Spf1 P5-ATPase remains functional after proteolytic cleavage at the C-terminal region

GERARDO R. CORRADI; HUGO P. ADAMO; GUIDO D. PETROVICH

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Biofísica; 2019

Spf1 P5-ATPase remains functional after proteolytic cleavage at the C-terminal regionPetrovich GD, Corradi GR, Adamo HPP-ATPases are active transporters essential for cellular homeostasis. Loss of function of the P-ATPases of group 5 or P5-ATPases in humans has been associated with early-onset Parkinsonism (Kufor-Rakeb syndrome) and other neurodegenerative diseases. P5-ATPases are still poorly characterized. We aimed to learn about the structure and function of the Spf1 P5-ATPase from yeast by partial proteolysis. Recombinant Spf1 was purified in mixed micelles of C12E10 detergent. In this condition, low amounts of chymotrypsin rapidly decreased Spf1 ATPase activity. In contrast, when the purified protein was supplemented with soybean asolectine the ATPase activity was only marginally affected by the protease despite the fact that SDS-PAGE and mass spectrometry of the proteolyzed sample indicated that the Spf1 (Mr 135 kDa) was being cut at a QT1 site located in the C-terminal portion of the molecule producing a fragment of about 110 kDa (Petrovich et al. SAB2018). After QT1 cleavage the mayor Spf1 fragment exhibited normal phosphoenzyme formation and turnover. Current homology models of the Spf1 structure place the QT1 site near the M5 transmembrane segment, a critical region for the function of the protein. Thus our observations light up unanticipated features of the P5-ATPases. With grants from UBA, CONICET and ANPCyT