Multiplicity of the Amino Acid Permeases in Saccharomyces cerevisiae IV. Evidence for a General Amino Acid Permease

Kinetic and genetic evidences are presented to show that, in addition to specific amino acid permeases, Saccharomyces cerevisiae has a general amino acid permease which catalyzes the transport of basic and neutral amino acids, but most probably not that of proline. The general amino acid permease appears to be constitutive, and its activity is inhibited when ammonium ions are added to the culture medium. A mutant which has lost the general amino acid permease activity was isolated. Its mutation, named gap (general amino acid permease), is not allelic to the aap (amino acid permease) mutation of Surdin et al., which has a quite different phenotype and cannot be considered as having selectively lost the general amino acid permease activity.     

Uptake of the β-Lactam Precursor α-Aminoadipic Acid in Penicillium chrysogenum Is Mediated by the Acidic and the General Amino Acid Permease

External addition of the β-lactam precursor α-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular α-aminoadipic acid concentration and an increase in penicillin production. The exact route for α-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of α-aminoadipic acid, although PcGap1 showed a higher affinity for α-aminoadipic acid than PcDip5 (K_m values, 230 and 800 μM, respectively). Leucine strongly inhibits α-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the α-aminoadipic acid flux in β-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the α-aminoadipic acid flux.     

Loss of Gibberellin Production in Fusarium verticillioides (Gibberella fujikuroi MP-A) Is Due to a Deletion in the Gibberellic Acid Gene Cluster

Fusarium verticillioides (Gibberella fujikuroi mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the Gibberella fujikuroi species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to Fusarium fujikuroi (G. fujikuroi MP-C) and Fusarium konzum (MP-I), although most members of the G. fujikuroi species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in F. verticillioides (MP-A) is due to the loss of a majority of the GA gene cluster as found in F. fujikuroi. The remaining part of the cluster consists of the full-length F. verticillioides des gene (Fvdes), encoding the GA₄ desaturase, and the coding region of FvP450-4, encoding the ent-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of F. fujikuroi. The GA production capacity of F. verticillioides was restored by transforming a cosmid with the entire GA gene cluster from F. fujikuroi, indicating the existence of an active regulation system in F. verticillioides. Furthermore, the GA₄ desaturase gene des from F. verticillioides encodes an active enzyme which was able to restore the GA production in a corresponding des deletion mutant of F. fujikuroi.     

Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation.     

Agp2, a Member of the Yeast Amino Acid Permease Family,Positively Regulates Polyamine Transport at the Transcriptional Level

Agp2 is a plasma membrane protein of the Saccharomyces cerevisiae amino acid transporter family, involved in high-affinity uptake of various substrates including L-carnitine and polyamines. The discovery of two high affinity polyamine permeases, Dur3 and Sam3, prompted us to investigate whether Agp2 directly transports polyamines or acts instead as a regulator. Herein, we show that neither dur3Δ nor sam3Δ single mutant is defective in polyamine transport, while the dur3Δ sam3Δ double mutant exhibits a sharp decrease in polyamine uptake and an increased resistance to polyamine toxicity similar to the agp2Δ mutant. Studies of Agp2 localization indicate that in the double mutant dur3Δ sam3Δ, Agp2-GFP remains plasma membrane-localized, even though transport of polyamines is strongly reduced. We further demonstrate that Agp2 controls the expression of several transporter genes including DUR3 and SAM3, the carnitine transporter HNM1 and several hexose, nucleoside and vitamin permease genes, in addition to SKY1 encoding a SR kinase that positively regulates low-affinity polyamine uptake. Furthermore, gene expression analysis clearly suggests that Agp2 is a strong positive regulator of additional biological processes. Collectively, our data suggest that Agp2 might respond to environmental cues and thus regulate the expression of several genes including those involved in polyamine transport.     

The Saccharomyces cerevisiae YCC5 (YCL025c) Gene Encodes an Amino Acid Permease, Agp1, Which Transports Asparagine and Glutamine

The yeast YCC5 gene encodes a putative amino acid permease and is homologous to GNP1 (encoding a high-affinity glutamine permease). Using strains with disruptions in the genes for multiple permeases, we demonstrated that Ycc5 (which we have renamed Agp1) is involved in the transport of asparagine and glutamine, performed a kinetic analysis of this activity, and showed that AGP1 expression is subject to nitrogen repression.     

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 (Gly⁵⁶, Thr⁵⁷), TMS3 (Glu¹³⁸), and TMS6 (Phe²⁴⁸), 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 K_m 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.     

Analysis of the global regulator Lae1 uncovers a connection between Lae1 and the histone acetyltransferase HAT1 in Fusarium fujikuroi

Applied Microbiology and Biotechnology - The fungus Fusarium fujikuroi causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), a family of plant hormones....     

