The binding mechanism of the yeast F1-ATPase inhibitory peptide: role of catalytic intermediates and enzyme turnover.

The mechanism of inhibition of yeast mitochondrial F(1)-ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (beta-Y345W). Fluorescence quenching of a beta-Trp(345) indicates that the binding of MgADP to F(1) can be described as 3 binding sites with dissociation constants of K(d)(1) = 10 +/- 2 nm, K(d2) = 0.22 +/- 0.03 microm, and K(d3) = 16.3 +/- 0.2 microm. In addition, the ATPase activity of the beta-Trp(345) enzyme followed simple Michaelis-Menten kinetics with a corresponding K(m) of 55 microm. Values for the K(d) for MgATP were estimated and indicate that the K(m) (55 microm) for ATP hydrolysis corresponds to filling the third catalytic site on F(1). IF1 binds very slowly to F(1)-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 mum, but decreased thereafter. The rate of inhibition was half-maximal at 5 microm MgATP, which is 10-fold lower than the K(m) for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F(1) with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.

Functional transitions of F0F1-ATPase mediated by the inhibitory peptide IF1 in yeast coupled submitochondrial particles.

The mechanism of inhibition of yeast F(0)F(1)-ATPase by its naturally occurring protein inhibitor (IF1) was investigated in submitochondrial particles by studying the IF1-mediated ATPase inhibition in the presence and absence of a protonmotive force. In the presence of protonmotive force, IF1 added during net NTP hydrolysis almost completely inhibited NTPase activity. At moderate IF1 concentration, subsequent uncoupler addition unexpectedly caused a burst of NTP hydrolysis. We propose that the protonmotive force induces the conversion of IF1-inhibited F(0)F(1)-ATPase into a new form having a lower affinity for IF1. This form remains inactive for ATP hydrolysis after IF1 release. Uncoupling simultaneously releases ATP hydrolysis and converts the latent form of IF1-free F(0)F(1)-ATPase back to the active form. The relationship between the different steps of the catalytic cycle, the mechanism of inhibition by IF1 and the interconversion process is discussed.

Functional characterization of a neuropeptide F-like receptor from Drosophila melanogaster.

A cDNA clone encoding a seven-transmembrane domain, G-protein-coupled receptor (NPFR76F, also called GPCR60), has been isolated from Drosophila melanogaster. Deletion mapping showed that the gene encoding this receptor is located on the left arm of the third chromosome at position 76F. Northern blotting and whole mount in situ hybridization have shown that this receptor is expressed in a limited number of neurons in the central and peripheral nervous systems of embryos and adults. Analysis of the deduced amino acid sequence suggests that this receptor is related to vertebrate neuropeptide Y receptors. This Drosophila receptor shows 62-66% similarity and 32-34% identity to type 2 neuropeptide Y receptors cloned from a variety of vertebrate sources. Coexpression in Xenopus oocytes of NPFR76F with the promiscuous G-protein Galpha16 showed that this receptor is activated by the vertebrate neuropeptide Y family to produce inward currents due to the activation of an endogenous oocyte calcium-dependent chloride current. Maximum receptor activation was achieved with short, putative Drosophila neuropeptide F peptides (Drm-sNPF-1, 2 and 2s). Neuropeptide F-like peptides in Drosophila have been implicated in a signalling system that modulates food response and social behaviour. The identification of this neuropeptide F-like receptor and its endogenous ligand by reverse pharmacology will facilitate genetic and behavioural studies of neuropeptide functions in Drosophila.

Investigation of the role and mechanism of IF1 and STF1 proteins, twin inhibitory peptides which interact with the yeast mitochondrial ATP synthase.

Inhibition of the yeast F(0)F(1)-ATP synthase by the regulatory peptides IF1 and STF1 was studied using intact mitochondria and submitochondrial particles from wild-type cells or from mutants lacking one or both peptides. In intact mitochondria, endogenous IF1 only inhibited uncoupled ATP hydrolysis and endogenous STF1 had no effect. Addition of alamethicin to mitochondria readily made the mitochondrial membranes permeable to nucleotides, and bypassed the kinetic control exerted on ATP hydrolysis by the substrate carriers. In addition, alamethicin made the regulatory peptides able to cross mitochondrial membranes. At pH 7.3, F(0)F(1)-ATPase, initially inactivated by either endogenous IF1 or endogenous STF1, was completely reactivated hours or minutes after alamethicin addition, respectively. Previous application of a membrane potential favored the release of endogenous IF1 and STF1. These observations showed that IF1 and STF1 can fully inhibit ATP hydrolysis at physiological concentrations and are sensitive to the same effectors. However, ATP synthase has a much lower affinity for STF1 than for IF1, as demonstrated by kinetic studies of ATPase inhibition in submitochondrial particles by externally added IF1 and STF1 at pHs ranging from 5.5 to 8.0. Our data do not support previously proposed effects of STF1, like the stabilization of the IF1-F(0)F(1) complex or the replacement of IF1 on its binding site in the presence of the proton-motive force or at high pH, and raise the question of the conditions under which STF1 could regulate ATPase activity in vivo.