Tail-anchored protein C-terminal domains stimulate ATP hydrolysis by the P5A-ATPase Spf1p

P5A-ATPases constitute a distinct branch of the P-type ATPase superfamily implicated in endoplasmic reticulum (ER) quality control through the removal or reorientation of transmembrane segments. Although genetic and structural studies have suggested a role for these enzymes in transmembrane-helix handling, biochemical evidence linking substrate interaction to catalytic activity has remained limited. Here, we show that purified Saccharomyces cerevisiae Spf1p, a prototypical P5A-ATPase, is stimulated by C-terminal domains of tail-anchored (TA) proteins. Fusion constructs containing the transmembrane and C-terminal regions of either Fis1p (FIS) or the bacterial TA protein YgiM (YGIM) increased ATP hydrolysis approximately twofold in a lipid-dependent and vanadate-sensitive manner. Disruption of membrane-proximal basic residues, either by deletion of the FIS C-terminal KKR motif or by alanine substitution in the YGIM C-terminal tail, markedly reduced stimulation, indicating that C-terminal positive charges contribute to activation. YGIM stimulated Spf1p ATPase activity in a concentration-dependent and saturable manner (Km ≈ 0.12 μM) and increased the relative contribution of a high-affinity component in vanadate inhibition. In pulldown assays, binding of YGIM to Spf1p was enhanced when the enzyme was stabilized in an E₂P-like conformation using beryllium fluoride. Structural inspection of the E₂P state revealed a conserved acidic patch at the luminal entrance of the central cavity, forming a vestibular ring positioned to interact electrostatically with basic C-terminal tails of TA proteins. Altogether, these results support the role of TA proteins as transported substrates of P5A-ATPases and highlight charge-based interactions at the luminal cavity entrance as a key determinant of substrate-dependent activation.