Thesis presented August 23, 2002
Abstract:
Cu+-ATPases have a common characteristic in all transporting ATPases: the existence of membranous domains which form the passage of ions. Mutation on specific important amino acids for Cu+ transport may cause human copper deficiencies as Menkes and Wilson diseases.
In the present work we decided to investigate the role of highly conserved amino acids in yeast and mammals Cu+-ATPase. Using the
Saccharomyces cerevisiae Cu+-ATPase (Ccc2), 9 mutants found in those diseases were obtained to perform
in vivo (complementation assays and yeast growth curves) and
in vitro (phosphorylation and dephosphorization) assays.
All mutants were phosphorylated by ATP with the same rate, except for mutant C583S, because its phosphorylation rate was drastically reduced. The successive phosphorylation steps (coupled to membrane Cu+ transport) were affected by different ways. Mutations in cysteines 583 and 585 result in a Cu+-ATPase expression that is not able to accomplish its cycle and prevent yeast growth. The mutation of threonines 375, 541, 591 and 593 express a Cu+-ATPase with lower capacity to accomplish dephosphorization step, therefore they can not catalyse Cu+ transfer through membrane.
Thus, it shows that different mutated amino acids contribute in various manners to form a cation transmembrane way passage and the different degrees in both diseases which affect human copper homeostasis could be related to the role of each mutated amino acid in the global of Cu+ active transport.
Keywords:
Copper homeostasis, Cu+-ATPase, phosphorylation, Menkes and Wilson diseases