71. "Oligomerization of the UapA purine transporter is critical for ER-exit, plasma membrane localization and turnover."
O. Martzoukou, M. Karachaliou, V. Yalelis, J. Leung, B. Byrne, S. Amillis and G. Diallinas.
Journal of Molecular Biology, Vol. , pages , (2015).  View at publisher's site  Request copy
Abstract: Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated BiFC assay, we provide evidence that a UapA oligomerization is associated with ER exit and turnover, as ER-retained mutants, either due to modification of a Tyr-based N-terminal motif or partial misfolding, physically associate, but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells.

70. "Minos as a novel Tc1/mariner-type transposable element for functional genomic analysis in Aspergillus nidulans."
M. Evangelinos, G. Anagnostopoulos, I. Karvela-Kalogeraki, PM. Stathopoulou, C. Scazzocchio and G. Diallinas.
Fungal Genetics and Biology, Vol. , pages , (2015).  View at publisher's site  Request copy
Abstract: Transposons constitute powerful genetic tools for gene inactivation, exon or promoter trapping and genome analyses. The Minos element from Drosophila hydei, a Tc1/mariner-like transposon, has proved as a very efficient tool for heterologous transposition in several metazoa. In filamentous fungi, only a handful of fungal-specific transposable elements have been exploited as genetic tools, with the impala Tc1/mariner element from Fusarium oxysporum being the most successful. Here, we developed a two-component transposition system to manipulate Minos transposition in Aspergillus nidulans (AnMinos). Our system allows direct selection of transposition events based on re-activation of niaD, a gene necessary for growth on nitrate as a nitrogen source. On average, among 10(8) conidiospores, we obtain up to ∼0.8×10(2) transposition events leading to the expected revertant phenotype (niaD(+)), while ∼16% of excision events lead to AnMinos loss. Characterized excision footprints consisted of the four terminal bases of the transposon flanked by the TA target duplication and led to no major DNA rearrangements. AnMinos transposition depends on the presence of its homologous transposase. Its frequency was not significantly affected by temperature, UV irradiation or the transcription status of the original integration locus (niaD). Importantly, transposition is dependent on nkuA, encoding an enzyme essential for non-homologous end joining of DNA in double-strand break repair. AnMinos proved to be an efficient tool for functional analysis as it seems to transpose in different genomic loci positions in all chromosomes, including a high proportion of integration events within or close to genes. We have used Minos to obtain morphological and toxic analogue resistant mutants. Interestingly, among morphological mutants some seem to be due to Minos-elicited over-expression of specific genes, rather than gene inactivation.

69. "Sul1 and Sul2 sulfate transceptors signal to protein kinase A upon exit of sulfur starvation."
HN. Kankipati, M. Rubio-Texeira, D. Castermans, G. Diallinas and JM. Thevelein.
Journal of Biological Chemistry, Vol. 290, pages 10430-46, (2015).  View at publisher's site  Request copy
Abstract: Sulfate is an essential nutrient with pronounced regulatory effects on cellular metabolism and proliferation. Little is known, however, about how sulfate is sensed by cells. Sul1 and Sul2 are sulfate transporters in the yeast Saccharomyces cerevisiae, strongly induced upon sulfur starvation and endocytosed upon the addition of sulfate. We reveal Sul1,2-dependent activation of PKA targets upon sulfate-induced exit from growth arrest after sulfur starvation. We provide two major arguments in favor of Sul1 and Sul2 acting as transceptors for signaling to PKA. First, the sulfate analogue, d-glucosamine 2-sulfate, acted as a non-transported agonist of signaling by Sul1 and Sul2. Second, mutagenesis to Gln of putative H(+)-binding residues, Glu-427 in Sul1 or Glu-443 in Sul2, abolished transport without affecting signaling. Hence, Sul1,2 can function as pure sulfate sensors. Sul1(E427Q) and Sul2(E443Q) are also deficient in sulfate-induced endocytosis, which can therefore be uncoupled from signaling. Overall, our data suggest that transceptors can undergo independent conformational changes, each responsible for triggering different downstream processes. The Sul1 and Sul2 transceptors are the first identified plasma membrane sensors for extracellular sulfate. High affinity transporters induced upon starvation for their substrate may generally act as transceptors during exit from starvation.

