A limited proteolysis study of a Parkinson?s associated P5-ATPase

PETROVICH GUIDO DANIEL; CORRADI GERARDO RAUL; ADAMO HUGO PEDRO

Rio de Janeiro Campus of the Federal University of Rio de Janeiro

Congreso; V Latin American Protein Society Meeting; 2016

Latin American Protein Society

A limitedproteolysis study of  a Parkinson?sassociated P5-ATPase.Guido  D. Petrovich,Gerardo R. Corradi,  and Hugo P.Adamo.From Instituto de Quimica yFisicoquimica Biologicas-Facultad de Farmacia y Bioquimica Universidad deBuenos Aires-CONICET, Junin 956, 1113 Ciudad de Buenos Aires, Argentina.  Thefamily of active transporters so-called P-ATPases (P1 to P5) is of principalimportance for cell homeostasis. The known transported substrates of P-ATPasesinclude ions and lipids. The P5 is the lesser studied subgroup and is presumedto be involved in the transport of a still unidentified substrate. Loss ofP5-ATPase function affects a wide range of cellular functions, and mutations inthe human P5-ATPase ATP13a2 has been associated with the early onset ofParkinson?s disease.  Here we advancedthe knowledge of the structural arrangement of P5-ATPases by limitedproteolysis experiments. We used a purified preparation of the recombinantP5-ATPase Spf1p from Saccharomycescerevisiae and its fluorescent version GFP-Spf1p. Spf1p and GFP-Spf1p wereexposed to the action of trypsin, chymotrypsin or proteinase K, and theresulting fragments were analyzed by SDS-PAGE followed by Coomasie Bluestaining , fluorescence intensity measurements  and mass spectrometry of the isolatedfragments. The results show that there is a proteolytic sensitive hot spotbetween putative transmembrane segments M4 and M5 of Spf1p leading to the rapidremoval of the C-terminal one third of the molecule. ATP and Ca2+reduced the proteolytic fragmentation suggesting that these ligands promote amore compact, protease resistant conformation, while vanadate, a well-knowninhibitor of the P-ATPases had the opposite effect. The integration of theseresults in a model by homology with other P-ATPases suggests the presence of anunanticipated highly exposed region of the Spf1p P5-ATPase near the nucleotidebinding-phosphorylation domains.