The Saccharomyces cerevisiae v-SNARE Vti1p Is Required for Multiple Membrane Transport Pathways to the Vacuole

The interaction between v-SNAREs on transport vesicles and t-SNAREs on target membranes is required for membrane traffic in eukaryotic cells. Here we identify Vti1p as the first v-SNARE protein found to be required for biosynthetic traffic into the yeast vacuole, the equivalent of the mammalian lysosome. Certain vti1-ts yeast mutants are defective in alkaline phosphatase transport from the Golgi to the vacuole and in targeting of aminopeptidase I from the cytosol to the vacuole. VTI1 interacts genetically with the vacuolar t-SNARE VAM3, which is required for transport of both alkaline phosphatase and aminopeptidase I to the vacuole. The v-SNARE Nyv1p forms a SNARE complex with Vam3p in homotypic vacuolar fusion; however, we find that Nyv1p is not required for any of the three biosynthetic pathways to the vacuole. v-SNAREs were thought to ensure specificity in membrane traffic. However, Vti1p also functions in two additional membrane traffic pathways: Vti1p interacts with the t-SNAREs Pep12p in traffic from the TGN to the prevacuolar compartment and with Sed5p in retrograde traffic to the cis-Golgi. The ability of Vti1p to mediate multiple fusion steps requires additional proteins to ensure specificity in membrane traffic.     

The Membrane Protein Alkaline Phosphatase Is Delivered to the Vacuole by a Route That Is Distinct from the VPS-dependent Pathway

Membrane trafficking intermediates involved in the transport of proteins between the TGN and the lysosome-like vacuole in the yeast Saccharomyces cerevisiae can be accumulated in various vps mutants. Loss of function of Vps45p, an Sec1p-like protein required for the fusion of Golgi-derived transport vesicles with the prevacuolar/endosomal compartment (PVC), results in an accumulation of post-Golgi transport vesicles. Similarly, loss of VPS27 function results in an accumulation of the PVC since this gene is required for traffic out of this compartment.

The vacuolar ATPase subunit Vph1p transits to the vacuole in the Golgi-derived transport vesicles, as defined by mutations in VPS45, and through the PVC, as defined by mutations in VPS27. In this study we demonstrate that, whereas VPS45 and VPS27 are required for the vacuolar delivery of several membrane proteins, the vacuolar membrane protein alkaline phosphatase (ALP) reaches its final destination without the function of these two genes. Using a series of ALP derivatives, we find that the information to specify the entry of ALP into this alternative pathway to the vacuole is contained within its cytosolic tail, in the 13 residues adjacent to the transmembrane domain, and loss of this sorting determinant results in a protein that follows the VPS-dependent pathway to the vacuole.

Using a combination of immunofluorescence localization and pulse/chase immunoprecipitation analysis, we demonstrate that, in addition to ALP, the vacuolar syntaxin Vam3p also follows this VPS45/27-independent pathway to the vacuole. In addition, the function of Vam3p is required for membrane traffic along the VPS-independent pathway.

     

Fusarium Species from the Fusarium fujikuroi Species Complex Involved in Mixed Infections of Maize in Northern Sinaloa,Mexico

Fusarium species belonging to the Fusarium fujikuroi species complex (FFSC) are associated with maize in northern Mexico and cause Fusarium ear and root rot. In order to assess the diversity of FFSC fungal species involved in this destructive disease in Sinaloa, Mexico, a collection of 108 fungal isolates was obtained from maize plants in 2007–2011. DNA sequence analysis of the calmodulin and elongation factor 1α genes identified four species: Fusarium verticillioides, F. nygamai, F. andiyazi and F. thapsinum (comprising 79, 23, 4 and 2 isolates, respectively). Differential distribution of Fusarium species in maize organs was observed, that is F. verticillioides was the most frequently isolated species from maize seeds, while F. nygamai predominated on maize roots. Mixed infections with F. verticillioides/F. thapsinum and F. verticillioides/F. nygamai were detected in maize seeds and roots, respectively. Pathogenicity assay demonstrated the ability of the four species to infect maize seedlings and induce different levels of disease severity, reflecting variation in aggressiveness, plant height and root biomass. Isolates of F. verticillioides and F. nygamai were the most aggressive. These species were able to colonize all root tissues, from the epidermis to the vascular vessels, while infection by F. andiyazi and F. thapsinum was restricted to the epidermis and adjacent cortical cells. This is the first report of F. nygamai, F. andiyazi and F. thapsinum infecting maize in Mexico and co‐infecting with F. verticillioides. Mixed infections should be taken into consideration due to the production and/or accumulation of diverse mycotoxins in maize grain.     

The White Collar protein WcoA of Fusarium fujikuroi is not essential for photocarotenogenesis, but is involved in the regulation of secondary metabolism and conidiation

The fungal proteins of the White Collar photoreceptor family, represented by WC-1 from Neurospora crassa, mediate the control by light of different biochemical and developmental processes, such as carotenogenesis or sporulation. Carotenoid biosynthesis is induced by light in the gibberellin-producing fungus Fusarium fujikuroi. In an attempt to identify the photoreceptor for this response, we cloned the only WC-1-like gene present in the available Fusarium genomes, that we called wcoA. The predicted WcoA polypeptide is highly similar to WC-1 and contains the relevant functional domains of this protein. In contrast to the Neurospora counterpart, wcoA expression is not affected by light. Unexpectedly, targeted wcoA disruptant strains maintain the light-induced carotenogenesis. Furthermore, the wcoA mutants show a drastic reduction of fusarin production in the light, and produce less gibberellins and more bikaverins than the parental strain under nitrogen-limiting conditions. The changes in the production of the different products indicate a key regulatory role for WcoA in secondary metabolism of this fungus. Additionally, the mutants are severely affected in conidiation rates under different culture conditions, indicating a more general regulatory role for this protein.