INVESTIGADORES
CORRADI Gerardo Raul
congresos y reuniones científicas
Título:
The P5-ATPasa Spf1 contains a highly exposed region near the transmembrane segment M5
Autor/es:
PETROVICH GD, CORRADI GR, ADAMO HP
Reunión:
Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2018
Resumen:
The P-ATPases are active transporters essentialfor cellular homeostasis. P-ATPasesare known to transport ions or lipids. The P5 subgroup is not very well characterized and its transported substrate has not yet been identified. Loss of function of P5-ATPases in humans has been associated with early-onset Parkinsonism (Kufor-Rakeb syndrome) and other neurodegenerative diseases. With the aim of advancing the knowledge of the structural organization of P5- ATPases we have performed experiments of limited of the recombinant Spf1 P5- ATPasa of Saccharomycescerevisiae and of its fluorescent versions containing GFP at the N or at the C terminus.The products were characterizedby SDS-PAGE, fluorescence, mass spectrometry andsequencing. The results show that a short exposure of Spf1 to chymotrypsin results in the split of the 135 kDa protein in a larger N-terminalfragment of about 110 kDa and a smaller peptideof 25 kDa containing the C-terminal end of the protein.This cut does not apparently affect the ability of the enzyme tohydrolyze ATPand the formation ofthe catalyticphosphoenzyme. N-terminal sequencing ofthe C-terminal fragment identified two possible cleavagesites at Ala769 (QT1A)and Ala996 (QT1B) both at a segmentof the protein predicted to be exposed to the cytosol near the transmembranesegment M5. The 25 kDa size of the fragment suggest that the main cut is at QT1B. However, analysisof the amino acidsequence by PeptideCutter estimates aprobability for cleavage of 54.5% for QT1B compared to 91.8% for QT1A.Moreover, secondary structure predictionand homology modelingindicates that M5 is part of a long helix starting at Ser972. Since the M5 segment ispart of the transported substratebinding site in other P-ATPases it is tempting to speculate that it has a similar role in Spf1.