Substrate- and ubiquitin-dependent trafficking of the yeast siderophore transporter Sit1

Eukaryotic plasma membrane transporters are subjected to a tightly regulated intracellular trafficking. The yeast siderophore iron transporter1 (Sit1) displays substrate-regulated trafficking. It is targeted to the plasma membrane or to a vacuolar degradative pathway when synthesized in the presence or absence of external substrate, respectively. Sorting of Sit1 to the vacuolar pathway is dependent on the clathrin adaptor Gga2, and more specifically on its C-GAT subdomain. Plasma membrane undergoes substrate-induced ubiquitylation dependent on the Rsp5 ubiquitin protein ligase. Sit1 is also ubiquitylated in an Rsp5-dependent manner in internal compartments when expressed in the absence of substrate. In several rsp5 mutants including cells deleted for RSP5 , Sit1 expressed in the absence of substrate is correctly targeted to the endosomal pathway but its sorting to multivesicular bodies (MVBs) is impaired. Consequently, it displays endosome to plasma membrane targeting, with kinetics similar to those observed in vps mutants defective for MVB sorting. Plasma membrane Sit1 is modified by Lys63-linked ubiquitin chains. We also show for the first time in yeast that modification by this latter type of ubiquitin chains is required directly or indirectly for efficient MVB sorting, as it is for efficient internalization at the plasma membrane.     

Ferrichrome induces endosome to plasma membrane cycling of the ferrichrome transporter,Arn1p,in Saccharomyces cerevisiae

Siderophores are small iron-binding molecules that are synthesized and secreted in the iron-free form by microorganisms. Saccharomyces cerevisiae takes up iron bound to siderophores by two separate systems, one of which requires the ARN family of sidero phore-iron transporters. Arn1p and Arn3p are expressed in endosome-like intracellular vesicles. Here we present evidence that, in the absence of its specific substrate, ferrichrome, Arn1p is sorted directly from the Golgi to the endosomal compartment and does not cycle to the plasma membrane. When cells are exposed to ferrichrome at low concentrations, Arn1p stably relocalizes to the plasma membrane. At higher concentrations of ferrichrome, Arn1p relocalizes to the plasma membrane and rapidly undergoes endocytosis. Plasma membrane localization of Arn1p occurs only in the presence of its specific substrate, and not in the presence of other siderophores. Despite expression of Arn1p on the plasma membrane, mutant strains with defects in endocytosis exhibit reduced uptake of ferrichrome-iron. Thus, siderophores influence the trafficking of the Arn transporters within the cell and this trafficking is important for transporter function.     

Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily

Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 fet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with ⁵⁵Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi.     

Phosphatidylserine is involved in the ferrichrome-induced plasma membrane trafficking of Arn1 in Saccharomyces cerevisiae

Arn1 is an integral membrane protein that mediates the uptake of ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.     

Siderophore reduction catalyzed by higher plant NADH:nitrate reductase

Squash cotyledon NADH:nitrate reductase catalyzes the reduction of the siderophore ferrioxamine B. The enzyme also reduced ferric ion in a buffer system containing the chelators oxalate and maleate. Ferrioxamine B reduction was maximal at pH 4; ferric ion reduction was maximal at pH 8. The present study indicates that iron assimilation by higher plants may occur with microbial siderophores serving as ferric ion sources and nitrate reductase functioning as the siderophore reductase.     

