Modification of the mitochondrial F1-ATPase ∊ subunit, enhancement of the ATPase activity of the IF1–F1 complex and IF1-binding dependence of the conformation of the ∊ subunit

Abstract

Treatment of bovine heart submitochondrial particles with a low concentration of 2-hydroxy-5-nitrobenzyl bromide (HNB), a selective reagent for the Trp residue of the epsilon subunit [Baracca, Barogi, Lenaz and Solaini (1993) Int. J. Biochem. 25, 1269-1275], enhances the ATP hydrolytic activity of the particles exclusively when the natural inhibitor protein IF1 is present. Similarly, isolated F1 [the catalytic sector of the mitochondrial H+-ATPase complex (ATP synthase)] treated with the reagent has the ATPase activity enhanced exclusively if IF1 is bound to it. These experiments suggest that the modification of the epsilon subunit decreases the inhibitory activity of IF1, eliciting the search for a relationship between the epsilon subunit and the inhibitory protein. Certainly, a reverse relationship exists because HNB binds covalently to the isolated F1 exclusively when the inhibitory protein is present. This finding is consistent with the existence of the epsilon subunit in different conformational states depending on whether IF1 is bound to F1 or not. Support for this assertion is obtained by measurements of the intrinsic phosphorescence decay rate of F1, a probe of the Trp epsilon subunit conformation in situ [Solaini, Baracca, Parenti-Castelli and Strambini (1993) Eur. J. Biochem. 214, 729-734]. A significant difference in phosphorescence decay rate is detected when IF1 is added to preparations of F1 previously devoid of the inhibitory protein. These studies indicate that IF1 and the epsilon subunit of the mitochondrial F1-ATPase complex are related, suggesting a possible role of the epsilon subunit in the mechanism of regulation of the mitochondrial ATP synthase.

The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

“…6). As expected, the oligomycin-sensitive ATPase activity of mitochondria was pH sensitive (31,38), being at pH 6.7 about half than at pH 7.4. As expected and in accord with previous data obtained from organ extracts and the isolated enzyme, the oligomycin-sensitive ATPase activity of mitochondria from IF 1 -silenced cells resulted in being 2-3-and 4 -5-fold higher than in controls at pH 7.4 and 6.7, respectively.…”

“…3D). On the basis of earlier studies on both isolated mitochondria and submitochondrial particles (30,31), the above results were expected because cells were grown under conditions that did not favor binding and inhibition of IF 1 to the F 1 F 0 -ATPase complex. Indeed, most of the above data agree with those recently reported by Fujikawa et al (32), who analyzed the only stable IF 1 -depleted cell line (HeLa cells) as yet studied.…”

ATP Synthase and the Actions of Inhibitors Utilized To Study Its Roles in Human Health, Disease, and Other Scientific Areas

“…IF 1 is considered to play its inhibitory role by impeding the closure of the ␣ DP -␤ DP catalytic interface to prevent the hydrolysis of bound ATP (61,141). Cross-linking and intrinsic phosphorescence decay studies implicate IF 1 as being functionally associated with the mitochondrial ε subunit (260,373). Both proteins are in close proximity in the crystal structure of the F 1 -IF 1 complex (141).…”

The Small Protein CP12: A Protein Linker for Supramolecular Complex Assembly

“…These proteins are often involved in protein-protein interactions and play major roles in cellular functions, such as DNA and RNA binding, transcriptional regulation, signal transduction, and cell cycle regulation (30). They are found in a wide variety of macromolecular complexes: Nup2p and the nuclear pore complex (33), Securin and cyclin B (34), complexins and SNARE complexes (35)(36)(37), IF1 and the regulation of ATPases (38)(39)(40), p21 or p27kip1 and regulation of CDK2, cyclin A or PCNA (41,42). Like CP12, their flexibility favors the binding of several partners and the possibility of presenting larger interaction surfaces, compared to rigid partners.…”