Mitochondrial mislocalization and altered assembly of a cluster of Barth syndrome mutant tafazzins

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215–232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets

Stimulus Threat and Exposure Context Modulate the Effect of Mere Exposure on Approach Behaviors

Mere-exposure (ME) research has found that initially neutral objects made familiar are preferred relative to novel objects. Recent work extends these preference judgments into the behavioral domain by illustrating that mere exposure prompts approachoriented behavior toward familiar stimuli. However, no investigations have examined the effect of mere exposure on approach-oriented behavior toward threatening stimuli. The current work examines this issue and also explores how exposure context interacts with stimulus threat to influence behavioral tendencies. In two experiments participants were presented with both mere-exposed and novel stimuli and approach speed was assessed. In the first experiment, when stimulus threat was presented in a homogeneous format (i.e., participants viewed exclusively neutral or threatening stimuli), ME potentiated approach behaviors for both neutral and threatening stimuli. However, in the second experiment, in which stimulus threat was presented in a heterogeneous fashion (i.e., participants viewed both neutral and threatening stimuli), mere exposure facilitated approach only for initially neutral stimuli. These results suggest that ME effects on approach behaviors are highly context sensitive and depend on both stimulus valence and exposure context. Further implications of these findings for the ME literature are discussed

Rapid degradation of mutant SLC25A46 by the ubiquitin-proteasome system results in MFN1/2-mediated hyperfusion of mitochondria.

SCL25A46 is a mitochondrial carrier protein that surprisingly localizes to the outer membrane and is distantly related to Ugo1. Here we show that a subset of SLC25A46 interacts with mitochondrial dynamics components and the MICOS complex. Decreased expression of SLC25A46 results in increased stability and oligomerization of MFN1 and MFN2 on mitochondria, promoting mitochondrial hyperfusion. A mutation at L341P causes rapid degradation of SLC25A46, which manifests as a rare disease, pontocerebellar hypoplasia. The E3 ubiquitin ligases MULAN and MARCH5 coordinate ubiquitylation of SLC25A46 L341P, leading to degradation by organized activities of P97 and the proteasome. Whereas outer mitochondrial membrane-associated degradation is typically associated with apoptosis or a specialized type of autophagy termed mitophagy, SLC25A46 degradation operates independently of activation of outer membrane stress pathways. Thus SLC25A46 is a new component in mitochondrial dynamics that serves as a regulator for MFN1/2 oligomerization. Moreover, SLC25A46 is selectively degraded from the outer membrane independently of mitophagy and apoptosis, providing a framework for mechanistic studies in the proteolysis of outer membrane proteins

Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane

Defined mutations in the mitochondrial ADP/ATP carrier (AAC) are associated with certain types of progressive external ophthalmoplegia. AAC is required for oxidative phosphorylation (OXPHOS), and dysregulation of AAC has been implicated in apoptosis. Little is known about the AAC interactome, aside from a known requirement for the phospholipid cardiolipin (CL) and that it is thought to function as a homodimer. Using a newly developed dual affinity tag, we demonstrate that yeast AAC2 physically participates in several protein complexes of distinct size and composition. The respiratory supercomplex and several smaller AAC2-containing complexes, including other members of the mitochondrial carrier family, are identified here. In the absence of CL, most of the defined interactions are destabilized or undetectable. The absence of CL and/or AAC2 results in distinct yet additive alterations in respiratory supercomplex structure and respiratory function. Thus, a single lipid can significantly alter the functional interactome of an individual protein

Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion

Tim54p, a component of the inner membrane TIM22 complex, does not directly mediate the import of inner membrane substrates but is required for assembly/stability of the 300-kD TIM22 complex. In addition, Δtim54 yeast exhibit a petite-negative phenotype (also observed in yeast harboring mutations in the F1Fo ATPase, the ADP/ATP carrier, mitochondrial morphology components, or the i–AAA protease, Yme1p). Interestingly, other import mutants in our strain background are not petite-negative. We report that Tim54p is not involved in maintenance of mitochondrial DNA or mitochondrial morphology. Rather, Tim54p mediates assembly of an active Yme1p complex, after Yme1p is imported via the TIM23 pathway. Defective Yme1p assembly is likely the major contributing factor for the petite-negativity in strains lacking functional Tim54p. Thus, Tim54p has two independent functions: scaffolding/stability for the TIM22 membrane complex and assembly of Yme1p into a proteolytically active complex. As such, Tim54p links protein import, assembly, and turnover pathways in the mitochondrion

Natural and Induced Mitochondrial Phosphate Carrier Loss: DIFFERENTIAL DEPENDENCE OF MITOCHONDRIAL METABOLISM AND DYNAMICS AND CELL SURVIVAL ON THE EXTENT OF DEPLETION.