68. "Origin, diversification and substrate specificity in the family of NCS1/FUR transporters."
E. Krypotou, T. Evangelidis, J. Bobonis, AA. Pittis, T. Gabaldón, C. Scazzocchio, E. Mikros and G. Diallinas.
Molecular Microbiology, Vol. 96, pages 927-50, (2015).  View at publisher's site  Request copy
Abstract: NCS1 proteins are H(+) /Na(+) symporters specific for the uptake of purines, pyrimidines and related metabolites. In this article, we study the origin, diversification and substrate specificity of fungal NCS1 transporters. We show that the two fungal NCS1 sub-families, Fur and Fcy, and plant homologues originate through independent horizontal transfers from prokaryotes and that expansion by gene duplication led to the functional diversification of fungal NCS1. We characterised all Fur proteins of the model fungus Aspergillus nidulans and discovered novel functions and specificities. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. Most importantly, we predict and confirm that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward-facing selective gate. Our evolutionary and structure-function analysis contributes in the understanding of the molecular mechanisms underlying the functional diversification of eukaryotic NCS1 transporters, and in particular, forward the concept that selective channel-like gates might contribute to substrate specificity.

67. "Expression and specificity profile of the major acetate transporter AcpA in Aspergillus nidulans."
J. Sá-Pessoa, S. Amillis, M. Casal and G. Diallinas.
Fungal Genetics and Biology, Vol. 75, pages 56-63, (2015).  View at publisher's site  Request copy
Abstract: AcpA has been previously characterized as a high-affinity transporter essential for the uptake and use of acetate as sole carbon source in Aspergillus nidulans. Here, we follow the expression profile of AcpA and define its substrate specificity. AcpA-mediated acetate transport is detected from the onset of conidiospore germination, peaks at the time of germ tube emergence, and drops to low basal levels in germlings and young mycelia, where a second acetate transporter is also becoming apparent. AcpA activity also responds to acetate presence in the growth medium, but is not subject to either carbon or nitrogen catabolite repression. Short-chain monocarboxylates (benzoate, formate, butyrate and propionate) inhibit AcpA-mediated acetate transport with apparent inhibition constants (Ki) of 16.89±2.12, 9.25±1.01, 12.06±3.29 and 1.44±0.13mM, respectively. AcpA is also shown not to be directly involved in ammonia export, as proposed for its Saccharomyces cerevisiae homologue Ady2p. In the second part of this work, we search for the unknown acetate transporter expressed in mycelia, and for other transporters that might contribute to acetate uptake. In silico analysis, genetic construction of relevant null mutants, and uptake assays, reveal that the closest AcpA homologue (AN1839), named AcpB, is the 'missing' secondary acetate transporter in mycelia. We also identify two major short-chain carboxylate (lactate, succinate, pyruvate and malate) transporters, named JenA (AN6095) and JenB (AN6703), which however are not involved in acetate uptake. This work establishes a framework for further exploiting acetate and carboxylate transport in filamentous ascomycetes.

66. "Functional characterization of NAT/NCS2 proteins of Aspergillus brasiliensis reveals a genuine xanthine-uric acid transporter and an intrinsically misfolded polypeptide."
E. Krypotou, C. Scazzocchio and G. Diallinas.
Fungal Genetics and Biology, Vol. 75, pages 56-63, (2015).  View at publisher's site  Request copy
Abstract: The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterized NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterized mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high-capacity, H(+)/xanthine-uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed.

65. "Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters."
G. Diallinas.
Frontiers in Pharmacology, Vol. 5, pages 207, (2014).  View at publisher's site  Request copy
Abstract: Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action.

