Timothy E. Shutt

The intracellular redox state is a core determinant of mitochondrial fusion

Mitochondrial hyperfusion has recently been shown to function as a cellular stress response, providing transient protection against apoptosis and mitophagy. However, the mechanisms that mediate this response remain poorly understood. In this study, we demonstrate that oxidized glutathione (GSSG), the core cellular stress indicator, strongly induces mitochondrial fusion. Biochemical and functional experiments show that GSSG induces the generation of disulphide‐mediated mitofusin oligomers, in a process that also requires GTP hydrolysis. Our data outline the molecular events that prime the fusion machinery, providing new insights into the coupling of mitochondrial fusion with the cellular stress response.

Ancient mtDNA genetic variants modulate mtDNA transcription and replication.

Although the functional consequences of mitochondrial DNA (mtDNA) genetic backgrounds (haplotypes, haplogroups) have been demonstrated by both disease association studies and cell culture experiments, it is not clear which of the mutations within the haplogroup carry functional implications and which are “evolutionary silent hitchhikers”. We set forth to study the functionality of haplogroup-defining mutations within the mtDNA transcription/replication regulatory region by in vitro transcription, hypothesizing that haplogroup-defining mutations occurring within regulatory motifs of mtDNA could affect these processes. We thus screened >2500 complete human mtDNAs representing all major populations worldwide for natural variation in experimentally established protein binding sites and regulatory regions comprising a total of 241 bp in each mtDNA. Our screen revealed 77/241 sites showing point mutations that could be divided into non-fixed (57/77, 74%) and haplogroup/sub-haplogroup-defining changes (i.e., population fixed changes, 20/77, 26%). The variant defining Caucasian haplogroup J (C295T) increased the binding of TFAM (Electro Mobility Shift Assay) and the capacity of in vitro L-strand transcription, especially of a shorter transcript that maps immediately upstream of conserved sequence block 1 (CSB1), a region associated with RNA priming of mtDNA replication. Consistent with this finding, cybrids (i.e., cells sharing the same nuclear genetic background but differing in their mtDNA backgrounds) harboring haplogroup J mtDNA had a >2 fold increase in mtDNA copy number, as compared to cybrids containing haplogroup H, with no apparent differences in steady state levels of mtDNA-encoded transcripts. Hence, a haplogroup J regulatory region mutation affects mtDNA replication or stability, which may partially account for the phenotypic impact of this haplogroup. Our analysis thus demonstrates, for the first time, the functional impact of particular mtDNA haplogroup-defining control region mutations, paving the path towards assessing the functionality of both fixed and un-fixed genetic variants in the mitochondrial genome.

Mitochondrial Stress Engages E2F1 Apoptotic Signaling to Cause Deafness

Mitochondrial dysfunction causes poorly understood tissue-specific pathology stemming from primary defects in respiration, coupled with altered reactive oxygen species (ROS), metabolic signaling, and apoptosis. The A1555G mtDNA mutation that causes maternally inherited deafness disrupts mitochondrial ribosome function, in part, via increased methylation of the mitochondrial 12S rRNA by the methyltransferase mtTFB1. In patient-derived A1555G cells, we show that 12S rRNA hypermethylation causes ROS-dependent activation of AMP kinase and the proapoptotic nuclear transcription factor E2F1. This retrograde mitochondrial-stress relay is operative in vivo, as transgenic-mtTFB1 mice exhibit enhanced 12S rRNA methylation in multiple tissues, increased E2F1 and apoptosis in the stria vascularis and spiral ganglion neurons of the inner ear, and progressive E2F1-dependent hearing loss. This mouse mitochondrial disease model provides a robust platform for deciphering the complex tissue specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of maternally inherited deafness and its exacerbation by environmental factors.

A compendium of human mitochondrial gene expression machinery with links to disease.

Mammalian mitochondrial DNA encodes 37 essential genes required for ATP production via oxidative phosphorylation, instability or misregulation of which is associated with human diseases and aging. Other than the mtDNA‐encoded RNA species (13 mRNAs, 12S and 16S rRNAs, and 22 tRNAs), the remaining factors needed for mitochondrial gene expression (i.e., transcription, RNA processing/modification, and translation), including a dedicated set of mitochondrial ribosomal proteins, are products of nuclear genes that are imported into the mitochondrial matrix. Herein, we inventory the human mitochondrial gene expression machinery, and, while doing so, we highlight specific associations of these regulatory factors with human disease. Major new breakthroughs have been made recently in this burgeoning area that set the stage for exciting future studies on the key outstanding issue of how mitochondrial gene expression is regulated differentially in vivo. This should promote a greater understanding of why mtDNA mutations and dysfunction cause the complex and tissue‐specific pathology characteristic of mitochondrial disease states and how mitochondrial dysfunction contributes to more common human pathology and aging. Environ. Mol. Mutagen., 2010.

Twinkle, the mitochondrial replicative DNA helicase, is widespread in the eukaryotic radiation and may also be the mitochondrial DNA primase in most eukaryotes.

