Uptake of exogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae.

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.

Conformation-dependent partitioning of yeast nutrient transporters into starvation-protective membrane domains.

The eukaryotic plasma membrane is compartmentalized into domains enriched in specific lipids and proteins. However, our understanding of the molecular bases and biological roles of this partitioning remains incomplete. The best-studied domain in yeast is the membrane compartment containing the arginine permease Can1 (MCC) and later found to cluster additional transporters. MCCs correspond to static, furrow-like invaginations of the plasma membrane and associate with subcortical structures named "eisosomes" that include upstream regulators of the target of rapamycin complex 2 (TORC2) in the sensing of sphingolipids and membrane stress. However, how and why Can1 and other nutrient transporters preferentially segregate in MCCs remains unknown. In this study we report that the clustering of Can1 in MCCs is dictated by its conformation, requires proper sphingolipid biosynthesis, and controls its ubiquitin-dependent endocytosis. In the substrate-free outward-open conformation, Can1 accumulates in MCCs in a manner dependent on sustained biogenesis of complex sphingolipids. An arginine transport-elicited shift to an inward-facing conformation promotes its cell-surface dissipation and makes it accessible to the ubiquitylation machinery triggering its endocytosis. We further show that under starvation conditions MCCs increase in number and size, this being dependent on the BAR domain-containing Lsp1 eisosome component. This expansion of MCCs provides protection for nutrient transporters from bulk endocytosis occurring in parallel with autophagy upon TORC1 inhibition. Our study reveals nutrient-regulated protection from endocytosis as an important role for protein partitioning into membrane domains.

The Bul1/2 Alpha-Arrestins Promote Ubiquitylation and Endocytosis of the Can1 Permease upon Cycloheximide-Induced TORC1-Hyperactivation.

Selective endocytosis followed by degradation is a major mechanism for downregulating plasma membrane transporters in response to specific environmental cues. In Saccharomyces cerevisiae, this endocytosis is promoted by ubiquitylation catalyzed by the Rsp5 ubiquitin-ligase, targeted to transporters via adaptors of the alpha-arrestin family. However, the molecular mechanisms of this targeting and their control according to conditions remain incompletely understood. In this work, we dissect the molecular mechanisms eliciting the endocytosis of Can1, the arginine permease, in response to cycloheximide-induced TORC1 hyperactivation. We show that cycloheximide promotes Rsp5-dependent Can1 ubiquitylation and endocytosis in a manner dependent on the Bul1/2 alpha-arrestins. Also crucial for this downregulation is a short acidic patch sequence in the N-terminus of Can1 likely acting as a binding site for Bul1/2. The previously reported inhibition by cycloheximide of transporter recycling, from the trans-Golgi network to the plasma membrane, seems to additionally contribute to efficient Can1 downregulation. Our results also indicate that, contrary to the previously described substrate-transport elicited Can1 endocytosis mediated by the Art1 alpha-arrestin, Bul1/2-mediated Can1 ubiquitylation occurs independently of the conformation of the transporter. This study provides further insights into how distinct alpha-arrestins control the ubiquitin-dependent downregulation of a specific amino acid transporter under different conditions.

On the Evolution of Specificity in Members of the Yeast Amino Acid Transporter Family as Parts of Specific Metabolic Pathways.

In the recent years, molecular modeling and substrate docking, coupled with biochemical and genetic analyses have identified the substrate-binding residues of several amino acid transporters of the yeast amino acid transporter (YAT) family. These consist of (a) residues conserved across YATs that interact with the invariable part of amino acid substrates and (b) variable residues that interact with the side chain of the amino acid substrate and thus define specificity. Secondary structure sequence alignments showed that the positions of these residues are conserved across YATs and could thus be used to predict the specificity of YATs. Here, we discuss the potential of combining molecular modeling and structural alignments with intra-species phylogenetic comparisons of transporters, in order to predict the function of uncharacterized members of the family. We additionally define some orphan branches which include transporters with potentially novel, and to be characterized specificities. In addition, we discuss the particular case of the highly specific l-proline transporter, PrnB, of Aspergillus nidulans, whose gene is part of a cluster of genes required for the utilization of proline as a carbon and/or nitrogen source. This clustering correlates with transcriptional regulation of these genes, potentially leading to the efficient coordination of the uptake of externally provided l-Pro via PrnB and its enzymatic degradation in the cell.

The Aspergillus nidulans proline permease as a model for understanding the factors determining substrate binding and specificity of fungal amino acid transporters.

Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly(56), Thr(57)), TMS3 (Glu(138)), and TMS6 (Phe(248)), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent Km for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle.

Function and Regulation of Fungal Amino Acid Transporters: Insights from Predicted Structure.