64. "Purine utilization proteins in the Eurotiales: cellular compartmentalization, phylogenetic conservation and divergence."
K. Galanopoulou K, C. Scazzocchio, ME. Galinou, W. Liu, F. Borbolis, M. Karachaliou, N. Oestreicher, DG. Hatzinikolaou DG, G. Diallinas and S. Amillis.
Fungal Genetics and Biology, Vol. 69, pages 96-108, (2014).  View at publisher's site  Request copy
Abstract: The purine utilization pathway has been thoroughly characterized in Aspergillus nidulans. We establish here the subcellular distribution of seven key intracellular enzymes, xanthine dehydrogenase (HxA), urate oxidase (UaZ), 5-hydroxy-isourate hydrolase (UaX), 2-oxo-4-hydroxy-4-carboxy ureido imidazoline decarboxylase (UaW), allantoinase (AlX), allantoicase (AaX), ureidoglycolate lyase (UglA), and the fungal-specific α-ketoglutarate Fe(II)-dependent dioxygenase (XanA). HxA, AlX, AaX, UaW and XanA are cytosolic, while UaZ, UaX and UglA are peroxisomal. Peroxisomal localization was confirmed by using appropriate pex mutants. The pathway is largely, but not completely conserved in the Eurotiomycetes, noticeably in some species AaX is substituted by an alternative enzyme of probable bacterial origin. UaZ and the urate-xanthine UapA and UapC transporters, are also localized in specific cells of the conidiophore. We show that metabolic accumulation of uric acid occurring in uaZ null mutations is associated with an increased frequency of appearance of morphologically distinct colony sectors, diminished conidiospore production, UV resistance and an altered response to oxidation stress, which may provide a rationale for the conidiophore-specific localization. The pathway-specific transcription factor UaY is localized in both the cytoplasm and nuclei under non-inducing conditions, but it rapidly accumulates exclusively to the nuclei upon induction by uric acid.

63. "Modelling, substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA."
E. Krypotou, G. Lambrinidis, T. Evangelidis, E. Mikros and G. Diallinas.
Molecular Microbiology, Vol. 93, pages 129-145, (2014).  View at publisher's site  Request copy
Abstract: The AzgA purine/H(+) symporter of Aspergillus nidulans is the founding member of a functionally and phylogenetically distinct transporter family present in fungi, bacteria and plants. Here a valid AzgA topological model is built based on the crystal structure of the Escherichia coli uracil transporter UraA, a member of the nucleobase-ascorbate transporter (NAT/NCS2) family. The model consists of 14 transmembrane, mostly α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct compact core of 8 TMSs, made of two intertwined inverted repeats (TMSs 1-4 and 8-11), is topologically distinct from a flexible domain (TMSs 5-7 and 12-14). A putative substrate binding cavity is visible between the core and the gate domains. Substrate docking, molecular dynamics and mutational analysis identified several residues critical for purine binding and/or transport in TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339 (TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates, while Asp342 (TMS8) might be involved in subsequent substrate translocation, through H(+) binding and symport. Thus, AzgA and other NAT transporters use topologically similar TMSs and amino acid residues for substrate binding and transport, which in turn implies that AzgA-like proteins constitute a distant subgroup of the ubiquitous NAT family.

62. "Transport assays in filamentous fungi: Kinetic characterization of the UapC purine transporter of Aspergillus nidulans."
E. Krypotou and G. Diallinas.
Fungal Genetics and Biology, Vol. 63, pages 1-8, (2014).  View at publisher's site  Request copy
Abstract: Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. Our method makes use of A. nidulans germinating conidiospores at a defined morphological stage in which most transporters show maximal expression, avoiding technical difficulties associated with the use of mycelia. In combination with the ease of construction of genetic null mutants in A. nidulans, our method allows the rigorous characterization of any transporter in genetic backgrounds that are devoid of other transporters of similar specificity. Here, we use this method to characterize the kinetic parameters and the specificity profile of UapC, a uric acid-xanthine transporter present in all ascomycetes and member of the ubiquitous Nucleobase-Ascorbate Transporter family, in specific genetic backgrounds lacking other relevant transporters.

