The proline transport protein of Aspergillus nidulans is very similar to amino acid transporters of Saccharomyces cerevisiae

Abstract

In Aspergillus nidulans, the gene prnB encoding the major proline transport system is one of a cluster of four genes necessary and sufficient for the utilization of proline as sole nitrogen and/or carbon source. The prn cluster has been cloned and the sequence and transcript map of the prnB gene are presented in this paper. The predicted translated sequence consists of 570 amino acids, resulting in a molecular weight of 63,028 Daltons. Its hydropathy profile shows 10 hydrophobic segments typical of integral membrane proteins. No N-terminal hydrophobic signal peptide is present, the N-terminal and C-terminal ends of the protein being hydrophilic. Similar results were previously found for the arginine and histidine transporters of Saccharomyces cerevisiae, with which the prnB transporter shares regions of highly conserved amino acid sequences. Using S1 mapping and Northern blot analyses, we confirm the presence of a unique inducible prnB transcript of 1.9 kb.

The gene encoding the major proline transporter of Aspergillus nidulans is upregulated during conidiospore germination and in response to proline induction and amino acid starvation

“…Cazelle and C. Scazzocchio, unpublished). It is very likely that amino acid starvation acts at the level of transcription, and that it is further supported by the effect of the mutation of a CpcA putative binding site, which is upstream of the prnB transcription start point (Sophianopoulou and Scazzocchio, 1989). No similar evidence exists for the PrnAindependent, CpcA-independent mRNA steady-state levels found in germinating conidiospores.…”

“…Extracted RNA was size-fractionated by electrophoresis on agarose gels after denaturation by glyoxal (Sambrook et al, 1989) and blotted on Hybond-N membranes (Amersham International). Hybridization was carried out in 0.5 M sodium orthophosphate, pH 7, 1 mM EDTA, 7% SDS and 1% bovine serum albumin at 65ЊC for at least 14 h. The DNA fragments used as probes to detect transcription of genes of interest were as follows: the 1.8 kb Pst I-Pst I fragment of pAN225 (Sophianopoulou and Scazzocchio, 1989) was used for the detection of prnB transcription, the 0.4 kb Hin dIII fragment of pAN910 (Gavrias 1992) was used for prnD transcription, the 0.7 kb Bgl II fragment of pAN914 (Gavrias 1992) was used for prnC transcription, and the 1.1 kb SphI-Hin dIII fragment of pFB39 (Berse et al, 1983) was used for argB transcription. In all Northern blots a 2.5 kb BamHI-KpnI restriction fragment of plasmid pSF5 (Fidel et al, 1988) was used to detect the A. nidulans actin (acnA) mRNA steady-state level as an internal control to monitor the amount of loaded mRNA.…”

“…Site-directed mutagenesis and construction of the mutant Aspergillus strain Site-directed mutagenesis (Kunkel et al, 1987) was performed on a pBluescript-II KSþ plasmid (Stratagene) containing a 0.5 kb EcoRI-Pst I restriction fragment of pAN225 (Sophianopoulou and Scazzocchio, 1989) cloned in the multiple cloning site. The oligonucleotide used to change the base situated at position ¹106 of the prnB start codon was 5Ј-GATAAGCCG-TAGACTCAGCAATCTGGGG-3Ј.…”

“…The prnA gene codes for a proline-responsive transcriptional activator, prnX is a proline-inducible gene of unknown function, prnD codes for proline oxidase, prnB for the proline transporter and prnC for ⌬ 1 -pyrroline-5-carboxylate dehydrogenase. prnA, prnX and prnD are transcribed divergently from prnB and prnC and thus there is a region between the prnD and prnB genes which may involve cisacting elements affecting the transcription of both genes (Arst and MacDonald, 1975;Arst et al, 1980;Sharma and Arst, 1985;Sophianopoulou and Scazzocchio, 1989;Gavrias et al, 1994;Sophianopoulou et al, 1993;Cubero and Scazzocchio, 1994).…”

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

“…Numerous YAT transporters can be identified in all sequenced genomes of fungi. Characterized members include 15 of the 16 characterized amino acid transporters of Saccharomyces cerevisiae (5,6), two proteins of Aspergillus nidulans, PrnB and AgtA (7,8), as well as transporters from Candida glabrata, Neurospora crassa, and Penicillium chrysogenum (9 -11). These proteins display different specificities and are subject to tight transcriptional and post-translational regulation (12)(13)(14).…”