ATP-binding cassette B4, an auxin-efflux transporter, stably associates with the plasma membrane and shows distinctive intracellular trafficking from that of PIN-FORMED proteins

Intracellular trafficking of auxin transporters has been implicated in diverse developmental processes in plants. Although the dynamic trafficking pathways of PIN-FORMED auxin efflux proteins have been studied intensively, the trafficking of ATP-binding cassette protein subfamily B proteins (ABCBs; another group of auxin efflux carriers) still remains largely uncharacterized. In this study, we address the intracellular trafficking of ABCB4 in Arabidopsis (Arabidopsis thaliana) root epidermal cells. Pharmacological analysis showed that ABCB4 barely recycled between the plasma membrane and endosomes, although it slowly endocytosed via the lytic vacuolar pathway. Fluorescence recovery after photobleaching analysis revealed that ABCB4 is strongly retained in the plasma membrane, further supporting ABCB4's nonrecycling property. The endocytosis of ABCB4 was not dependent on the GNOM-LIKE1 function, and the sensitivity of ABCB4 to brefeldin A required guanine nucleotide exchange factors for adenosyl ribosylation factor other than GNOM. These characteristics of intracellular trafficking of ABCB4 are well contrasted with those of PIN-FORMED proteins, suggesting that ABCB4 may be a basic and constitutive auxin efflux transporter for cellular auxin homeostasis.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Vacuolar import of phosphatidylcholine requires the ATP-binding cassette transporter Ybt1

ATP-binding cassette (ABC) transporters are well known for their roles as multidrug resistance determinants but also play important roles in regulation of lipid levels. In the yeast Saccharomyces cerevisiae, the plasma membrane ABC transporter proteins Pdr5 and Yor1 are required for normal rates of transport of phosphatidyethanolamine to the surface of the cell. Loss of these ABC transporters causes a defect in phospholipid asymmetry across the plasma membrane and has been linked with slowed rates of trafficking of other membrane proteins. Four ABC transporter proteins are found on the limiting membrane of the yeast vacuole and loss of one of these vacuolar ABC transporters, Ybt1, caused a major defect in the normal delivery of the phosphatidylcholine (PC) analog NBD-PC (7-nitro-2,1,3-benzoxadiazol-PC) to the lumen of the vacuole. NBD-PC accumulates on cytosolic membranes in an ybt1Δ strain. We demonstrated that Ybt1 is required to import NBD-PC into vacuoles in the presence of ATP in vitro. Loss of Ybt1 prevented vacuolar remodeling of PC analogs. Turnover of Ybt1 was reduced under conditions in which function of this vacuolar remodeling pathway was required. Our data describe a novel vacuolar route for lipid remodeling and reutilization in addition to previously described enzymatic avenues in the cytoplasm.     

跨膜离子转运蛋白与植物耐盐的分子生物学

植物抵御盐害的主要方式是增加Na 的外排、减少Na 的吸入和Na 的区隔化,而Na 的跨膜运输主要由质膜和液泡膜上的离子转运蛋白完成。对质膜和液泡膜跨膜离子转运蛋白包括K /Na 离子转运蛋白,Na /H 逆向转运蛋白以及液泡膜H -PPase的分子生物学研究及应用进展进行了综述。     

Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters

K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant "halotolerance" protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+ -pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves.     

Glucose-induced Ubiquitylation and Endocytosis of the Yeast Jen1 Transporter: ROLE OF LYSINE 63-LINKED UBIQUITIN CHAINS*

