José J. García-Trejo

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.

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c10 ring and the other stator subunits at the F0-F0 dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356–12358). In the ATP synthase dimer, the two peripheral stalks are located near the F1-F1 interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis 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

What limits the allotopic expression of nucleus-encoded mitochondrial genes? The case of the chimeric Cox3 and Atp6 genes.

Allotopic expression is potentially a gene therapy for mtDNA-related diseases. Some OXPHOS proteins like ATP6 (subunit a of complex V) and COX3 (subunit III of complex IV) that are typically mtDNA-encoded, are naturally nucleus-encoded in the alga Chlamydomonas reinhardtii. The mitochondrial proteins whose genes have been relocated to the nucleus exhibit long mitochondrial targeting sequences ranging from 100 to 140 residues and a diminished overall mean hydrophobicity when compared with their mtDNA-encoded counterparts. We explored the allotopic expression of the human gene products COX3 and ATP6 that were re-designed for mitochondrial import by emulating the structural properties of the corresponding algal proteins. In vivo and in vitro data in homoplasmic human mutant cells carrying either a T8993G mutation in the mitochondrial atp6 gene or a 15bp deletion in the mtDNA-encoded cox3 gene suggest that these human mitochondrial proteins re-designed for nuclear expression are targeted to the mitochondria, but fail to functionally integrate into their corresponding OXPHOS complexes.

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

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

The F1F0-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the ε subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two α-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF1) interferes with the intrinsic rotational mechanism of the central γ subunit and with the opening and closing of the catalytic β-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF1 protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F1F0 mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F1 portion. In addition, F1F0 dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.

Novel Mutations in the Transcriptional Activator Domain of the Human TBX20 in Patients with Atrial Septal Defect

Background. The relevance of TBX20 gene in heart development has been demonstrated in many animal models, but there are few works that try to elucidate the effect of TBX20 mutations in human congenital heart diseases. In these studies, all missense mutations associated with atrial septal defect (ASD) were found in the DNA-binding T-box domain, none in the transcriptional activator domain. Methods. We search for TBX20 mutations in a group of patients with ASD or ventricular septal defect (VSD) using the High Resolution Melting (HRM) method and DNA sequencing. Results. We report three missense mutations (Y309D, T370O, and M395R) within the transcriptional activator domain of human TBX20 that were associated with ASD. Conclusions. This is the first association of TBX20 transcriptional activator domain missense mutations with ASD. These findings could have implications for diagnosis, genetic screening, and patient follow-up.

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.

Structure of a catalytic dimer of the α- and β-subunits of the F-ATPase from Paracoccus denitrificans at 2.3 Å resolution

The structure of the αβ heterodimer of the F-ATPase from the α-proteobacterium P. denitrificans has been determined at 2.3 Å resolution. It corresponds to the ‘open’ or ‘empty’ catalytic interface found in other F-ATPases.

A Deletion in the PRKAR1A Gene is Associated with Carney Complex

Mutations of the PRKAR1A gene are an important cause of Carney complex (CC). The PRKAR1A gene encodes the type 1A regulatory subunit of cAMP-dependent protein kinase A. We have identified one mutation of PRKAR1A (553delG) in three members of the same family affected by CC. This mutation was not identified in six unaffected family members, 12 patients with sporadic cardiac myxoma and 100 non-related healthy individuals. The novel mutation (553delG) is predicted to produce a frameshift leading to a premature stop codon. RNA analysis in the index patient showed normal size transcripts in RT-PCR amplicons of several exons, but an overall tendency to lower amounts of transcripts in relation to GAPDH controls. In Western blot analyses only full-length protein was present without any evidence of truncated product. These data suggest that the mutant allele might be a null allele due to degradation of the mutant mRNA via nonsense-mediated decay.