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.     

Functional characterization of a maize purine transporter by expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.     

A novel improved method for Aspergillus nidulans transformation

We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods.     

Amino acid residues N450 and Q449 are critical for the uptake capacity and specificity of UapA, a prototype of a nucleobase-ascorbate transporter family

Specific carrier-mediated transport of purine and pyrimidine nucleobases across cell membranes is a basic biological process in both prokaryotes and eukaryotes. Recent in silico analysis has shown that the Aspergillus nidulans (UapA, UapC) and bacterial (PbuX, UraA, PyrP) nucleobase transporters, and a group of mammalian L-ascorbic acid transporters (SVCT1 and SVCT2), constitute a unique protein family which includes putative homologues from archea, bacteria, plants and metazoans. The construction and functional analysis of chimeric purine transporters (UapA-UapC) and UapA-specific missense mutations in A. nidulans has previously shown that the region including amino acid residues 378-446 in UapA is critical for purine recognition and transport. Here, we extend our studies on UapA structure-function relationships by studying missense mutations constructed within a 'signature' sequence motif [(F/Y/S)X(Q/E/P)NXGXXXXT(K/R/G)] which is conserved in the putative functional region of all members of the nucleobase/ascorbate transporter family. Residues Q449 and N450 were found to be critical for purine recognition and transport. The results suggest that these residues might directly or indirectly be involved in specific interactions with the purine ring. In particular, interaction of residue 449 with C-2 groups of purines might act as a critical molecular filter involved in the selection of transported substrates. The present and previous mutagenic analyses in UapA suggest that specific polar or charged amino acid residues on either side of an amphipathic alpha-helical transmembrane segment are critical for purine binding and transport.     

Transport-dependent endocytosis and turnover of a uric acid-xanthine permease

In this work we unmask a novel downregulation mechanism of the uric acid/xanthine transporter UapA, the prototype member of the ubiquitous Nucleobase-Ascorbate Transporter family, directly related to its function. In the presence of substrates, UapA is endocytosed, sorted into the multivesicular body pathway and degraded in vacuoles. Substrate-induced endocytosis, unlike ammonium-induced turnover, is absolutely dependent on UapA activity and several lines of evidence showed that the signal for increased endocytosis is the actual translocation of substrates through the UapA protein. The use of several UapA functional mutants with altered kinetics and specificity has further shown that transport-dependent UapA endocytosis occurs through a mechanism, which senses subtle conformational changes associated with the transport cycle. We also show that distinct mechanisms of UapA endocytosis necessitate ubiquitination of a single Lys residue (K572) by HulA^Rsp5. Finally, we demonstrate that in the presence of substrates, non-functional UapA versions can be endocytosed in trans if expressed in the simultaneous presence of active UapA versions, even if the latter cannot be endocytosed themselves.     

The nucleobase-ascorbate transporter (NAT) signature motif in UapA defines the function of the purine translocation pathway

UapA, a member of the NAT/NCS2 family, is a high affinity, high capacity, uric acid-xanthine/H+ symporter of Aspergillus nidulans. We have previously presented evidence showing that a highly conserved signature motif ([Q/E/P]408-N-X-G-X-X-X-X-T-[R/K/G])417 is involved in UapA function. Here, we present a systematic mutational analysis of conserved residues in or close to the signature motif of UapA. We show that even the most conservative substitutions of residues Q408, N409 and G411 modify the kinetics and specificity of UapA, without affecting targeting in the plasma membrane. Q408 substitutions show that this residue determines both substrate binding and transport catalysis, possibly via interactions with position N9 of the imidazole ring of purines. Residue N409 is an irreplaceable residue necessary for transport catalysis, but is not involved in substrate binding. Residue G411 determines, indirectly, both the kinetics (K(m), V) and specificity of UapA, probably due to its particular property to confer local flexibility in the binding site of UapA. In silico predictions and a search in structural databases strongly suggest that the first part of the NAT signature motif of UapA (Q(408)NNG(411)) should form a loop, the structure of which is mostly affected by mutations in G411. Finally, substitutions of residues T416 and R417, despite being much better tolerated, can also affect the kinetics or the specificity of UapA. Our results show that the NAT signature motif defines the function of the UapA purine translocation pathway and strongly suggest that this might occur by determining the interactions of UapA with the imidazole part of purines.