Protein ubiquitylation is essential for many events linked to intracellular protein trafficking. Despite the significance of this process, the molecular mechanisms that govern the regulation of ubiquitylation remain largely unknown. Plasma membrane transporters are subjected to tightly regulated endocytosis, and ubiquitylation is a key signal at several stages of the endocytic pathway. The yeast monocarboxylate transporter Jen1 displays glucose-regulated endocytosis. We show here that casein kinase 1-dependent phosphorylation and HECT-ubiquitin ligase Rsp5-dependent ubiquitylation are required for Jen1 endocytosis. Ubiquitylation and endocytosis of Jen1 are induced within minutes in response to glucose addition. Jen1 is modified at the cell surface by oligo-ubiquitylation with ubiquitin-Lys⁶³ linked chain(s), and Jen1-Lys³³⁸ is one of the target residues. Ubiquitin-Lys⁶³-linked chain(s) are also required directly or indirectly to sort Jen1 into multivesicular bodies. Jen1 is one of the few examples for which ubiquitin-Lys⁶³-linked chain(s) was shown to be required for correct trafficking at two stages of endocytosis: endocytic internalization and sorting at multivesicular bodies.Ubiquitylation is one of the most prevalent protein post-translational modifications in eukaryotes. In addition to its role in promoting proteasomal degradation of target proteins, ubiquitylation has been shown to regulate multiple processes, including DNA repair, signaling, and intracellular trafficking. Ubiquitylation serves as a key signal mediating the internalization of plasma membrane receptors and transporters, followed by their intracellular transport and subsequent recycling or lysosomal/vacuolar degradation (1, 2). In Saccharomyces cerevisiae, transporters usually display both constitutive and accelerated endocytosis regulated by factors such as excess substrate, changes in nutrient availability, and stress conditions. Ubiquitylation of these cell surface proteins acts as a signal triggering their internalization (1). A single essential E3⁴ ubiquitin ligase, Rsp5, has been implicated in the internalization of most, if not all, endocytosed proteins (3). Rsp5 is the unique member in S. cerevisiae of the HECT (homologous to E6AP COOH terminus)-ubiquitin ligases of the Nedd4/Rsp5 family (4). In a few cases, Rsp5-dependent cell surface ubiquitylation was shown to involve PY-containing adapters that bind to Rsp5 (5–7). Rsp5-mediated ubiquitylation is also required for sorting into multivesicular bodies (MVBs) of endosomal membrane proteins that come from either the plasma membrane (through endocytosis) or the Golgi (through vacuolar protein sorting (VPS) pathway) (8). Although much progress has been made in elucidating the mechanistic basis of various steps in protein trafficking, the precise requirement for a specific type and length of Ub chains at various stages of the endocytic pathway remains to be addressed.The ubiquitin profile needed for proper internalization has been established for some yeast membrane proteins (1). The α-factor receptor Ste2 was described as undergoing monoubiquitylation on several lysines (multimonoubiquitylation). The a-factor receptor, Ste3p; the general transporter of amino acids, Gap1; the zinc transporter, Ztr1; and the uracil transporter, Fur4, have been shown to be modified by short chains of two to three ubiquitins, each attached to one, two, or more target lysine residues (oligo-ubiquitylation). Among them, Fur4 and Gap1 were the only transporters demonstrated to undergo plasma membrane oligo-ubiquitylation with ubiquitin residues linked via ubiquitin-Lys⁶³ (9, 10). In addition, the two siderophore transporters Arn1 and Sit1 were also shown to undergo Lys⁶³-linked cell surface ubiquitylation (11, 12). Whether these four transporters are representative of a larger class of plasma membrane substrates remains to be determined. Little is known about the type of ubiquitylation involved and/or required for sorting to MVBs. Some MVB cargoes appear to undergo monoubiquitylation (8), whereas Sna3, an MVB cargo of unknown function, undergoes Lys⁶³-linked ubiquitylation (13). Lys⁶³-linked ubiquitin chains were also recently reported to be required, directly or indirectly, for MVB sorting of the siderophore transporter, Sit1, when trafficking through the VPS pathway in the absence of its external substrate (11). In agreement with the possibility that additional membrane-bound proteins might undergo Lys⁶³-linked ubiquitylation, a proteomic study aiming to uncover ubiquitylated yeast proteins showed that Lys⁶³-ubiquitin chains are far more abundant than previously thought (14).The transport of monocarboxylates, such as lactate and pyruvate, as well as ketone bodies across the plasma membrane is essential for the metabolism of cells of various organisms. A family of monocarboxylate transporters has been reported that includes mainly mammalian members (15). In S. cerevisiae, two monocarboxylate-proton symporters have been described, Jen1 and Ady2 (16, 17). These transporters exhibit differences in their mechanisms of regulation and specificity. Jen1 is a lactate-pyruvate-acetate-propionate transporter induced in lactic or pyruvic acid-grown cells (18). Ady2, which accepts acetate, propionate, or formate, is present in cells grown in non-fermentable carbon sources (19). Jen1 has unique regulatory characteristics and has been extensively studied. It was the first secondary porter of S. cerevisiae characterized by heterologous expression in Pichia pastoris at both the cell and the membrane vesicle levels (20). The addition of glucose to lactic acid-grown cells very rapidly triggers loss of Jen1 activity and repression of JEN1 gene expression (21, 22). Newly synthesized Jen1-GFP fusion protein is sorted to the plasma membrane in an active and stable form, and loss of Jen1-GFP activity upon glucose addition is the result of its endocytosis followed by vacuolar degradation (23). Data from large scale analyses based on mass spectrometry approaches led to the detection of two sites of ubiquitylation for Jen1, one located in the N terminus of the protein and the second in the central loop (14), and several sites of phosphorylation in the N terminus, central loop, and C terminus of the protein (14, 24). In the present study, we aimed at further characterizing the internalization step of endocytosis of the transporter Jen1 and the potential role of the phosphorylation and ubiquitylation events required for its correct endocytic trafficking.     

