Dissertation committee:
Αναπλ. Καθηγητής Γεώργιος Διαλλινάς (Επιβλέπων), Καθηγήτρια Αμαλία Καραγκούνη, Καθηγητής Οικονόμου Αναστάσιος
Summary:
The role of three conserved polar residues, Glu356, Gln382 and K439, in the
translocation mechanism of the UapA uric acid transporter of Aspergillus
nidulans, a eukaryotic model system, was examined. These residues were
substituted and the mutant alleles were physiologically, microscopically and
kinetically studied. The results showed that Glu356 is necessary for transport
and seems to contribute to substrate binding. In regard to the
post-translational regulation of expression of the transporter, the phenomenon
of its down-regulation by its substrates was studied. It was shown that UapA is
endocytosed upon substrate addition, sorted to endosomes and finally degraded
in the vacuole. The substrate-induced endocytosis was shown to be independent
of its intracellular concentration, but absolutely dependent on transporter
function. The signal for the transporter’s endocytosis seems to rely on subtle
dynamic conformational changes of the transporter during the completation of
its transport cycle. Mechanistically, UapA endocytosis is achieved through
ubiquitinylation of Lys572 from the HulARsp5 ubiquitin ligase. Finally,
endocytosis can occur in trans, as non functional or non-ubiquitinylated UapA
molecules can be endocytosed when co-expressed with functional UapA molecules,
a result that suggests UapA oligomerisation.
Keywords:
Purine transporters, Aspergillus nidulans, uric acid, endocytosis, ubiquitinylation
