Screens for mutants defective in UapA trafficking highlight the importance of ER-exit as a primary control point in transporter biogenesis.

Most transmembrane membrane proteins are thought to traffic to the plasma membrane (PM) via the conventional secretory pathway through sorting from the Golgi. However, our recent work has shown that in the filamentous fungus Aspergillus nidulans several nutrient transporters and other major membrane proteins traffic to the PM via Golgi-bypass and independently of known post-Golgi secretory mechanisms. Here in an effort to dissect the molecular mechanism underlying membrane cargo trafficking via Golgi-bypass we design and use unbiased genetic screens, based on the UapA uric acid-xanthine transporter, which allowed the isolation of mutants defective in UapA translocation to the plasma membrane.

Distinct trafficking routes of polarized and non-polarized membrane cargoes in .

Membrane proteins are sorted to the plasma membrane via Golgi-dependent trafficking. However, our recent studies challenged the essentiality of Golgi in the biogenesis of specific transporters. Here, we investigate the trafficking mechanisms of membrane proteins by following the localization of the polarized R-SNARE SynA versus the non-polarized transporter UapA, synchronously co-expressed in wild-type or isogenic genetic backgrounds repressible for conventional cargo secretion.

High-resolution structures of the UapA purine transporter reveal unprecedented aspects of the elevator-type transport mechanism.

UapA is an extensively studied elevator-type purine transporter from the model fungus . Determination of a 3.6Å inward-facing crystal structure lacking the cytoplasmic N-and C-tails, molecular dynamics (MD), and functional studies have led to speculative models of its transport mechanism and determination of substrate specificity.

The last two transmembrane helices in the APC-type FurE transporter act as an intramolecular chaperone essential for concentrative ER-exit.

FurE is a H symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus nidulans. Being member of the NCS1 protein family, FurE is structurally related to the APC-superfamily of transporters. APC-type transporters are characterised by a 5+5 inverted repeat fold made of ten transmembrane segments (TMS1-10) and function through the rocking-bundle mechanism.

The interactome of the UapA transporter reveals putative new players in anterograde membrane cargo trafficking.

Neosynthesized plasma membrane (PM) proteins co-translationally translocate to the ER, concentrate at regions called ER-exit sites (ERes) and pack into COPII secretory vesicles which are sorted to the early-Golgi through membrane fusion. Following Golgi maturation, membrane cargoes reach the late-Golgi, from where they exit in clathrin-coated vesicles destined to the PM, directly or through endosomes. Post-Golgi membrane cargo trafficking also involves the cytoskeleton and the exocyst.

Phytochemical investigation of Pistacia lentiscus L. var. Chia leaves: A byproduct with antimicrobial potential.

Pistacia lentiscus L. var. Chia belongs to the Anacardiaceae family, and it is cultivated only in the south part of Chios island, in Greece.

Uracil/H Symport by FurE Refines Aspects of the Rocking-bundle Mechanism of APC-type Transporters.

Transporters mediate the uptake of solutes, metabolites and drugs across the cell membrane. The eukaryotic FurE nucleobase/H symporter of Aspergillus nidulans has been used as a model protein to address structure-function relationships in the APC transporter superfamily, members of which are characterized by the LeuT-fold and seem to operate by the so-called 'rocking-bundle' mechanism. In this study, we reveal the binding mode, translocation and release pathway of uracil/H by FurE using path collective variable, funnel metadynamics and rational mutational analysis.

Genetic and cellular characterization of MscS-like putative channels in the filamentous fungus .

Mechanosensitive ion channels are integral membrane proteins ubiquitously present in bacteria, archaea, and eukarya. They act as molecular sensors of mechanical stress to serve vital functions such as touch, hearing, osmotic pressure, proprioception and balance, while their malfunction is often associated with pathologies. Amongst them, the structurally distinct MscL and MscS channels from bacteria are the most extensively studied.

Nucleoside Transport and Nucleobase Uptake Null Mutants in for the Routine Expression and Characterization of Purine and Pyrimidine Transporters.

The study of transporters is highly challenging, as they cannot be isolated or studied in suspension, requiring a cellular or vesicular system, and, when mediated by more than one carrier, difficult to interpret. Nucleoside analogues are important drug candidates, and all protozoan pathogens express multiple equilibrative nucleoside transporter (ENT) genes. We have therefore developed a system for the routine expression of nucleoside transporters, using CRISPR/cas9 to delete both copies of all three nucleoside transporters from (ΔNT1.

Transmembrane helices 5 and 12 control transport dynamics, substrate affinity, and specificity in the elevator-type UapA transporter.

An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences, all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best-studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans.

Golgi-Bypass Is a Major Unconventional Route for Translocation to the Plasma Membrane of Non-Apical Membrane Cargoes in .

Nutrient transporters have been shown to translocate to the plasma membrane (PM) of the filamentous fungus an unconventional trafficking route that bypasses the Golgi. This finding strongly suggests the existence of distinct COPII vesicle subpopulations, one following Golgi-dependent conventional secretion and the other directed towards the PM. Here, we address whether Golgi-bypass concerns cargoes other than nutrient transporters and whether Golgi-bypass is related to cargo structure, size, abundance, physiological function, or polar vs.

Interactions of cytosolic tails in the Jen1 carboxylate transporter are critical for trafficking and transport activity.

