Functional expression of human PARK9-ATP13A2 P5-ATPase in Saccharomyces cerevisiae.

ADAMO HUGO P.; CORRADI, GERARDO R.; MARCOS, ALEJANDRA LUCÍA; DE TEZANOS PINTO, FELICITAS

Ciudad Autónoma de Buenos Aires

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Sociedad Argentina de Investigación Clínica, Sociedad Argentina de Biofísica y otras

The P-type ion pumps are membrane transporters energized by ATP-hydrolysis. They were classified into five subfamilies termed P1-P5; the substrate specificity of P5 subfamily is still unknown. Five genes named ATP13A1-ATP13A5 that belong to the P5-ATPases are present in humans, while two named Spf1p and Ypk9p were found in the yeast S. cerevisiae. By DNA sequence alignment it was shown that P5-ATPases were classified in groups P5A and P5B; the mouse gene Atp13a1 and the yeast gene coding Spfp1 (Yel031w) are members of the first group, while the mouse genes Atp13a2-Atp13a5 and the yeast gene coding Ypk9p (Yor291w) are clustered into the second one. Mutations of the ATP13A2 gene, also known as PARK9, are associated with a form of Parkinson?s Disease (Kufor-Rakeb syndrome), a form of Neuronal Ceroid Lipofuscinosis (CNL12) and hereditary spastic paraplegia (SPG78). ATP13A2 is localized in lysosomes and late endosomes (LEs). Dysfunction of this protein diminishes the lysosomal degradation, the autophagic flux and the exosome externalization. In order to advance the biochemical characterization of ATP13A2, S.C cells were transformed with the pMP625 vector coding for GFP tagged-ATP13A2 protein and the clone exhibiting the highest GFP-fluorescence intensity selected. Yeasts expressing GFP-ATP13A2 showed a reduced growing rate and an autophagic phenotype characterized by vacuolar multivescularization. The recombinant fusion protein was purified from microsomal membranes by pseudo-affinity chromatography. Analysis of the eluate by SDS-PAGE and western blot indicated that ATP13A2 was successfully expressed and had the expected molecular size. Preliminary experiments measuring the release of free phosphate from ATP by the Baginsky?s assay showed that the purified GFP-ATP13A2 is a functional ATPase.