Amino acids constitute a major nutritional source for probably all fungi. Studies of model species such as the yeast Saccharomyces cerevisiae and the filamentous fungus Aspergillus nidulans have shown that they possess multiple amino acid transporters. These proteins belong to a limited number of superfamilies, now defined according to protein fold in addition to sequence criteria, and differ in subcellular location, substrate specificity range, and regulation. Structural models of several of these transporters have recently been built, and the detailed molecular mechanisms of amino acid recognition and translocation are now being unveiled. Furthermore, the particular conformations adopted by some of these transporters in response to amino acid binding appear crucial to promoting their ubiquitin-dependent endocytosis and/or to triggering signaling responses. We here summarize current knowledge, derived mainly from studies on S. cerevisiae and A. nidulans, about the transport activities, regulation, and sensing role of fungal amino acid transporters, in relation to predicted structure.

Ferroptosis-protective membrane domains in quiescence.

Quiescence is a common cellular state, required for stem cell maintenance and microorganismal survival under stress conditions or starvation. However, the mechanisms promoting quiescence maintenance remain poorly known. Plasma membrane components segregate into distinct microdomains, yet the role of this compartmentalization in quiescence remains unexplored. Here, we show that flavodoxin-like proteins (FLPs), ubiquinone reductases of the yeast eisosome membrane compartment, protect quiescent cells from lipid peroxidation and ferroptosis. Eisosomes and FLPs expand specifically in respiratory-active quiescent cells, and mutants lacking either show accelerated aging and defective quiescence maintenance and accumulate peroxidized phospholipids with monounsaturated or polyunsaturated fatty acids (PUFAs). FLPs are essential for the extramitochondrial regeneration of the lipophilic antioxidant ubiquinol. FLPs, alongside the Gpx1/2/3 glutathione peroxidases, prevent iron-driven, PUFA-dependent ferroptotic cell death. Our work describes ferroptosis-protective mechanisms in yeast and introduces plasma membrane compartmentalization as an important factor in the long-term survival of quiescent cells.

Hypertonic conditions trigger transient plasmolysis, growth arrest and blockage of transporter endocytosis in Aspergillus nidulans and Saccharomyces cerevisiae.

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.

Transport-dependent endocytosis and turnover of a uric acid-xanthine permease.

In this work we unmask a novel downregulation mechanism of the uric acid/xanthine transporter UapA, the prototype member of the ubiquitous Nucleobase-Ascorbate Transporter family, directly related to its function. In the presence of substrates, UapA is endocytosed, sorted into the multivesicular body pathway and degraded in vacuoles. Substrate-induced endocytosis, unlike ammonium-induced turnover, is absolutely dependent on UapA activity and several lines of evidence showed that the signal for increased endocytosis is the actual translocation of substrates through the UapA protein. The use of several UapA functional mutants with altered kinetics and specificity has further shown that transport-dependent UapA endocytosis occurs through a mechanism, which senses subtle conformational changes associated with the transport cycle. We also show that distinct mechanisms of UapA endocytosis necessitate ubiquitination of a single Lys residue (K572) by HulA(Rsp5). Finally, we demonstrate that in the presence of substrates, non-functional UapA versions can be endocytosed in trans if expressed in the simultaneous presence of active UapA versions, even if the latter cannot be endocytosed themselves.

Completing the purine utilisation pathway of Aspergillus nidulans.

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.

Eisosome organization in the filamentous ascomycete Aspergillus nidulans.

Eisosomes are subcortical organelles implicated in endocytosis and have hitherto been described only in Saccharomyces cerevisiae. They comprise two homologue proteins, Pil1 and Lsp1, which colocalize with the transmembrane protein Sur7. These proteins are universally conserved in the ascomycetes. We identify in Aspergillus nidulans (and in all members of the subphylum Pezizomycotina) two homologues of Pil1/Lsp1, PilA and PilB, originating from a duplication independent from that extant in the subphylum Saccharomycotina. In the aspergilli there are several Sur7-like proteins in each species, including one strict Sur7 orthologue (SurG in A. nidulans). In A. nidulans conidiospores, but not in hyphae, the three proteins colocalize at the cell cortex and form tightly packed punctate structures that appear different from the clearly distinct eisosome patches observed in S. cerevisiae. These structures are assembled late during the maturation of conidia. In mycelia, punctate structures are present, but they are composed only of PilA, while PilB is diffused in the cytoplasm and SurG is located in vacuoles and endosomes. Deletion of each of the genes does not lead to any obvious growth phenotype, except for moderate resistance to itraconazole. We could not find any obvious association between mycelial (PilA) eisosome-like structures and endocytosis. PilA and SurG are necessary for conidial eisosome organization in ways that differ from those for their S. cerevisiae homologues. These data illustrate that conservation of eisosomal proteins within the ascomycetes is accompanied by a striking functional divergence.

