IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
V Latin American Protein Society Meeting (LAPSM)
Autor/es:
GERARDO R. CORRADI ; GUIDO D. PETROVICH; HUGO P. ADAMO
Lugar:
Río de Janeiro
Reunión:
Congreso; V Latin American Protein Society Meeting (LAPSM); 2016
Institución organizadora:
The Protein Society
Resumen:
The family of active transporters so-called P-ATPases (P1 to P5) is of principal importance for cell homeostasis. The known transported substrates of P-ATPases include ions and lipids. The P5 is the lesser studied subgroup and is presumed to be involved in the transport of a still unidentified substrate. Loss of P5-ATPase function affects a wide range of cellular functions, and mutations in the human P5-ATPase ATP13a2 has been associated with the early onset of Parkinson?s disease. Here we advanced the knowledge of the structural arrangement of P5-ATPases by limited proteolysis experiments. We used a purified preparation of the recombinant P5-ATPase Spf1p from Saccharomyces cerevisiae and its fluorescent version GFP-Spf1p. Spf1p and GFP-Spf1p were exposed to the action of trypsin, chymotrypsin or proteinase K, and the resulting fragments were analyzed by SDS-PAGE followed by Coomassie Blue staining , fluorescence intensity measurements and mass spectrometry of the isolated fragments. The results show that there is a proteolytic sensitive hot spot between putative transmembrane segments M4 and M5 of Spf1p leading to the rapid removal of the C-terminal one third of the molecule. ATP and Ca2+ reduced the proteolytic fragmentation suggesting that these ligands promote a more compact, protease resistant conformation,while vanadate, a well-known inhibitor of the P-ATPases had the opposite effect. The integration of these results in a model by homology with other P-ATPases suggests the presence of an unanticipated highly exposed region of the Spf1p P5-ATPase near the nucleotide binding-phosphorylation domains. With grants from UBACyT, CONICET and ANPCyT.