61. "Allopurinol and xanthine use different translocation mechanisms and trajectories in the fungal UapA transporter."
G. Diallinas.
Biochimie, Vol. 95, pages 1755-1764, (2013).  View at publisher's site  Request copy
Abstract: In Aspergillus nidulans UapA is a H(+)-driven transporter specific for xanthine, uric acid and several analogues. Here, genetic and physiological evidence is provided showing that allopurinol is a high-affinity, low-capacity, substrate for UapA. Surprisingly however, transport kinetic measurements showed that, uniquely among all recognized UapA substrates, allopurinol is transported by apparent facilitated diffusion and exhibits a paradoxical effect on the transport of physiological substrates. Specifically, excess xanthine or other UapA substrates inhibit allopurinol uptake, as expected, but the presence of excess allopurinol results in a concentration-dependent enhancement of xanthine binding and transport. Flexible docking approaches failed to detect allopurinol binding in the major UapA substrate binding site, which was recently identified by mutational analysis and substrate docking using all other UapA substrates. These results and genetic evidence suggest that the allopurinol translocation pathway is distinct from, but probably overlapping with, that of physiological UapA substrates. Furthermore, although the stimulating effect of allopurinol on xanthine transport could, in principle, be rationalized by a cryptic allopurinol-specific allosteric site, evidence was obtained supporting that accelerated influx of xanthine is triggered through exchange with cytoplasmically accumulated allopurinol. Our results are in line with recently accumulating evidence revealing atypical and complex mechanisms underlying transport systems.

60. "The arrestin-like protein ArtA is essential for ubiquitination and endocytosis of the UapA transporter in response to both broad-range and specific signals."
M. Karachaliou, S. Amillis, M. Evangelinos, A.C. Kokotos, V. Yialelis and G. Diallinas.
Molecular Microbiology, Vol. 88, pages 301-317, (2013).  View at publisher's site  Request copy
Abstract: We investigated the role of all arrestin-like proteins of Aspergillus nidulans in respect to growth, morphology, sensitivity to drugs and specifically for the endocytosis and turnover of the uric acid-xanthine transporter UapA. A single arrestin-like protein, ArtA, is essential for HulA(Rsp) (5) -dependent ubiquitination and endocytosis of UapA in response to ammonium or substrates. Mutational analysis showed that residues 545-563 of the UapA C-terminal region are required for efficient UapA endocytosis, whereas the N-terminal region (residues 2-123) and both PPxY motives are essential for ArtA function. We further show that ArtA undergoes HulA-dependent ubiquitination at residue Lys-343 and that this modification is critical for UapA ubiquitination and endocytosis. Lastly, we show that ArtA is essential for vacuolar turnover of transporters specific for purines (AzgA) or l-proline (PrnB), but not for an aspartate/glutamate transporter (AgtA). Our results are discussed within the frame of recently proposed mechanisms on how arrestin-like proteins are activated and recruited for ubiquitination of transporters in response to broad range signals, but also put the basis for understanding how arrestin-like proteins, such as ArtA, regulate the turnover of a specific transporter in the presence of its substrates.

59. "Modeling, Substrate Docking, and Mutational Analysis Identify Residues Essential for the Function and Specificity of a Eukaryotic Purine-Cytosine NCS1 Transporter."
E. Krypotou, V. Kosti, S. Amillis, V. Myrianthopoulos, E. Mikros and G. Diallinas.
Journal of Biological Chemistry, Vol. 287, pages 36792-36803, (2012).  View at publisher's site  Request copy
Abstract: The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a three-dimensional model of the Aspergillus nidulans purine-cytosine/H(+) FcyB symporter. The model consists of 12 transmembrane α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct core of 10 TMSs is made of two intertwined inverted repeats (TMS1-5 and TMS6-10) that are followed by two additional TMSs. TMS1, TMS3, TMS6, and TMS8 form an open cavity that is predicted to host the substrate binding site. Based on primary sequence alignment, three-dimensional topology, and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; Ser-85 (TMS1), Trp-159, Asn-163 (TMS3), Trp-259 (TMS6), and Asn-354 (TMS8). To validate the role of these and other putatively critical residues, we performed a systematic functional analysis of relevant mutants. We show that the proposed substrate binding residues, plus Asn-350, Asn-351, and Pro-353 are irreplaceable for FcyB function. Among these residues, Ser-85, Asn-163, Asn-350, Asn-351, and Asn-354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that Ser-85, Asn-163, and Asn-354 directly interact with substrates, Trp-159 and Trp-259 stabilize binding through π-π stacking interactions, and Pro-353 affects the local architecture of substrate binding site, whereas Asn-350 and Asn-351 probably affect substrate binding indirectly. Our work is the first systematic approach to address structure-function-specificity relationships in a eukaryotic member of NCS1 family by combining genetic and computational approaches.

