource-use strategies Science of the Total Environment 672, 583-592, /doi.org/10.1016/j.scitotenv.2019.04.030

Abstract: Vascular plants have been found to align along globally-recognised resource-allocation trade-offs among specific functional traits. Genetic constrains and environmental pressures limit the spectrum of viable resource-use strategies employed by plant species. While conspecific plants have often been described as identical, intraspecific variation facilitates species coexistence and evolutionary potential. This study attempts to link an individual's phenotype to its environmental tolerance and ecosystem function. We hypothesised that: (1) seasonal variation in water availability has selected for tight phenotypic integration patterns that shape Mediterranean vegetation; however, (2) coexisting species employ alternative resource-use strategies to avoid competitive exclusion; specifically (3) species with smaller climatic niches (i.e. potential distributions) display higher functional diversity. We examined the interdependence among and the sources of variation within 11 functional traits, reflecting whole-plant economics (e.g. construction costs, hydraulics, defenses, water storage capacity), from nine dominant, thermo-Mediterranean species measured across a wide environmental and geographic gradient. Furthermore, we delineated the phenotypic and climatic hypervolumes of each studied species to test for climatic niche overlap and functional distinctiveness. By adopting this multidimensional trait-based approach we detected fundamental phenotypic integration patterns that define thermo-Mediterranean species regardless of life history strategy. The studied traits emerged intercorrelated shaping a resource-allocation spectrum. Significant intraspecific variability in most measured traits allowed for functional distinctiveness among the measured species. Higher functional diversity was observed in species restricted within narrower climatic niches. Our results support our initial hypotheses. The studied functional traits collectively formed an integrated space of viable phenotypic expressions; however, phenotypic plasticity enables functionally distinctive species to succeed complementary in a given set of environmental conditions. Functional variability among coexisting individuals defined species' climatic niches within the trait-spectrum permitted by Mediterranean conditions. Ultimately, a species establishment in a locality depends on the extent that it can shift its trait values.

Mikros E, Diallinas G. 2019 Tales of tails in transporters. Open Biol. 2019 Jun 28;9(6):190083. doi: 10.1098/rsob.190083. Epub 2019 Jun 19. PubMed PMID: 31213137; PubMed Central PMCID: PMC6597760.

Abstract: Cell nutrition, detoxification, signalling, homeostasis and response to drugs, processes related to cell growth, differentiation and survival are all mediated by plasma membrane (PM) proteins called transporters. Despite their distinct fine structures, mechanism of function, energetic requirements, kinetics and substrate specificities, all transporters are characterized by a main hydrophobic body embedded in the PM as a series of tightly packed, often intertwined, α-helices that traverse the lipid bilayer in a zigzag mode, connected with intracellular or extracellular loops and hydrophilic N- and C-termini. Whereas longstanding genetic, biochemical and biophysical evidence suggests that specific transmembrane segments, and also their connecting loops, are responsible for substrate recognition and transport dynamics, emerging evidence also reveals the functional importance of transporter N- and C-termini, in respect to transport catalysis, substrate specificity, subcellular expression, stability and signalling. This review highlights selected prototypic examples of transporters in which their termini play important roles in their functioning.

Papadaki GF, Lambrinidis G, Zamanos A, Mikros E, Diallinas G. 209 Cytosolic N- and C-Termini of the Aspergillus nidulans FurE Transporter Contain Distinct Elements that Regulate by Long-Range Effects Function and Specificity. J Mol Biol. 2019 Sep 6;431(19):3827-3844. doi: 10.1016/j.jmb.2019.07.013. Epub 2019 Jul 12. PubMed PMID: 31306663.

Abstract: FurE, a member of the NCS1 family, is an Aspergillus nidulans transporter specific for uracil, allantoin and uric acid. Recently, we showed that C- or N-terminally truncated FurE versions are blocked for endocytosis and surprisingly show modified substrate specificities. Bifluorescence complementation assays and genetic analyses supported the idea that C- and N-termini interact dynamically and through this interaction regulate selective substrate translocation. Here we functionally dissect and define distinct motifs crucial for endocytosis, transport activity, substrate specificity and folding, in both cytosolic termini of FurE. Subsequently, we obtain novel genetic and in silico evidence indicating that the molecular dynamics of specific N- and C-terminal regions exert long-range effects on the gating mechanism responsible for substrate selection, via pH-dependent interactions with other internal cytosolic loops and membrane lipids. Our work shows that expanded cytoplasmic termini, acquired through evolution mostly in eukaryotic transporters, provide novel specific functional roles.

