Inhibitory to non-inhibitory evolution of the ζ subunit of the F1FO-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis

Introduction: The ζ subunit is a potent inhibitor of the F1FO-ATPase of Paracoccus denitrificans (PdF1FO-ATPase) and related α-proteobacteria different from the other two canonical inhibitors of bacterial (ε) and mitochondrial (IF1) F1FO-ATPases. ζ mimics mitochondrial IF1 in its inhibitory N-terminus, blocking the PdF1FO-ATPase activity as a unidirectional pawl-ratchet and allowing the PdF1FO-ATP synthase turnover. ζ is essential for the respiratory growth of P. denitrificans, as we showed by a Δζ knockout. Given the vital role of ζ in the physiology of P. denitrificans, here, we assessed the evolution of ζ across the α-proteobacteria class.Methods: Through bioinformatic, biochemical, molecular biology, functional, and structural analyses of several ζ subunits, we confirmed the conservation of the inhibitory N-terminus of ζ and its divergence toward its C-terminus. We reconstituted homologously or heterologously the recombinant ζ subunits from several α-proteobacteria into the respective F-ATPases, including free-living photosynthetic, facultative symbiont, and intracellular facultative or obligate parasitic α-proteobacteria.Results and discussion: The results show that ζ evolved, preserving its inhibitory function in free-living α-proteobacteria exposed to broad environmental changes that could compromise the cellular ATP pools. However, the ζ inhibitory function was diminished or lost in some symbiotic α-proteobacteria where ζ is non-essential given the possible exchange of nutrients and ATP from hosts. Accordingly, the ζ gene is absent in some strictly parasitic pathogenic Rickettsiales, which may obtain ATP from the parasitized hosts. We also resolved the NMR structure of the ζ subunit of Sinorhizobium meliloti (Sm-ζ) and compared it with its structure modeled in AlphaFold. We found a transition from a compact ordered non-inhibitory conformation into an extended α-helical inhibitory N-terminus conformation, thus explaining why the Sm-ζ cannot exert homologous inhibition. However, it is still able to inhibit the PdF1FO-ATPase heterologously. Together with the loss of the inhibitory function of α-proteobacterial ε, the data confirm that the primary inhibitory function of the α-proteobacterial F1FO-ATPase was transferred from ε to ζ and that ζ, ε, and IF1 evolved by convergent evolution. Some key evolutionary implications on the endosymbiotic origin of mitochondria, as most likely derived from α-proteobacteria, are also discussed

Response to Arsenate treatment in Schizosaccharomyces pombe and the role of its Arsenate reductase activity

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast.Spanish Science and Innovation Ministry (CTQ2008-01031/BQU, BFU2006/01767, BFU2009/09116)Peer Reviewe

Response to arsenate treatment in Schizosaccharomyces pombe and the role of its arsenate reductase activity.

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast

Response to arsenate treatment in Schizosaccharomyces pombe and the role of its arsenate reductase activity.

Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast

Arsenic speciation in different fission yeast mutants.

<p>Total cell extracts from 5×10⁷ cells and growth media from wild type, <i>spc1</i>Δ, <i>cdc2-3w</i> and <i>cdc25</i>Δ <i>cdc2-3w</i> strains were obtained after treatment for 3 or 9 hours with 100 µM sodium arsenate. Graph shows the amount of As (V) (A) or As (III) (B) present in the extracts or growth media.</p

Instrumental parameters for As determination by LC/ICP/MS.

<p>Instrumental parameters for As determination by LC/ICP/MS.</p

Spc1 MAPK pathway and the response to arsenate.

<p>A. Serial dilutions of wild type, <i>wis1</i>Δ, <i>mcs4</i>Δ, <i>wis4</i>Δ, <i>win1-1</i> and <i>wis4</i>Δ <i>win1-1</i> strains were plated in rich media (YES) or rich media containing 50 µM sodium arsenate. Pictures were taken after incubation at 30°C for 48 hours. B. Western blotting of purified Spc1 extracts from wild type, <i>wis1</i>Δ, <i>wis1-AA</i>, <i>win1-1, wis4</i>Δ, and <i>win1-1 wis4</i>Δ treated with 100 µM sodium arsenate for 0 to 30 minutes. Antibodies against phosphorylated p38 were used. As a control, antibodies against HA epitope were used. C. Western blotting of purified Spc1 extracts from wild type, <i>wis1</i>Δ, <i>win1-1 wis4</i>Δ, <i>win1-1 wis4</i>Δ <i>pyp1</i>Δ and <i>win1-1 wis4</i>Δ <i>pyp2</i>Δ treated with 100 µM sodium arsenate for 0 to 30 minutes. Antibodies against phosphorylated p38 were used. As a control, antibodies against HA epitope were used.</p

Genotypes of <i>Schizosaccharomyces pombe</i> strains used in this work.

<p>Genotypes of <i>Schizosaccharomyces pombe</i> strains used in this work.</p

Cdc25 is essential for the response to arsenate.

<p>A. Arsenate to arsenite conversion in fission yeast. Cell extracts from cells treated with 100 µM sodium arsenate were analyzed for the presence of As (III) at different time points. Graph represents parts per million (ppm) As (III). B. Protein alignment of a fragment of <i>S. pombe</i> Cdc25, rice Cdc25 and <i>S. cerevisiae</i> Acr2 and human Cdc25. Asterisks indicate full conservation. C. Serial dilutions of wild type,<i>cdc2-3w</i> and <i>cdc2-3w cdc25</i>Δ strains were plated in rich media (YES) or rich media containing 25 µM sodium arsenate. Pictures were taken after incubation at 30°C for 48 hours. D. Western blotting of whole cell extracts from Cdc25:myc strains treated with 100 µM sodium arsenate for 0 to 180 minutes. Anti-myc antibodies were used to detect Cdc25:myc and anti-actin as a control. E. Total RNA from the experiment presented in (D) was purified and the total amount of Cdc25 mRNA quantified by qPCR. Actin mRNA was used as an internal control.</p

Typical Chromatogram obtained for a standard solution of As species at 2.5 µg L⁻¹ using the experimental parameters summarized in Table 3.

<p>Peak 1: As (III); Peak 2: DMA; Peak 3: MMA; Peak 4: As (V).</p