Producción CyT
Libro de Resumenes de LII Reunion anual de la SAB - ATPase Activity of Spf1p in the Presence of Putative Transmembrane Helix Substrates
Congreso
Fecha:
2024Editorial y Lugar de Edición:
SABResumen *
The precise targeting and insertion of membrane proteins into their designated organellesis a fundamental cellular process. The endoplasmic reticulum (ER) membrane isparticularly vulnerable to the misinsertion of proteins synthesized in the cytosol and post-translationally inserted as is the case of tail-anchored (TA) proteins. Recent studies haveimplicated P5A-ATPases in the retrieval of mistargeted TA mitochondrial outer membraneproteins from the ER membrane. However, the molecular mechanisms underlyingsubstrate recognition and ATP hydrolysis-coupled dislocation remain elusive. Toinvestigate the influence of specific substrate proteins on the ATPase activity of the yeastP5A-ATPase Spf1p, we purified recombinant His-tagged Spf1p-GFP and two putativesubstrates, GFP-FIS and mTFP-YGIM, in C12E10 micelles using Ni-NTA chromatography.Under optimal conditions (28°C, 3 mM ATP), Spf1p exhibited a basal ATPase activity of0.51 μmol Pi/mg/min. The addition of 478 nM mTFP-YGIM or 91 nM GFP-FIS significantlystimulated Spf1p ATPase activity by 30% and 75%, respectively. Conversely, the additionof 150 nM GFP-FIST, a truncated version of FIS lacking the positively charged C-terminalRNKRR motif, had no effect on Spf1p activity. These findings strongly suggest that FISand YGIM are indeed substrates of Spf1p and that the positively charged amino acids inthe C-terminal region are crucial for the dislocation process Información suministrada por el agente en SIGEVAPalabras Clave
FIS1ATP13A1SPF1P5A-ATPASES