58. "Identification of the substrate recognition and transport pathway in a eukaryotic member of the nucleobase-ascorbate transporter (NAT) family."
V. Kosti, G. Lambrinidis, V. Myrianthopoulos, G. Diallinas and E. Mikros.
PLOS One, Vol. 7, pages e41939, (2012).  View at publisher's site  Request copy
Abstract: Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H(+) symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site.

57. "Stabilizing the heterologously expressed uric acid-xanthine transporter UapA from the lower eukaryote Aspergillus nidulans."
J. Leung, A.D. Cameron, G. Diallinas and B. Byrne.
Molecular Membrane Biology, doi:10.3109/09687688.2012.690572, (2012).  View at publisher's site  Request copy
Abstract: Despite detailed genetic and mutagenic analysis and a recent high-resolution structure of a bacterial member of the nucleobase-ascorbate transporter (NAT) family, understanding of the mechanism of action of eukaryotic NATs is limited. Preliminary studies successfully expressed and purified wild-type UapA to high homogeneity; however, the protein was extremely unstable, degrading almost completely after 48 h at 4°C. In an attempt to increase UapA stability we generated a number of single point mutants (E356D, E356Q, N409A, N409D, Q408E and G411V) previously shown to have reduced or no transport activity, but correct targeting to the membrane. The mutant UapA constructs expressed well as GFP fusions in Saccharomyces cerevisiae and exhibited similar fluorescent size exclusion chromatography (FSEC) profiles to the wild-type protein, following solubilization in 1% DDM, LDAO or OM + 1 mM xanthine. In order to assess the relative stabilities of the mutants, solubilized fractions prepared in 1% DDM + 1 mM xanthine were heated at 45°C for 10 min prior to FSEC. The Q408E and G411V mutants gave markedly better profiles than either wild-type or the other mutants. Further FSEC analysis following solubilization of the mutants in 1% NG ± xanthine confirmed that G411V is more stable than the other mutants, but showed that Q408E is unstable under these conditions. G411V and an N-terminally truncated construct G411VΔ1-11 were submitted to large-scale expression and purification. Long-term stability analysis revealed that G411VΔ1-11 was the most stable construct and the most suited to downstream structural studies.

56. "The role of flotillin FloA and stomatin StoA in the maintenance of apical sterol-rich membrane domains and polarity in the filamentous fungus Aspergillus nidulans."
N. Takeshita, G. Diallinas and R. Fischer.
Molecular Microbiology, Vol. 83, pages 1136-1152, (2012).  View at publisher's site  Request copy
Abstract: Apical sterol-rich plasma membrane domains (SRDs), which can be viewed using the sterol-binding fluorescent dye filipin, are gaining attention for their important roles in polarized growth of filamentous fungi. The microdomain scaffolding protein flotillin/reggie and related stomatin were thought to be good candidates involved in the formation of SRDs. Here, we show that the flotillin/reggie orthologue FloA tagged with GFP localized as stable dots along the plasma membrane except hyphal tips. Deletion of floA reduced the growth rate, often resulted in irregularly shaped hyphae and impaired SRDs. In contrast, the stomatin orthologue StoA, tagged with GFP, localized at the cortex of young branch tips and at the subapical cortex in long hyphal tips, and was transported bi-directionally along microtubules on endosomes. Deletion of stoA resulted in irregular hyphal morphology and increased branching especially in young hyphae, but did not obviously affect SRDs. Double deletion of floA and stoA enhanced the defects of growth and hyphal morphology. Our data suggest that the plasma membrane of hyphal tips and in subapical regions are distinct and that FloA is involved in membrane compartmentalization and probably indirectly in SRD maintenance.

