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

PETROVICH GUIDO DANIEL; CORRADI GERARDO RAUL; ADAMO HUGO PEDRO

La Plata, Buenos Aires

Congreso; XLVII Reunión Anual de la Sociedad Argentina de BiofísicaXLVII 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  wellcharacterized and its transported substrate has  not  yet been identified.  Loss offunction of  P5-ATPases in humans has  been  associated with early-onsetParkinsonism   (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 Saccharomycescerevisiaeand 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 resultsshow 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  kDacontaining the C-terminal end  of the  protein.This cut  does  not  apparentlyaffect the ability of the enzyme tohydrolyze ATPand the  formationofthe  catalyticphosphoenzyme. N-terminal sequencing ofthe  C-terminal fragment identified two possible cleavagesites  at Ala769  (QT1A)and Ala996  (QT1B) both  at a segmentofthe   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 thatM5 is part  of a long helix  starting at  Ser972.  Since  the  M5segment ispart  ofthe transported substratebinding site  in other P-ATPases it is temptingto  speculate that  ithas a similar role in Spf1.