Plasma membrane (PM) transporters of the major facilitator superfamily (MFS) are essential for cell metabolism, growth and response to stress or drugs. In Saccharomyces cerevisiae, Jen1 is a monocarboxylate/H+ symporter that provides a model to dissect the molecular details underlying cellular expression, transport mechanism and turnover of MFS transporters. Here, we present evidence revealing novel roles of the cytosolic N- and C-termini of Jen1 in its biogenesis, PM stability and transport activity, using functional analyses of Jen1 truncations and chimeric constructs with UapA, an endocytosis-insensitive transporter of Aspergillus nidulans.

On the Evidence Supporting That AN11127 Encodes an Aspergillus Nidulans Sec12 Orthologous Protein. Reply to Bravo-Plaza et al. Comment on "Dimou et al. Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in . 2021, , 560".

Prof. Peñalva and co-workers provided evidence that AN11127 is related by sequence and function to Sec12 [..

Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens.

Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in .

Solute and ion transporters are proteins essential for cell nutrition, detoxification, signaling, homeostasis and drug resistance. Being polytopic transmembrane proteins, they are co-translationally inserted and folded into the endoplasmic reticulum (ER) of eukaryotic cells and subsequently sorted to their final membrane destination via vesicular secretion. During their trafficking and in response to physiological/stress signals or prolonged activity, transporters undergo multiple quality control processes and regulated turnover.

Impact of Membrane Lipids on UapA and AzgA Transporter Subcellular Localization and Activity in .

Recent biochemical and biophysical evidence have established that membrane lipids, namely phospholipids, sphingolipids and sterols, are critical for the function of eukaryotic plasma membrane transporters. Here, we study the effect of selected membrane lipid biosynthesis mutations and of the ergosterol-related antifungal itraconazole on the subcellular localization, stability and transport kinetics of two well-studied purine transporters, UapA and AzgA, in . We show that genetic reduction in biosynthesis of ergosterol, sphingolipids or phosphoinositides arrest growth after germling formation, but solely blocks in early steps of ergosterol (Erg11) or sphingolipid (BasA) synthesis have a negative effect on plasma membrane (PM) localization and stability of transporters before growth arrest.

Transporter Specificity: A Tale of Loosened Elevator-Sliding.

Elevator-type transporters are a group of proteins translocating nutrients and metabolites across cell membranes. Despite structural and functional differences, elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator), which includes the substrate binding site, along a rigid scaffold domain, stably anchored in the plasma membrane. How substrate specificity is determined in elevator transporters remains elusive.

Context-dependent Cryptic Roles of Specific Residues in Substrate Selectivity of the UapA Purine Transporter.

Members of the ubiquitous Nucleobase Ascorbate Transporter (NAT) family are H or Na symporters specific for the cellular uptake of either purines and pyrimidines or L-ascorbic acid. Despite the fact that several bacterial and fungal members have been extensively characterised at a genetic, biochemical or cellular level, and crystal structures of NAT members from Escherichia coli and Aspergillus nidulans have been determined pointing to a mechanism of transport, we have little insight on how substrate selectivity is determined. Here, we present systematic mutational analyses, rational combination of mutations, and novel genetic screens that reveal cryptic context-dependent roles of partially conserved residues in the so-called NAT signature motif in determining the specificity of the UapA transporter of A.

Life and Death of Fungal Transporters under the Challenge of Polarity.

Eukaryotic plasma membrane (PM) transporters face critical challenges that are not widely present in prokaryotes. The two most important issues are proper subcellular traffic and targeting to the PM, and regulated endocytosis in response to physiological, developmental, or stress signals. Sorting of transporters from their site of synthesis, the endoplasmic reticulum (ER), to the PM has been long thought, but not formally shown, to occur via the conventional Golgi-dependent vesicular secretory pathway.

Untargeted metabolomics as a hypothesis-generation tool in plant protection product discovery: Highlighting the potential of trehalose and glycerol metabolism of fungal conidiospores as novel targets.

Introduction: The production of high quality and safe food represents a main priority for the agri-food sector in the effort to sustain the exponentially growing human population. Nonetheless, there are major challenges that require the discovery of new, alternative, and improved plant protection products (PPPs). Focusing on fungal plant pathogens, the dissection of mechanisms that are essential for their survival provides insights that could be exploited towards the achievement of the aforementioned aim.

Translocation of nutrient transporters to cell membrane via Golgi bypass in Aspergillus nidulans.

Nutrient transporters, being polytopic membrane proteins, are believed, but not formally shown, to traffic from their site of synthesis, the ER, to the plasma membrane through Golgi-dependent vesicular trafficking. Here, we develop a novel genetic system to investigate the trafficking of a neosynthesized model transporter, the well-studied UapA purine transporter of Aspergillus nidulans. We show that sorting of neosynthesized UapA to the plasma membrane (PM) bypasses the Golgi and does not necessitate key Rab GTPases, AP adaptors, microtubules or endosomes.

mRNA decapping is an evolutionarily conserved modulator of neuroendocrine signaling that controls development and ageing.

Eukaryotic 5'-3' mRNA decay plays important roles during development and in response to stress, regulating gene expression post-transcriptionally. In , deficiency of DCAP-1/DCP1, the essential co-factor of the major cytoplasmic mRNA decapping enzyme, impacts normal development, stress survival and ageing. Here, we show that overexpression of in neurons of worms is sufficient to increase lifespan through the function of the insulin/IGF-like signaling and its effector DAF-16/FOXO transcription factor.

Specific Residues in a Purine Transporter Are Critical for Dimerization, ER Exit, and Function.

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 formation of functional dimers in the ER, or ER exit and further subcellular trafficking.