55. "Mutational analysis and modeling reveal functionally critical residues in transmembrane segments 1 and 3 of the UapA transporter."
S. Amillis, V. Kosti, A. Pantazopoulou, E. Mikros and G. Diallinas.
Journal of Molecular Biology, Vol. 411, pages 567-580, (2011).  View at publisher's site  Request copy
Abstract: Earlier, we identified mutations in the first transmembrane segment (TMS1) of UapA, a uric acid-xanthine transporter in Aspergillus nidulans, that affect its turnover and subcellular localization. Here, we use one of these mutations (H86D) and a novel mutation (I74D) as well as genetic suppressors of them, to show that TMS1 is a key domain for proper folding, trafficking and turnover. Kinetic analysis of mutants further revealed that partial misfolding and deficient trafficking of UapA does not affect its affinity for xanthine transport, but reduces that of uric acid and confers a degree of promiscuity towards the binding of other purines. This result strengthens the idea that subtle interactions among domains not directly involved in substrate binding refine the selectivity of UapA. Characterization of second-site suppressors of H86D revealed a genetic interaction of TMS1 with TMS3, the latter segment shown for the first time to be important for UapA function. Systematic mutational analysis of polar and conserved residues in TMS3 showed that Ser154 is crucial for UapA transport activity. Our results are in agreement with a topological model of UapA built on the recently published structure of UraA, a bacterial homolog of UapA.

54. "A substrate translocation trajectory in a cytoplasm-facing topological model of the monocarboxylate/H⁺ symporter Jen1p."
I. Soares-Silva, J. Sá-Pessoa, V. Myrianthopoulos, E. Mikros, M. Casal and G. Diallinas.
Molecular Microbiology, Vol. 81, pages 805-817, (2011).  View at publisher's site  Request copy
Abstract:Previous mutational analysis of Jen1p, a Saccharomyces cerevisiae monocarboxylate/H+ symporter of the Major Facilitator Superfamily, has suggested that the consensus sequence 379NXX[S/T]HX[S/T]QD387 in transmembrane segment VII (TMS-VII) is part of the substrate translocation pathway. Here, we rationally design, analyse and show that several novel mutations in TMS-V and TMS-XI directly modify Jen1p function. Among the residues studied, F270 (TMS-V) and Q498 (TMS-XI) are critical specificity determinants for the distinction of mono- from dicarboxylates, and N501 (TMS-XI) is a critical residue for function. Using a model created on the basis of Jen1p similarity with the GlpT permease, we show that all polar residues critical for function within TMS-VII and TMS-XI (N379, H383, D387, Q498, N501) are perfectly aligned in an imaginary axis that lies parallel to the protein pore. This model and subsequent mutational analysis further reveal that an additional polar residue facing the pore, R188 (TMS-II), is irreplaceable for function. Our model also justifies the role of F270 and Q498 in substrate specificity. Finally, docking calculations reveal a 'trajectory-like' substrate displacement within the Jen1p pore, where R188 plays a major dynamic role mediating the orderly relocation of the substrate by subsequent H-bond interactions involving itself and residues H383, N501 and Q498.

53. "Completing the purine utilisation pathway of Aspergillus nidulans."
C. Gournas, N. Oestreicher, S. Amillis, G. Diallinas and C. Scazzocchio.
Fungal Genetics and Biology, Vol. 48, pages 840-848, (2011).  View at publisher's site  Request copy
Abstract: We have previously identified by classical genetics and biochemistry, all the genes of Aspergillus nidulans predicted to be involved in purine utilisation, together with cognate regulatory genes and one gene encoding a novel xanthine hydroxylation activity. In this article we complete the description of the purine utilisation pathway with the identification of the two genes (uaX and uaW) encoding the enzymes catalysing the conversion of the product of urate oxidation by urate oxidase, 5-hydroxyisourate, to optically active allantoin. The identification of these additional genes confirms the complete absence of clustering of the genes involved in purine utilisation in A. nidulans.

