The P5-ATPasa Spf1 contains a highly exposed region near the transmembrane segment M5

PETROVICH GD, CORRADI GR, ADAMO HP

La Plata

Congreso; Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

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.