P-glycoprotein4 displays auxin efflux transporter-like action in Arabidopsis root hair cells and tobacco cells

ATP binding cassette (ABC) transporters transport diverse substrates across membranes in various organisms. However, plant ABC transporters have only been scantily characterized. By taking advantage of the auxin-sensitive Arabidopsis thaliana root hair cell and tobacco (Nicotiana tabacum) suspension cell systems, we show here that Arabidopsis P-glycoprotein4 (PGP4) displays auxin efflux activity in plant cells. Root hair cell-specific overexpression of PGP4 (PGP4ox) and known auxin efflux transporters, such as PGP1, PGP19, and PIN-FORMEDs, decreased root hair elongation, whereas overexpression of the influx transporter AUXIN-RESISTANT1 enhanced root hair length. PGP4ox-mediated root hair shortening was rescued by the application of auxin or an auxin efflux inhibitor. These results indicate that the increased auxin efflux activity conferred by PGP4 reduces auxin levels in the root hair cell and consequently inhibits root hair elongation. PGP4ox in tobacco suspension cells also increased auxin efflux. PGP4 proteins were targeted to the plasma membrane of Arabidopsis root hair cells and tobacco cells without any clear subcellular polarity. Brefeldin A partially interfered with the trafficking of PGP4 reversibly, and this was rescued by pretreatment with auxin. These results suggest that PGP4 is an auxin efflux transporter in plants and that its trafficking to the plasma membrane involves both BFA-sensitive and -insensitive pathways.     

Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes

Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe³⁺-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe³⁺-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe³⁺ bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe³⁺ or Fe²⁺ forms or Fe³⁺ associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.     

Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean (Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.     

Identification of a vacuolar sucrose transporter in barley and Arabidopsis mesophyll cells by a tonoplast proteomic approach

The vacuole is the main cellular storage pool, where sucrose (Suc) accumulates to high concentrations. While a limited number of vacuolar membrane proteins, such as V-type H(+)-ATPases and H(+)-pyrophosphatases, are well characterized, the majority of vacuolar transporters are still unidentified, among them the transporter(s) responsible for vacuolar Suc uptake and release. In search of novel tonoplast transporters, we used a proteomic approach, analyzing the tonoplast fraction of highly purified mesophyll vacuoles of the crop plant barley (Hordeum vulgare). We identified 101 proteins, including 88 vacuolar and putative vacuolar proteins. The Suc transporter (SUT) HvSUT2 was discovered among the 40 vacuolar proteins, which were previously not reported in Arabidopsis (Arabidopsis thaliana) vacuolar proteomic studies. To confirm the tonoplast localization of this Suc transporter, we constructed and expressed green fluorescent protein (GFP) fusion proteins with HvSUT2 and its closest Arabidopsis homolog, AtSUT4. Transient expression of HvSUT2-GFP and AtSUT4-GFP in Arabidopsis leaves and onion (Allium cepa) epidermal cells resulted in green fluorescence at the tonoplast, indicating that these Suc transporters are indeed located at the vacuolar membrane. Using a microcapillary, we selected mesophyll protoplasts from a leaf protoplast preparation and demonstrated unequivocally that, in contrast to the companion cell-specific AtSUC2, HvSUT2 and AtSUT4 are expressed in mesophyll protoplasts, suggesting that HvSUT2 and AtSUT4 are involved in transport and vacuolar storage of photosynthetically derived Suc.     

