Mutational analysis of the major proline transporter (PrnB) of Aspergillus nidulans.

PrnB, the l-proline transporter of Aspergillus nidulans, belongs to the Amino acid Polyamine Organocation (APC) transporter family conserved in prokaryotes and eukaryotes. In silico analysis and limited biochemical evidence suggest that APC transporters comprise 12 transmembrane segments (TMS) connected with relatively short hydrophilic loops (L). However, very little is known on the structure-function relationships in APC transporters. This work makes use of the A. nidulans PrnB transporter to address structure-function relationships by selecting, constructing and analysing several prnB mutations. In the sample, most isolated missense mutations affecting PrnB function map in the borders of cytoplasmic loops with transmembrane domains. These are I119N and G120W in L2-TMS3, F278V in L6-TMS7, NRT378NRTNRT and PY382PYPY in L8-TMS9 and T456N in L10-TMS11. A single mutation (G403E) causing, however, a very weak phenotype, maps in the borders of an extracellular loop (L9-TMS10). An important role of helix TMS6 for proline binding and transport is supported by mutations K245L and, especially, F248L that clearly affect PrnB uptake kinetics. The critical role of these residues in proline binding and transport is further shown by constructing and analysing isogenic strains expressing selected prnB alleles fused to the gene encoding the Green Fluorescent Protein (GFP). It is shown that, while some prnB mutations affect proper translocation of PrnB in the membrane, at least two mutants, K245E and F248L, exhibit physiological cellular expression of PrnB and, thus, the corresponding mutations can be classified as mutations directly affecting proline binding and/or transport. Finally, comparison of these results with analogous studies strengthens conclusions concerning amino acid residues critical for function in APC transporters.

The mammary gland iodide transporter is expressed during lactation and in breast cancer.

The sodium/iodide symporter mediates active iodide transport in both healthy and cancerous thyroid tissue. By exploiting this activity, radioiodide has been used for decades with considerable success in the detection and treatment of thyroid cancer. Here we show that a specialized form of the sodium/iodide symporter in the mammary gland mediates active iodide transport in healthy lactating (but not in nonlactating) mammary gland and in mammary tumors. In addition to characterizing the hormonal regulation of the mammary gland sodium/iodide symporter, we demonstrate by scintigraphy that mammary adenocarcinomas in transgenic mice bearing Ras or Neu oncogenes actively accumulate iodide by this symporter in vivo. Moreover, more than 80% of the human breast cancer samples we analyzed by immunohistochemistry expressed the symporter, compared with none of the normal (nonlactating) samples from reductive mammoplasties. These results indicate that the mammary gland sodium/iodide symporter may be an essential breast cancer marker and that radioiodide should be studied as a possible option in the diagnosis and treatment of breast cancer.

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.

In Aspergillus nidulans a highly specific L-proline transporter is encoded by the prnB gene which is tightly linked to all other genes involved in proline catabolism. In mycelia, the expression of the prn structural genes is finely co-regulated in response to proline induction and nitrogen/carbon catabolite repression. In this study we establish that prnB expression is also activated during germination of conidiospores. This activation persists until the development of 6 h-old mycelia and it is independent of proline induction mediated by the pathway-specific prnA gene product. We then show that, in mycelia, prnB transcription is activated in response to proline or histidine starvation. This process has two components: a prnA-dependent and a prnA-independent component. A cis-acting element that conforms to the consensus target of the GCN4/CPC1 transcriptional activators mediating amino acid biosynthesis activation in other fungi is involved in the activation of prnB transcription in response to amino acid starvation. We also show that the stimulation of prnB expression in germinating conidiospores is not due exclusively to transient internal amino acid starvation occurring during the transition from conidiospore to mycelium. This is the first report that an amino acid transporter gene is upregulated during development and in response to amino acid starvation and specific amino acid induction.

Post-transcriptional control and kinetic characterization of proline transport in germinating conidiospores of Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, L-proline uptake is mediated by the product of the prnB gene which codes for a member of a family of amino acid transporters found both in pro- and eukaryotes. Regulation of prnB gene expression has previously been studied in great detail at the molecular level. However, no studies have addressed possible post-transcriptional controls or the kinetic characterisation of the PrnB transporter. Here we develop a rapid and efficient method for direct uptake measurements of proline in germinating conidiospores of A. nidulans. We make use of this method and Northern blot analyses in parallel to study the regulation of PrnB expression both at the level of prnB message accumulation and at a post-transcriptional level. These studies show that (i) pathway-specific and wide-domain regulatory systems, previously shown to control prnB gene expression in multicellular mycelia, also operate in unicellular conidia committed to germination; and (ii) PrnB activity is regulated in response to the nitrogen source present in the medium and the level of internally accumulated proline or other amino acids. We also characterise kinetically the PrnB transporter and a secondary proline transport system. Our results open new possibilities for studies using unicellular conidiospores of filamentous fungi and constitute a necessary first step for a subsequent structure-function analysis of the PrnB transporter.