52. "Homologous overexpression of xylanase in Fusarium oxysporum increases ethanol productivity during consolidated bioprocessing (CBP) of lignocellulosics."
G.E. Anasontzis, A. Zerva, P.M. Stathopoulou, K. Haralampidis, G. Diallinas, A.D. Karagouni and D.G. Hatzinikolaou.
Journal of Biotechnology, Vol. 152, pages 16-23, (2011).  View at publisher's site  Request copy
Abstract: In an effort to increase ethanol productivity during the consolidated bioprocessing (CBP) of lignocellulosics by Fusarium oxysporum, we attempted the constitutive homologous overexpression of one of the key process enzymes, namely an endo-xylanase. The endo-β-1,4-xylanase 2 gene, was incorporated into the F. oxysporum genome under the regulation of the gpdA promoter of Aspergillus nidulans. The transformation was effected through Agrobacterium tumefaciens and resulted in 12 transformants, two of which were selected for their high extracellular xylanase activities under normally repressing conditions (glucose as sole carbon source). During natural induction conditions (growth on xylan) though, the extracellular enzyme levels of the transformants were only marginally higher (5–10%) compared to the wild type despite the significantly stronger xylanase 2 mRNA signals. SDS-PAGE verified enzyme assay results that there was no intracellular xylanase 2 accumulation in the transformants, suggesting the potential regulation in a post transcriptional or translation level. The fermentative performance of the transformants was evaluated and compared to that of the wild type in simple CBP systems using either corn cob or wheat bran as sole carbon sources. Both transformants produced approximately 60% more ethanol compared to the wild type on corn cob, while for wheat bran this picture was repeated for only one of them. This result is attributed to the high extracellular xylanase activities in the transformants’ fermentation broths that were maintained 2–2.5 fold higher compared to the wild type.

51. "Hypertonic conditions trigger transient plasmolysis, growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae."
V. Bitsikas, M. Karachaliou, C. Gournas and G. Diallinas.
Molecular Membrane Biology, vol. 28, pages 54-68, (2011).  View at publisher's site  Request copy
Abstract: By using Aspergillus nidulans strains expressing functional GFP-tagged transporters under hypertonic conditions, we noticed the rapid appearance of cortical, relatively static, fluorescent patches (0.5-2.3 μm). These patches do not correspond to transporter microdomains as they co-localize with other plasma membrane-associated molecules, such as the pleckstrin homology (PH) domain and the SsoA t-Snare, or the lipophilic markers FM4-64 and filipin. In addition, they do not show characteristics of lipid rafts, MCCs or other membrane microdomains. Deconvoluted microscopic images showed that fluorescent patches correspond to plasma membrane invaginations. Transporters remain fully active during this phenomenon of localized plasmolysis. Plasmolysis was however associated with reduced growth rate and a dramatic blockage in transporter and FM4-64 endocytosis. These phenomena are transient and rapidly reversible upon wash-out of hypertonic media. Based on the observation that block in endocytosis by hypertonic treatment altered dramatically the cellular localization of tropomyosin (GFP-TpmA), although it did not affect the cortical appearance of upstream (SlaB-GFP) or downstream (AbpA-mRFP) endocytic components, we conclude that hypertonicity modifies actin dynamics and thus acts indirectly on endocytosis. This was further supported by the effect of latrunculin B, an actin depolymerization agent, on endocytosis. We show that the phenomena observed in A. nidulans also occur in Saccharomyces cerevisiae, suggesting that they constitute basic homeostatic responses of ascomycetes to hypertonic shock. Finally, our work shows that hypertonic treatments can be used as physiological tools to study the endocytic down-regulation of transporters in A. nidulans, as non-conditional genetic blocks affecting endocytic internalization are lethal or severely debilitating.