Constitutive Tor2 Activity Promotes Retention of the Amino Acid Transporter Agp3 at Trans-Golgi/Endosomes in Fission Yeast

Amino acid transporters are located at specific subcellular compartments, and their localizations are regulated by the extracellular availability of amino acids. In yeast, target of rapamycin (TOR) activation induces the internalization of amino acid transporters located at the plasma membrane. However, whether and how TOR signaling regulates other amino acid transporters located at intracellular compartments remains unknown. Here, we demonstrate that in the fission yeast, the TOR inhibitor Torin–1 induces the transfer of several yellow fluorescent protein (YFP)-fused intracellular amino acid transporters, including Agp3, Isp5, Aat1, and Put4, from trans-Golgi/endosomes into the vacuoles. By contrast, the localizations of YFP-fused Can1, Fnx1, and Fnx2 transporter proteins were unaffected upon Torin–1 treatment. There are two TOR isoforms in fission yeast, Tor1 and Tor2. Whereas tor1 deletion did not affect the Torin-1-induced transfer of Agp3-YFP, Tor2 inhibition using a temperature-sensitive mutant induced the transfer of Agp3-YFP to the vacuolar lumen, similar to the effects of Torin–1 treatment. Tor2 inhibition also induced the transfer of the YFP-fused Isp5, Aat1, and Put4 transporter proteins to the vacuoles, although only partial transfer of the latter two transporters was observed. Under nitrogen depletion accompanied by reduced Tor2 activity, Agp3-YFP was transferred from the trans-Golgi/endosomes to the plasma membrane and then to the vacuoles, where it was degraded by the vacuolar proteases Isp6 and Psp3. Mutants with constitutively active Tor2 showed delayed transfer of Agp3-YFP to the plasma membrane upon nitrogen depletion. Cells lacking Tsc2, a negative regulator of Tor2, also showed a delay in this process in a Tor2-dependent manner. Taken together, these findings suggest that constitutive Tor2 activity is critical for the retention of amino acid transporters at trans-Golgi/endosomes. Moreover, nitrogen depletion suppresses Tor2 activity through Tsc2, thereby promoting the surface expression of these transporters.     

Rapid evolution of a bacterial iron acquisition system

Under iron limitation, bacteria scavenge ferric (Fe³⁺) iron bound to siderophores or other chelates from the environment to fulfill their nutritional requirement. In gram‐negative bacteria, the siderophore uptake system prototype consists of an outer membrane transporter, a periplasmic binding protein and a cytoplasmic membrane transporter, each specific for a single ferric siderophore or siderophore family. Here, we show that spontaneous single gain‐of‐function missense mutations in outer membrane transporter genes of Bradyrhizobium japonicum were sufficient to confer on cells the ability to use synthetic or natural iron siderophores, suggesting that selectivity is limited primarily to the outer membrane and can be readily modified. Moreover, growth on natural or synthetic chelators required the cytoplasmic membrane ferrous (Fe²⁺) iron transporter FeoB, suggesting that iron is both dissociated from the chelate and reduced to the ferrous form within the periplasm prior to cytoplasmic entry. The data suggest rapid adaptation to environmental iron by facile mutation of selective outer membrane transporter genes and by non‐selective uptake components that do not require mutation to accommodate new iron sources.