The relevance of mitochondrial phosphate carrier (PiC), encoded by SLC25A3, in bioenergetics is well accepted. However, little is known about the mechanisms mediating the cellular impairments induced by pathological SLC25A3 variants. To this end, we investigated the pathogenicity of a novel compound heterozygous mutation in SLC25A3 First, each variant was modeled in yeast, revealing that substituting GSSAS for QIP within the fifth matrix loop is incompatible with survival on non-fermentable substrate, whereas the L200W variant is functionally neutral. Next, using skin fibroblasts from an individual expressing these variants and HeLa cells with varying degrees of PiC depletion, PiC loss of ∼60% was still compatible with uncompromised maximal oxidative phosphorylation (oxphos), whereas lower maximal oxphos was evident at ∼85% PiC depletion. Furthermore, intact mutant fibroblasts displayed suppressed mitochondrial bioenergetics consistent with a lower substrate availability rather than phosphate limitation. This was accompanied by slowed proliferation in glucose-replete medium; however, proliferation ceased when only mitochondrial substrate was provided. Both mutant fibroblasts and HeLa cells with 60% PiC loss showed a less interconnected mitochondrial network and a mitochondrial fusion defect that is not explained by altered abundance of OPA1 or MFN1/2 or relative amount of different OPA1 forms. Altogether these results indicate that PiC depletion may need to be profound (\u3e85%) to substantially affect maximal oxphos and that pathogenesis associated with PiC depletion or loss of function may be independent of phosphate limitation when ATP requirements are not high

Emerging Roles in the Biogenesis of Cytochrome c Oxidase for Members of the Mitochondrial Carrier Family

The mitochondrial carrier family (MCF) is a group of transport proteins that are mostly localized to the inner mitochondrial membrane where they facilitate the movement of various solutes across the membrane. Although these carriers represent potential targets for therapeutic application and are repeatedly associated with human disease, research on the MCF has not progressed commensurate to their physiologic and pathophysiologic importance. Many of the 53 MCF members in humans are orphans and lack known transport substrates. Even for the relatively well-studied members of this family, such as the ADP/ATP carrier and the uncoupling protein, there exist fundamental gaps in our understanding of their biological roles including a clear rationale for the existence of multiple isoforms. Here, we briefly review this important family of mitochondrial carriers, provide a few salient examples of their diverse metabolic roles and disease associations, and then focus on an emerging link between several distinct MCF members, including the ADP/ATP carrier, and cytochrome c oxidase biogenesis. As the ADP/ATP carrier is regarded as the paradigm of the entire MCF, its newly established role in regulating translation of the mitochondrial genome highlights that we still have a lot to learn about these metabolite transporters

Defining functional classes of Barth syndrome mutation in humans

The X-linked disease Barth syndrome (BTHS) is caused by mutations in TAZ; TAZ is the main determinant of the final acyl chain composition of the mitochondrial-specific phospholipid, cardiolipin. To date, a detailed characterization of endogenous TAZ has only been performed in yeast. Further, why a given BTHS-associated missense mutation impairs TAZ function has only been determined in a yeast model of this human disease. Presently, the detailed characterization of yeast tafazzin harboring individual BTHS mutations at evolutionarily conserved residues has identified seven distinct loss-of-function mechanisms caused by patient-associated missense alleles. However, whether the biochemical consequences associated with individual mutations also occur in the context of human TAZ in a validated mammalian model has not been demonstrated. Here, utilizing newly established monoclonal antibodies capable of detecting endogenous TAZ, we demonstrate that mammalian TAZ, like its yeast counterpart, is localized to the mitochondrion where it adopts an extremely protease-resistant fold, associates non-integrally with intermembrane space-facing membranes and assembles in a range of complexes. Even though multiple isoforms are expressed at the mRNA level, only a single polypeptide that co-migrates with the human isoform lacking exon 5 is expressed in human skin fibroblasts, HEK293 cells, and murine heart and liver mitochondria. Finally, using a new genome-edited mammalian BTHS cell culture model, we demonstrate that the loss-of-function mechanisms for two BTHS alleles that represent two of the seven functional classes of BTHS mutation as originally defined in yeast, are the same when modeled in human TAZ

Barth syndrome mutations that cause tafazzin complex lability

Complexes containing tafazzin, which remodels newly synthesized cardiolipin, are destabilized by mutations associated with Barth syndrome

Metalloprotease OMA1 Fine-tunes Mitochondrial Bioenergetic Function and Respiratory Supercomplex Stability

Mitochondria are involved in key cellular functions including energy production, metabolic homeostasis, and apoptosis. Normal mitochondrial function is preserved by several interrelated mechanisms. One mechanism – intramitochondrial quality control (IMQC) – is represented by conserved proteases distributed across mitochondrial compartments. Many aspects and physiological roles of IMQC components remain unclear. Here, we show that the IMQC protease Oma1 is required for the stability of the respiratory supercomplexes and thus balanced and tunable bioenergetic function. Loss of Oma1 activity leads to a specific destabilization of respiratory supercomplexes and consequently to unbalanced respiration and progressive respiratory decline in yeast. Similarly, experiments in cultured Oma1-deficient mouse embryonic fibroblasts link together impeded supercomplex stability and inability to maintain proper respiration under conditions that require maximal bioenergetic output. Finally, transient knockdown of OMA1 in zebrafish leads to impeded bioenergetics and morphological defects of the heart and eyes. Together, our biochemical and genetic studies in yeast, zebrafish and mammalian cells identify a novel and conserved physiological role for Oma1 protease in fine-tuning of respiratory function. We suggest that this unexpected physiological role is important for cellular bioenergetic plasticity and may contribute to Oma1- associated disease phenotypes in humans