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.
Institute of Biology National Center for Scientific Research Demokritos (NCSRD), Aghia Paraskevi 153 10 Athens, Greece.
S N
SN
Tavoularis
U H
UH
Tazebay
G
G
Diallinas
M
M
Sideridou
A
A
Rosa
C
C
Scazzocchio
V
V
Sophianopoulou
D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001233:Aspergillus nidulans / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009584:Nitrogen / Q000378:metabolism; D010641:Phenotype; D011487:Protein Conformation; D016415:Sequence Alignment; D013329:Structure-Activity Relationship
D026921:Amino Acid Transport Systems, Neutral; C033082:proline transporter; D009584:Nitrogen