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