Quantitative Analysis of Aspergillus nidulans Growth Rate using Live Microscopy and Open-Source Software.

It is well established that colony growth of filamentous fungi, mostly dependent on changes in hyphae/mycelia apical growth rate, is macroscopically estimated on solidified media by comparing colony size. However, to quantitatively measure the growth rate of genetically different fungal strains or strains under different environmental/growth conditions (pH, temperature, carbon and nitrogen sources, antibiotics, etc.) is challenging. Thus, the pursuit of complementary approaches to quantify growth kinetics becomes mandatory in order to better understand fungal cell growth. Furthermore, it is well-known that filamentous fungi, including Aspergillus spp., have distinct modes of growth and differentiation under sub-aerial conditions on solid media or submerged cultures. Here, we detail a quantitative microscopic method for analyzing growth kinetics of the model fungus Aspergillus nidulans, using live imaging in both submerged cultures and solid media. We capture images, analyze, and quantify growth rates of different fungal strains in a reproducible and reliable manner using an open source, free software for bio-images (e.g., Fiji), in a way that does not require any prior image analysis expertise from the user.

A highly conserved mechanism for the detoxification and assimilation of the toxic phytoproduct L-azetidine-2-carboxylic acid in Aspergillus nidulans.

Plants produce toxic secondary metabolites as defense mechanisms against phytopathogenic microorganisms and predators. L-azetidine-2-carboxylic acid (AZC), a toxic proline analogue produced by members of the Liliaceae and Agavaciae families, is part of such a mechanism. AZC causes a broad range of toxic, inflammatory and degenerative abnormalities in human and animal cells, while it is known that some microorganisms have evolved specialized strategies for AZC resistance. However, the mechanisms underlying these processes are poorly understood. Here, we identify a widespread mechanism for AZC resistance in fungi. We show that the filamentous ascomycete Aspergillus nidulans is able to not only resist AZC toxicity but also utilize it as a nitrogen source via GABA catabolism and the action of the AzhA hydrolase, a member of a large superfamily of detoxifying enzymes, the haloacid dehalogenase-like hydrolase (HAD) superfamily. This detoxification process is further assisted by the NgnA acetyltransferase, orthologue of Mpr1 of Saccharomyces cerevisiae. We additionally show that heterologous expression of AzhA protein can complement the AZC sensitivity of S. cerevisiae. Furthermore, a detailed phylogenetic analysis of AzhA homologues in Fungi, Archaea and Bacteria is provided. Overall, our results unravel a widespread mechanism for AZC resistance among microorganisms, including important human and plant pathogens.

Overlapping Roles of Yeast Transporters Aqr1, Qdr2, and Qdr3 in Amino Acid Excretion and Cross-Feeding of Lactic Acid Bacteria.

Microbial species occupying the same ecological niche or codeveloping during a fermentation process can exchange metabolites and mutualistically influence each other's metabolic states. For instance, yeast can excrete amino acids, thereby cross-feeding lactic acid bacteria unable to grow without an external amino acid supply. The yeast membrane transporters involved in amino acid excretion remain poorly known. Using a yeast mutant overproducing and excreting threonine (Thr) and its precursor homoserine (Hom), we show that excretion of both amino acids involves the Aqr1, Qdr2, and Qdr3 proteins of the Drug H+-Antiporter Family (DHA1) family. We further investigated Aqr1 as a representative of these closely related amino acid exporters. In particular, structural modeling and molecular docking coupled to mutagenesis experiments and excretion assays enabled us to identify residues in the Aqr1 substrate-binding pocket that are crucial for Thr and/or Hom export. We then co-cultivated yeast and Lactobacillus fermentum in an amino-acid-free medium and found a yeast mutant lacking Aqr1, Qdr2, and Qdr3 to display a reduced ability to sustain the growth of this lactic acid bacterium, a phenotype not observed with strains lacking only one of these transporters. This study highlights the importance of yeast DHA1 transporters in amino acid excretion and mutualistic interaction with lactic acid bacteria.

Fungal plasma membrane domains.

The plasma membrane (PM) performs a plethora of physiological processes, the coordination of which requires spatial and temporal organization into specialized domains of different sizes, stability, protein/lipid composition and overall architecture. Compartmentalization of the PM has been particularly well studied in the yeast Saccharomyces cerevisiae, where five non-overlapping domains have been described: The Membrane Compartments containing the arginine permease Can1 (MCC), the H+-ATPase Pma1 (MCP), the TORC2 kinase (MCT), the sterol transporters Ltc3/4 (MCL), and the cell wall stress mechanosensor Wsc1 (MCW). Additional cortical foci at the fungal PM are the sites where clathrin-dependent endocytosis occurs, the sites where the external pH sensing complex PAL/Rim localizes, and sterol-rich domains found in apically grown regions of fungal membranes. In this review, we summarize knowledge from several fungal species regarding the organization of the lateral PM segregation. We discuss the mechanisms of formation of these domains, and the mechanisms of partitioning of proteins there. Finally, we discuss the physiological roles of the best-known membrane compartments, including the regulation of membrane and cell wall homeostasis, apical growth of fungal cells and the newly emerging role of MCCs as starvation-protective membrane domains.

