Functional Expression and Cellular Localization of a Green Fluorescent Protein-Tagged Proline Transporter in Aspergillus nidulans

Background: PrnB, different from Put4p, its Saccharomyces cerevisiae orthologue, is a highly specific L-proline transporter of Aspergillus nidulans. Results: Identification of 12 residues important for PrnB activity and/or specificity. Put4p-mimicking mutants of PrnB recognize other Put4p substrates. Conclusion: Residues variable in the yeast amino acid transporter (YAT) family determine transporter specificity. Significance: Understanding the molecular mechanisms underlying amino acid recognition and translocation through YATs.

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“…These phenotypes were more prominent at 25°C (Fig. 8), consistent with the partial thermosensitivity of PrnB-GFP (50). Altogether (Fig.…”

Section: Table 4 Specificity Profile Of Functional Prnb Mutants Carrysupporting

confidence: 82%

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

Gournas

Evangelidis

Αθανασόπουλος

et al. 2015

Journal of Biological Chemistry

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“…Vacuole staining with 7-amino-4-chloromethyl coumarin (CMAC; Molecular Probes, Inc.) was as described by Tavoularis at al. (45). Coverslips with germinated conidia were placed on top of plastic covers, covered with 0.1 ml of 1/1,000 dilution of CMAC (5-mg/ml stock solution), incubated at 25°C for 30 min, washed twice with phosphate-buffered saline (PBS), and observed under a phase-contrast fluorescent microscope with a 4Ј,6-diamidino-2-phenylindole filter (excitation, 365/10 nm; UV-1A filter; model DM 400 digital microscope and model BA 400 light microscope [Leica]).…”

Section: Methodsmentioning

confidence: 99%

“…The VS172 (agtA::sgfp pilA⌬::Afribo ϩ ) strain was isolated by crossing the VS86 and VS125 strains. The VS186 strain was isolated by crossing the VS83 strain with the TpA4 strain (45). The nkuA⌬ pyrG89 riboB2 and nkuA pyrG89 pyroA4 strains were kindly provided by M. Peñalva and were used for the deletion and the in-locus fusions of the pilA, pilB, and surG genes.…”

Section: Methodsmentioning

confidence: 99%

“…Ten milliliters of a suspension of 5 ϫ 10 5 or 5 ϫ 10 7 conidia/ml was inoculated onto sterile coverslips embedded into appropriate liquid culture media, incubated for 4 h or 16 h at 25°C, and observed by fluorescence microscopy (FM) and laser scanning confocal microscopy (CSLM), as described previously (45,46). Two hundred microliters of a suspension of 5 ϫ 10 7 conidia/ml was inoculated into sterile Eppendorf tubes and observed by FM and CSLM, as described previously (45,46). To elicit AgtA-GFP endocytosis, ammonium L-(ϩ)-tartrate was added to a final concentration of 10 mM at 30, 60, and 120 min before observation.…”

Section: Methodsmentioning

confidence: 99%

“…nidulans is arguably, among the Pezizomycotina, the organism where membrane proteins and endocytosis have been better studied. In this organism, a number of transporters driven by their physiological promoters have been visualized in the cell membrane (UapC [48], PrnB [45], UapA and AzgA [35], AgtA [5], MstE [14], FcyB [51], UreA [M. Sanguinetti and A. Ramón, personal communication], and FurD [G. Diallinas and F. Borbolis, personal communication]). Load-and-chase experiments have shown that when the dye FM4-64 is endocytosed, it first appears in cortical punctate structures (36).…”

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confidence: 99%

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Eisosome Organization in the Filamentous AscomyceteAspergillus nidulans

et al. 2010

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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.

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Genomics of Protein Secretion and Hyphal Growth in Aspergillus

Archer

Turner

The Mycota

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a These sites provide an entry point to the sequence data but access requires agreement being reached with the respective company b Genome sequence data for A. oryzae are not yet available. A summary of the A. oryzae genome sequencing project and related topics has been published (Machida 2002) Table. 5.1. Websites for Aspergillus genome information (modified after Archer and Dyer 2004) Table. 5.2. Summary of information derived from the genome sequences of Aspergillus species a A. fumigatus b A. nidulans c A. niger d A. oryzae e Genome size (Mb) f 29 30 36 37 Predicted genes f 10,000 10,000 14,000 14,000 Genes with Pfam hits f 4,400 4,500 5,300 5,300 a This table was published in extended form elsewhere (Archer and Dyer 2004), and the data were obtained from the websites and contributions from William Nierman (TIGR, USA), James Galagan (Broad Institute, USA), Masa Machida (Tsukuba, Japan), and Gert Groot and Noel van Peij (DSM, The Netherlands) b

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Functional Expression and Cellular Localization of a Green Fluorescent Protein-Tagged Proline Transporter in Aspergillus nidulans

Cited by 27 publications

References 37 publications

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

Eisosome Organization in the Filamentous AscomyceteAspergillus nidulans

Genomics of Protein Secretion and Hyphal Growth in Aspergillus

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