Search

The unicellular green alga Chlamydomonas reinhardtii is a biflagellated cell with two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Here, we probe how actin redundancy contributes to flagellar assembly. Disrupting a single actin allows complete flagellar assembly. However, when disrupting both actins using latrunculin B (LatB) treatment on the nap1 mutant background, we find that actins are necessary for flagellar growth from newly synthesized limiting flagellar proteins. Under total actin disruption, transmission electron microscopy identified an accumulation of Golgi-adjacent vesicles. We also find that there is a mislocalization of a key transition zone gating and ciliopathy protein, NPHP-4. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in transport of Golgi-adjacent vesicles and flagellar incorporation of newly synthesized proteins. • At least one filamentous actin required for full flagellar assembly • Transition zone flagellar gate affected upon actin disruption • Actin required for normal flagellar protein synthesis • Golgi-adjacent vesicles accumulate upon total actin loss Prevailing knowledge tells us that intracellular trafficking of flagellar proteins occurs on microtubule tracks. In this study, Jack et al. discover that filamentous actin is required for full flagellar assembly, normal flagellar protein synthesis, an intact flagellar gating region, and vesicle transport in Chlamydomonas reinhardtii. [ABSTRACT FROM AUTHOR]

The ATP synthase is under a number of mechanisms of regulation. The chloroplast ATPase has a unique mode of regulation in which activity is controlled by the redox state in the organelle. This mode of regulation is determined by a small unique region within the γ-subunit and this region contains two cysteine residues. Introduction of this region within the yeast γ-subunit causes a defect in oxidative phosphorylation. Oxidative phosphorylation is restored if the cysteine residues are replaced with serine. Biochemical analysis of the chimeric mitochondrial ATPase indicates that the ATP synthase is not largely altered with the cysteine residues in either the oxidized or reduced states. However, the level and activity of cytochrome c oxidase are decreased by about 90%, whereas that of NADH dehydrogenase and cytochrome c reductase are unchanged as compared with the wild-type enzymes. The level and activity of cytochrome c oxidase are restored with replacement of the cysteine residues with serine in the regulatory region. These results indicate that the chimeric ATP synthase containing cysteine, but not serine, decreases the expression or assembly of cytochrome c oxidase with little effect on the activity of the ATP synthase. [ABSTRACT FROM AUTHOR]

The mitochondrial ATP synthase is a molecular motor, which couples the flow of protons with phosphorylation of ADP. Rotation of the central stalk within the core of ATP synthase effects conformational changes in the active sites driving the synthesis of ATP. Mitochondrial genome integrity (mgi) mutations have been previously identified in the α-, β-, and γ-subunits of ATP synthase in yeast Kluyveromyces lactis and trypanosome Trypanosoma brucei. These mutations reverse the lethality of the loss of mitochondrial DNA in petite negative strains. Introduction of the homologous mutations in Saccharomyces cerevisiae results in yeast strains that lose mitochondrial DNA at a high rate and accompanied decreases in the coupling of the ATP synthase. The structure of yeast F1-ATPase reveals that the mgi residues cluster around the γ-subunit and selectively around the collar region of F1. These results indicate that residues within the mgi complementation group are necessary for efficient coupling of ATP synthase, possibly acting as a support to fix the axis of rotation of the central stalk. [ABSTRACT FROM AUTHOR]

The mitochondrial ATP synthase couples the flow of protons with the phosphorylation of ADP. A class of mutations, the mitochondrial genome integrity (mgi) mutations, has been shown to uncouple this process in the yeast mitochondrial ATP synthase. Four mutant forms of the yeast F1 ATPase with mgi mutations were crystallized; the structures were solved and analyzed. The analysis identifies two mechanisms of structural uncoupling: one in which the empty catalytic site is altered and in doing so, apparently disrupts substrate (phosphate) binding, and a second where the steric hindrance predicted between γLeu83 and βDP residues, Leu-391 and Glu-395, located in Catch 2 region, is reduced allowing rotation of the γ-subunit with less impedance. Overall, the structures provide key insights into the critical interactions in the yeast ATP synthase involved in the coupling process. [ABSTRACT FROM AUTHOR]

The crystal structure of nucleotide-free yeast F1 ATPase has been determined at a resolution of 3.6 Å. The overall structure is very similar to that of the ground state enzyme, In particular, the βDP and βTP subunits both adopt the closed conformation found in the ground state structure despite the absence of bound nucleotides. This implies that interactions between the γ and β subunits are as important as nucleotide occupancy in determining the conformational state of the β subunits. Furthermore, this result suggests that for the mitochondrial enzyme, there is no state of nucleotide occupancy that would result in more than one of the β subunits adopting the open conformation. The adenine-binding pocket of the βTP subunit is disrupted in the apo-enzyme, suggesting that the βDP subunit is responsible for unisite catalytic activity. [ABSTRACT FROM AUTHOR]

The mitochondrial F1F0-ATP synthase is a multimeric enzyme complex composed of at least 16 unique peptides with an overall molecular mass of ∼600 kDa. F1-ATPase is composed of α3β3γδε with an overall molecular mass of 370 kDa. The genes encoding bovine F1-ATPase have been expressed in a quintuple yeast Saccharomyces cerevisiae deletion mutant (ΔαΔβΔγΔδΔε). This strain expressing bovine F1 is unable to grow on medium containing a non-fermentable carbon source (YPG), indicating that the enzyme is non-functional. However, daughter strains were easily selected for growth on YPG medium and these were evolved for improved growth on YPG medium. The evolution of the strains was presumably due to mutations, but mutations in the genes encoding the subunits of the bovine F1-ATPase were not required for the ability of the cell to grow on YPG medium. The bovine enzyme expressed in yeast was partially purified to a specific activity of about half of that of the enzyme purified from bovine heart mitochondria. These results indicate that the molecular machinery required for the assembly of the mitochondrial ATP synthase is conserved from bovine and yeast and suggest that yeast may be useful for the expression, mutagenesis, and analysis of the mammalian F1- or F1F0- ATP synthase. [ABSTRACT FROM AUTHOR]

The mechanism of inhibition of yeast mitochondrial F1-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 (β-Y345W). Fluorescence quenching of a β-Trp345 indicates that the binding of MgADP to F1 can be described as 3 binding sites with dissociation constants of Kd1 = 10 ± 2 nM, Kd2 = 0.22 ± 0.03 μM, and Kd3 = 16.3 ± 0.2 μM. In addition, the ATPase activity of the β-Trp345 enzyme followed simple Michaelis-Menten kinetics with a corresponding Km of 55 μM. Values for the Kd for MgATP were estimated and indicate that the Km (55 μM) for ATP hydrolysis corresponds to filling the third catalytic site on F1. IF1 binds very slowly to F1-ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 μM, but decreased thereafter. The rate of inhibition was half-maximal at 5 μM MgATP, which is 10-fold lower than the Km 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 F1 with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP. [ABSTRACT FROM AUTHOR]