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.     

Missense mutations that inactivate the Aspergillus nidulans nrtA gene encoding a high-affinity nitrate transporter

The transport of nitrate into prokaryotic and eukaryotic cells, of considerable interest to agriculture, ecology, and human health, is carried out by members of a distinct cluster of proteins within the major facilitator superfamily. To obtain structure/function information on this important class of nitrate permeases, a collection of chemically induced mutations in the nrtA gene encoding a 12-transmembrane domain, high-affinity nitrate transporter from the eukaryote Aspergillus nidulans was isolated and characterized. This mutational analysis, coupled with protein alignments, demonstrates the utility of the approach to predicting peptide motifs and individual residues important for the movement of nitrate across the membrane. These include the highly conserved nitrate signature motif (residues 166-173) in Tm 5, the conserved charged residues Arg87 (Tm 2) and Arg368 (Tm 8), as well as the aromatic residue Phe47 (Tm 1), all within transmembrane helices. No mutations were observed in the large central loop (Lp 6/7) between Tm 6 and Tm 7. Finally, the study of a strain with a conversion of Trp481 (Tm 12) to a stop codon suggests that all 12 transmembrane domains and/or the C-terminal tail are required for membrane insertion and/or stability of NrtA.     

The product of the SHR3 orthologue of Aspergillus nidulans has restricted range of amino acid transporter targets

The shrA gene of Aspergillus nidulans codes for a structural and functional homologue of Shr3p, a yeast ER membrane protein, which plays a crucial role in the secretory pathway of yeast amino acid permeases. shrA is a single-copy gene, whose expression is early activated during germination of A. nidulans conidiospores. ShrA is localized in the ER of the fungal cells and partially complements the shr3delta phenotype. Differently from Saccharomyces cerevisiae, where SHr3p is necessary for membrane localization of the majority of amino acid permeases, deletion of the shrA locus in A. nidulans impairs a limited number of amino acid uptake activities, including those responsible for proline and aspartate transport. Strongly reduced membrane levels of a PrnB-sGFP fusion in a shrAdelta background clearly suggest a direct role of ShrA in the topogenesis of the proline specific transporter.     

Metabolite repression and inducer exclusion in the proline utilization gene cluster of Aspergillus nidulans

The clustered prnB, prnC, and prnD genes are repressed by the simultaneous presence of glucose and ammonium. A derepressed mutation inactivating a CreA-binding site acts in cis only on the permease gene (prnB) while derepression of prnD and prnC is largely the result of reversal of inducer exclusion.     

Basic and neutral amino acid transport in Aspergillus nidulans.

Arginine and methionine transport by Aspergillus nidulans mycelium was investigated. A single uptake system is responsible for the transport of arginine, lysine and ornithine. Transport is energy-dependent and specific for these basic amino acids. The Km value for arginine is 1 X 10(-5) M, and Vmax is 2-8 nmol/mg dry wt/min; Km for lysine is 8 X 10(-6) M; Kt for lysine as inhibitor of arginine uptake is 12 muM, and Ki for ornithine is mM. On minimal medium, methionine is transported with a Km of 0-I mM and Vmax about I nmol/mg dry wt/min; transport is inhibited by azide. Neutral amnio acids such as serine, phenylalanine and leucine are probably transported by the same system, as indicated by their inhibition of methionine uptake and the existence of a mutant specifically impaired in their transport. The recessive mutant nap3, unable to transport neutral amino acids, was isolated as resistant to selenomethionine and p-fluorophenylanine. This mutant has unchanged transport of methionine by general and specific sulphur-regulated permeases.     

Endosomal microautophagy is an integrated part of the autophagic response to amino acid starvation

Starvation is a fundamental type of stress naturally occurring in biological systems. All organisms have therefore evolved different safeguard mechanisms to cope with deficiencies in various types of nutrients. Cells, from yeast to humans, typically respond to amino acid starvation by initiating degradation of cellular components by inducing autophagy. This degradation releases metabolic building blocks to sustain essential core cellular processes. Increasing evidence indicates that starvation-induced autophagy also acts to prepare cells for prolonged starvation by degrading key regulators of different cellular processes. In a recent study, we found that within the first hours of amino acid starvation cells elicit an autophagic response causing rapid degradation of specific proteins. The response is executed independently of both MTOR and canonical macroautophagy. Based on RNAi-mediated knockdown of essential components of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery and electron microscopy we conclude that the response relies on some sort of endosomal microautophagy, hence vesicle budding into endosomes. Substantiated by the different substrates that are selectively degraded by this novel pathway we propose that the response predominantly acts to prepare cells for prolonged starvation. Intriguingly, this includes shutting down selective macroautophagy in preparation for a massive induction of bulk macroautophagy.     

Functional expression and cellular localization of a green fluorescent protein-tagged proline transporter in Aspergillus nidulans.

The PrnB protein is a highly specific proline transporter that belongs to an amino acid transporter family conserved in both prokaryotes and eukaryotes. In this work, we detected and analyzed the cellular localization of PrnB in vivo by means of green fluorescent protein (GFP) fusions. Several prnB-gfp gene fusions, driven by prnB native promoter sequences, were constructed and targeted to the genomic locus of a prnB null mutant. Chimeric proteins containing GFP fused to the C terminus of PrnB through a linker of two, four, or eight amino acids, with low potential to form secondary structure elements, were shown to be functional in vivo. A two-linker fusion results in partial complementation at both 25 and 37 degrees C. A four-linker fusion affords almost full complementation at 25 degrees C but partial complementation at 37 degrees C, whereas the eight-linker fusion results in partial complementation at both temperatures but in no GFP fluorescence. These results show that the number of linker amino acids is critical for the correct expression and/or translocation of PrnB-GFP fused proteins to the plasma membrane and for the fluorescence of the GFP. The expression of the four-linker PrnB-GFP transporter was detected and analyzed in vivo by both conventional fluorescence and confocal laser microscopy. This chimeric protein is localized in the plasma membrane, secondarily in large vacuoles found in the swollen conidial end of the germlings, and in other small intracellular structures observed as fluorescent granules. A strong correlation between known patterns of PrnB expression and intensity of PrnB-GFP fluorescence was observed. This work also demonstrates that the GFP fusion technology is a unique tool with which to study the expression and cellular localization of low-abundance transmembrane transporters expressed from their native promoters.