title: Shadows of an absent partner: ATP hydrolysis and phosphoenzyme turnover of the Spf1 (sensitivity to Pichia farinosa killer toxin) P5-ATPase.
abstract: The P5-ATPases are important components of eukaryotic cells. They have been shown to influence protein biogenesis, folding, and transport. The knowledge of their biochemical properties is, however, limited, and the transported ions are still unknown. We expressed in Saccharomyces cerevisiae the yeast Spf1 P5A-ATPase containing the GFP fused at the N-terminal end. The GFP-Spf1 protein was localized in the yeast endoplasmic reticulum. Purified preparations of GFP-Spf1 hydrolyzed ATP at a rate of ~0.3-1 μmol of P(i)/mg/min and formed a phosphoenzyme in a simple reaction medium containing no added metal ions except Mg(2+). No significant differences were found between the ATPase activity of GFP-Spf1 and recombinant Spf1. Omission of protease inhibitors from the purification buffers resulted in a high level of endogenous proteolysis at the C-terminal portion of the GFP-Spf1 molecule that abolished phosphoenzyme formation. The Mg(2+) dependence of the GFP-Spf1 ATPase was similar to that of other P-ATPases where Mg(2+) acts as a cofactor. The addition of Mn(2+) to the reaction medium decreased the ATPase activity. The enzyme manifested optimal activity at a near neutral pH. When chased by the addition of cold ATP, 90% of the phosphoenzyme remained stable after 5 s. In contrast, the phosphoenzyme rapidly decayed to less than 20% when chased for 3 s by the addition of ADP. The greater effect of ADP accelerating the disappearance of EP suggests that GFP-Spf1 accumulated the E1~P phosphoenzyme. This behavior may reflect a limiting countertransported substrate needed to promote turnover or a missing regulatory factor.
authors: From the Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Ciudad Autónoma de Buenos Aires, Argentina.
authors: Gerardo R
authors: GR
authors: Corradi
authors: Felicitas
authors: F
authors: de Tezanos Pinto
authors: Luciana R
authors: LR
authors: Mazzitelli
authors: Hugo P
authors: HP
authors: Adamo
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D011993:Recombinant Fusion Proteins; C406793:SPF1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium