Mariel Zarco-Zavala

Atypical Cristae Morphology of Human Syncytiotrophoblast Mitochondria: ROLE FOR COMPLEX V

Mitochondrial complexes I, III2, and IV from human cytotrophoblast and syncytiotrophoblast associate to form supercomplexes or respirasomes, with the following stoichiometries: I1:(III2)1 and I1:(III2)1–2:IV1–4. The content of respirasomes was similar in both cell types after isolating mitochondria. However, syncytiotrophoblast mitochondria possess low levels of dimeric complex V and do not have orthodox cristae morphology. In contrast, cytotrophoblast mitochondria show normal cristae morphology and a higher content of ATP synthase dimer. Consistent with the dimerizing role of the ATPase inhibitory protein (IF1) (García, J. J., Morales-Ríos, E., Cortés-Hernandez, P., and Rodríguez-Zavala, J. S. (2006) Biochemistry 45, 12695–12703), higher relative amounts of IF1 were observed in cytotrophoblast when compared with syncytiotrophoblast mitochondria. Therefore, there is a correlation between dimerization of complex V, IF1 expression, and the morphology of mitochondrial cristae in human placental mitochondria. The possible relationship between cristae architecture and the physiological function of the syncytiotrophoblast mitochondria is discussed.

A novel 11-kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related α-proteobacteria

The F1FO and F1‐ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome‐reconstituted holoenzyme preserves its tight coupling between F1 and FO sectors, as evidenced by its high sensitivity to the FO inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N‐terminal sequencing of the band profile in SDS‐PAGE of the F1 and F1FO preparations showed a novel 11‐kDa protein in addition to the 5 canonical α, β, γ, δ, and ε subunits present in all known F1‐ATPase complexes. BN‐PAGE followed by 2D‐SDS‐PAGE confirmed the presence of this 11‐kDa protein bound to the native F1FO‐ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and ε subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F1FO and soluble F1‐ATPase complexes. The 11‐kDa protein, but not the ε subunit, inhibited the F1FO and F1‐ATPase activities of P. denitrificans. The 11‐kDa protein was also found in Rhodobacter sphaeroides associated to its native F1FO‐ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11‐kDa protein on the F1FO‐ATPase nanomotor of P. denitrificans and closely related α‐proteobacteria.—Morales‐Ríos, E., de la Rosa‐Morales, F., Mendoza‐Hernández, G., Rodríguez‐Zavala, J. S., Celis, H., Zarco‐Zavala, M., García‐Trejo, J. J. A novel 11‐kDa inhibitory subunit in the F1FO ATP synthase of Paracoccus denitrificans and related α‐proteobacteria. FASEB J. 24, 599–608 (2010). www.fasebj.org

The ζ subunit of the F1FO-ATP synthase of α-proteobacteria controls rotation of the nanomotor with a different structure

The ζ subunit is a novel natural inhibitor of the α‐proteobacterial F1FO‐ATPase described originally in Paracoccus denitrificans. To characterize the mechanism by which this subunit inhibits the F1FO nanomotor, the ζ subunit of Paracoccus denitrificans (Pd‐ζ was analyzed by the combination of kinetic, biochemical, bioinformatic, proteomic, and structural approaches. The ζ subunit causes full inhibition of the sulfite‐activated PdF1‐ATPase with an apparent IC50 of 270 nM by a mechanism independent of the 8 subunit. The inhibitory region of the ζ subunit resides in the first 14 N‐terminal residues of the protein, which protrude from the 4‐α‐helix bundle structure of the isolated ζ subunit, as resolved by NMR. Cross‐linking experiments show that the ζ subunit interacts with rotor (γ)and stator (α, β) subunits of the F1‐ATPase, indicating that the ζ subunit hinders rotation of the central stalk. In addition, a putatively regulatory nucleotide‐binding site was found in the ζ subunit by isothermal titration calorimetry. Together, the data show that the ζ subunit controls the rotation of F1FO‐ATPase by a mechanism reminiscent of, but different from, those described for mitochondrial IF1 and bacterial ε subunits where the 4‐α‐helix bundle of ζ seems to work as an anchoring domain that orients the N‐terminal inhibitory domain to hinder rotation of the central stalk.—Zarco‐Zavala, M., Morales‐Ríos, E., Mendoza‐Hernández, G., Ramírez‐Silva, L., Pérez‐Hernández, G., García‐Trejo, J. J. The ζ subunit of the F1FO‐ATP synthase of α‐proteobacteria controls rotation of the nanomotor with a different structure. FASEB J. 28, 2146–2157 (2014). www.fasebj.org

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria.

The ζ subunit is a novel inhibitor of the F1FO-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF1) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F1-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599–608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F1-ATPase and F1-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F1-ATPase at the open catalytic αE/βE interface, and two partial γ rotations lock the N terminus of ζ in an “inhibition-general core region,” blocking further γ rotation, while the ζ globular domain anchors it to the closed αDP/βDP interface. Heterologous inhibition of the F1-ATPase of P. denitrificans by the mitochondrial IF1 supported both the modeled ζ binding site at the αDP/βDP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF1 or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.

Unidirectional regulation of the F 1 F O -ATP synthase nanomotor by the ζ pawl-ratchet inhibitor protein of Paracoccus denitrificans and related α-proteobacteria

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF1 subunits, prevent the wasteful CCW F1FO-ATPase activity by blocking γ rotation at the αDP/βDP/γ interface of the F1 portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F1-ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF1, it also binds to the αDP/βDP/γ interface of the F1 of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the αDP/βDP/γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW F1FO-ATPase rotation without affecting the CW-F1FO-ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as a unidirectional pawl-ratchet PdF1FO-ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is a useful model that mimics mitochondrial IF1 but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF1) of this unique ATP synthase nanomotor, essential for life.

Control of rotation of the F1FO-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF1 proteins

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase. In bacteria and mitochondria, there are two known canonical natural inhibitor proteins, namely the ε and IF1 subunits. These proteins regulate the CCW F1FO-ATPase activity by blocking γ subunit rotation at the αDP/βDP/γ subunit interface in the F1 domain. Recently, we discovered a unique natural F1-ATPase inhibitor in Paracoccus denitrificans and related α-proteobacteria denoted the ζ subunit. Here, we compare the functional and structural mechanisms of ε, IF1, and ζ, and using the current data in the field, it is evident that all three regulatory proteins interact with the αDP/βDP/γ interface of the F1-ATPase. In order to exert inhibition, IF1 and ζ contain an intrinsically disordered N-terminal protein region (IDPr) that folds into an α-helix when inserted in the αDP/βDP/γ interface. In this context, we revised here the mechanism and role of the ζ subunit as a unidirectional F-ATPase inhibitor blocking exclusively the CCW F1FO-ATPase rotation, without affecting the CW-F1FO-ATP synthase turnover. In summary, the ζ subunit has a mode of action similar to mitochondrial IF1, but in α-proteobacteria. The structural and functional implications of these intrinsically disordered ζ and IF1 inhibitors are discussed to shed light on the control mechanisms of the ATP synthase nanomotor from an evolutionary perspective.