J. Peyton, A.F. Martinou, O. L. Pescott, M. Demetriou, T. Adriaens, M. Arianoutsou et al. 2019. Horizon scanning for invasive alien species with the potential to threaten biodiversity and human health on a Mediterranean island. Biological Invasions, https://doi.org/10.1007/s10530-019-01961-7

Abstract: Invasive alien species (IAS) are one of the major drivers of change that can negatively affect biodiversity, ecosystem functions and services and human health; islands are particularly vulnerable to biological invasions. Horizon scanning can lead to prioritisation of IAS to inform decision-making and action; its scale and scope can vary depending on the need. We focused on IAS likely to arrive, establish and affect biodiversity and human health on the Mediterranean island of Cyprus. The scope of the horizon scanning was the entire island of Cyprus. We used a two-step consensus-building process in which experts reviewed and scored lists of alien species on their likelihood of arrival, establishment and potential to affect biodiversity, ecosystems and/or human health in the next 10 years. We reviewed 225 alien species, considered to be currently absent on Cyprus, across taxa and environments. We agreed upon 100 species that constituted very high, high or medium biodiversity risk, often arriving through multiple pathways of introduction. The remaining 125 species were ranked as low risk. The potential impacts on human health were documented for all 225 species; 82 species were considered to have a potentially negative impact on human health ranging from nuisance to disease transmission. The scope of the horizon scanning was the entire island of Cyprus, but the thematic groups also considered the relevance of the top 100 species to the Sovereign Base Areas of Cyprus, given their differing governance. This horizon scan provides the first systematic exercise to identify invasive alien species of potential concern to biodiversity and ecosystems but also human health within the Mediterranean region. The process and outcomes should provide other islands in the region and beyond with baseline data to improve IAS prioritisation and management.

Pyle E, Kalli AC, Amillis S, Hall Z, Lau AM, Hanyaloglu AC, Diallinas G, Byrne B, Politis A. 2019 Structural Lipids Enable the Formation of Functional Oligomers of the Eukaryotic Purine Symporter UapA. Cell Chem Biol. 2018 Jul 19;25(7):840-848.e4. doi: 10.1016/j.chembiol.2018.03.011. Epub 2018 Apr 19. PubMed PMID: 29681524; PubMed Central PMCID: PMC6058078.

Abstract: The role of membrane lipids in modulating eukaryotic transporter assembly and function remains unclear. We investigated the effect of membrane lipids in the structure and transport activity of the purine transporter UapA from Aspergillus nidulans. We found that UapA exists mainly as a dimer and that two lipid molecules bind per UapA dimer. We identified three phospholipid classes that co-purified with UapA: phosphatidylcholine, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). UapA delipidation caused dissociation of the dimer into monomers. Subsequent addition of PI or PE rescued the UapA dimer and allowed recovery of bound lipids, suggesting a central role of these lipids in stabilizing the dimer. Molecular dynamics simulations predicted a lipid binding site near the UapA dimer interface. Mutational analyses established that lipid binding at this site is essential for formation of functional UapA dimers. We propose that structural lipids have a central role in the formation of functional, dimeric UapA.

Sanguinetti M, Iriarte A, Amillis S, Marín M, Musto H, Ramón A. 2019 A pair of non-optimal codons are necessary for the correct biosynthesis of the Aspergillus nidulans urea transporter, UreA. R Soc Open Sci. 6(11): 190773. doi: 10.1098/rsos.190773

Abstract: In both prokaryotic and eukaryotic genomes, synonymous codons are unevenly used. Such differential usage of optimal or non-optimal codons has been suggested to play a role in the control of translation initiation and elongation, as well as at the level of transcription and mRNA stability. In the case of membrane proteins, codon usage has been proposed to assist in the establishment of a pause necessary for the correct targeting of the nascent chains to the translocon. By using as a model UreA, the Aspergillus nidulans urea transporter, we revealed that a pair of non-optimal codons encoding amino acids situated at the boundary between the N-terminus and the first transmembrane segment are necessary for proper biogenesis of the protein at 37°C. These codons presumably regulate the translation rate in a previously undescribed fashion, possibly contributing to the correct interaction of ureA-translating ribosome-nascent chain complexes with the signal recognition particle and/or other factors, while the polypeptide has not yet emerged from the ribosomal tunnel. Our results suggest that the presence of the pair of non-optimal codons would not be functionally important in all cellular conditions. Whether this mechanism would affect other proteins remains to be determined.