The nucleobase-ascorbate transporter (NAT) family: genomics, evolution, structure-function relationships and physiological role.

This review summarizes knowledge concerning a ubiquitous plasma transmembrane protein family that mediates nucleobase or ascorbate secondary active transport (NAT). We show that prototype bacterial and mostly fungal members have become unique model systems to unravel structure-function relationships and regulation of expression, using classical and reverse genetics, as well as biochemical approaches. We discuss the importance of NAT-mediated ascorbate transport in mammals and how changes in substrate specificity, from different nucleobases to ascorbate, might have evolved at the molecular level. Finally, we also discuss how modelling NAT-purine interactions might constitute a step towards the use of NAT proteins as specific gateways for targeting pathogenic microbes.

The yeast H+-ATPase Pma1 promotes Rag/Gtr-dependent TORC1 activation in response to H+-coupled nutrient uptake.

The yeast Target of Rapamycin Complex 1 (TORC1) plays a central role in controlling growth. How amino acids and other nutrients stimulate its activity via the Rag/Gtr GTPases remains poorly understood. We here report that the signal triggering Rag/Gtr-dependent TORC1 activation upon amino-acid uptake is the coupled H+ influx catalyzed by amino-acid/H+ symporters. H+-dependent uptake of other nutrients, ionophore-mediated H+ diffusion, and inhibition of the vacuolar V-ATPase also activate TORC1. As the increase in cytosolic H+ elicited by these processes stimulates the compensating H+-export activity of the plasma membrane H+-ATPase (Pma1), we have examined whether this major ATP-consuming enzyme might be involved in TORC1 control. We find that when the endogenous Pma1 is replaced with a plant H+-ATPase, H+ influx or increase fails to activate TORC1. Our results show that H+ influx coupled to nutrient uptake stimulates TORC1 activity and that Pma1 is a key actor in this mechanism.

Transition of yeast Can1 transporter to the inward-facing state unveils an α-arrestin target sequence promoting its ubiquitylation and endocytosis.

Substrate-transport-elicited endocytosis is a common control mechanism of membrane transporters avoiding excess uptake of external compounds, though poorly understood at the molecular level. In yeast, endocytosis of transporters is triggered by their ubiquitylation mediated by the Rsp5 ubiquitin-ligase, recruited by α-arrestin-family adaptors. We here report that transport-elicited ubiquitylation of the arginine transporter Can1 is promoted by transition to an inward-facing state. This conformational change unveils a region of the N-terminal cytosolic tail targeted by the Art1 α-arrestin, which is activated via the TORC1 kinase complex upon arginine uptake. Can1 mutants altered in the arginine-binding site or a cytosolic tripeptide sequence permanently expose the α-arrestin-targeted region so that Art1 activation via TORC1 is sufficient to trigger their endocytosis. We also provide evidence that substrate-transport elicited endocytosis of other amino acid permeases similarly involves unmasking of a cytosolic Art1-target region coupled to activation of Art1 via TORC1. Our results unravel a mechanism likely involved in regulation of many other transporters by their own substrates. They also support the emerging view that transporter ubiquitylation relies on combinatorial interaction rules such that α-arrestins, stimulated via signaling cascades or in their basal state, recognize transporter regions permanently facing the cytosol or unveiled during transport.

The α-Arrestin Bul1p Mediates Lactate Transporter Endocytosis in Response to Alkalinization and Distinct Physiological Signals.

Eukaryotic α-arrestins connect environmental or stress signaling pathways to the endocytosis of plasma membrane transporters or receptors. The Saccharomyces cerevisiae lactate transporter Jen1p has been used as a model cargo for elucidating the mechanisms underlying endocytic turnover in response to carbon sources. Here, we discover a novel pathway of Jen1p endocytosis mediated by the α-arrestin Bul1p in response to the presence of cycloheximide or rapamycin, or prolonged growth in lactate. While cycloheximide or rapamycin modify cells pleiotropically, the major effect of prolonged growth in lactate was shown to be external pH alkalinization. Importantly, employment of specific inactive Jen1p versions showed that Bul1p-dependent endocytosis requires lactate transport, according to the signal imposed. Our results support a model where conformational changes of Jen1p, associated with substrate/H+ symport, are critical for the efficiency of Bul1p-dependent Jen1p turnover.

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.