Recently, the human protein responsible for replicative mtDNA helicase activity was identified and designated Twinkle. Twinkle has been implicated in autosomal dominant progressive external ophthalmoplegia (adPEO), a mitochondrial disorder characterized by mtDNA deletions. The Twinkle protein appears to have evolved from an ancestor shared with the bifunctional primase-helicase found in the T-odd bacteriophages. However, the question has been raised as to whether human Twinkle possesses primase activity, due to amino acid sequence divergence and absence of a zinc-finger motif thought to play an integral role in DNA binding. To date, a primase protein participating in mtDNA replication has not been identified in any eukaryote. Here we investigate the wider phylogenetic distribution of Twinkle by surveying and analyzing data from ongoing EST and genome sequencing projects. We identify Twinkle homologues in representatives from five of six major eukaryotic assemblages (“supergroups”) and present the sequence of the complete Twinkle gene from two members of Amoebozoa, a supergroup of amoeboid protists at the base of the opisthokont (fungal/metazoan) radiation. Notably, we identify conserved primase motifs including the zinc finger in all Twinkle sequences outside of Metazoa. Accordingly, we propose that Twinkle likely serves as the primase as well as the helicase for mtDNA replication in most eukaryotes whose genome encodes it, with the exception of Metazoa.

Core human mitochondrial transcription apparatus is a regulated two-component system in vitro

The core human mitochondrial transcription apparatus is currently regarded as an obligate three-component system comprising the bacteriophage T7-related mitochondrial RNA polymerase, the rRNA methyltransferase-related transcription factor, h-mtTFB2, and the high mobility group box transcription/DNA-packaging factor, h-mtTFA/TFAM. Using a faithful recombinant human mitochondrial transcription system from Escherichia coli, we demonstrate that specific initiation from the mtDNA promoters, LSP and HSP1, only requires mitochondrial RNA polymerase and h-mtTFB2 in vitro. When h-mtTFA is added to these basal components, LSP exhibits a much lower threshold for activation and a larger amplitude response than HSP1. In addition, when LSP and HSP1 are together on the same transcription template, h-mtTFA-independent transcription from HSP1 and h-mtTFA-dependent transcription from both promoters is enhanced and a higher concentration of h-mtTFA is required to stimulate HSP1. Promoter competition experiments revealed that, in addition to LSP competing transcription components away from HSP1, additional cis-acting signals are involved in these aspects of promoter regulation. Based on these results, we speculate that the human mitochondrial transcription system may have evolved to differentially regulate transcription initiation and transcription-primed mtDNA replication in response to the amount of h-mtTFA associated with nucleoids, which could begin to explain the heterogeneity of nucleoid structure and activity in vivo. Furthermore, this study sheds new light on the evolution of mitochondrial transcription components by showing that the human system is a regulated two-component system in vitro, and thus more akin to that of budding yeast than thought previously.

Staying cool in difficult times: mitochondrial dynamics, quality control and the stress response.

One of the critical problems with the combustion of sugar and fat is the generation of cellular oxidation. The ongoing consumption of oxygen results in damage to lipids, protein and mtDNA, which must be repaired through essential pathways in mitochondrial quality control. It has long been established that intrinsic protease pathways within the matrix and intermembrane space actively degrade unfolded and oxidized mitochondrial proteins. However, more recent work into the field of quality control has established distinct roles for both mitochondrial fragmentation and hyperfusion in different aspects of quality control and survival. In addition, mitochondrial derived vesicles have recently been shown to carry cargo directly to the lysosome, adding further insight into the integration of mitochondrial dynamics in cellular homeostasis. This review will focus on the mechanisms and emerging questions concerning the links between mitochondrial dynamics and quality control. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

ROMO1 Is an Essential Redox-Dependent Regulator of Mitochondrial Dynamics

ROMO1 links the oxidative state of the cell to changes in mitochondrial shape and function. Fueling Fusion Mitochondria are dynamic organelles that undergo fusion or fission. In response to cell death–inducing stimuli, mitochondria undergo fragmentation. OPA1 is a guanosine triphosphatase (GTPase) that is present as a transmembrane protein in the inner mitochondrial membrane and as a cleaved form in the intermembrane space; a balance in the abundance of both forms is required for OPA1 to promote mitochondrial fusion. Norton et al. identified ROMO1 as a regulator of mitochondrial morphology that, in response to reactive oxygen species, was oxidized and formed inactive oligomers. Cells lacking ROMO1 had more of the cleaved form of OPA1, showed an increase in fragmented mitochondria, and were more sensitive to cell death–inducing stimuli. Thus, ROMO1 acts as a link between the oxidative state of the cell and the changes in mitochondrial shape and function. The dynamics of mitochondria undergoing fusion and fragmentation govern many mitochondrial functions, including the regulation of cell survival. Although the machinery that catalyzes fusion and fragmentation has been well described, less is known about the signaling components that regulate these phenomena. We performed a genome-wide RNA interference (RNAi) screen and identified reactive oxygen species modulator 1 (ROMO1) as a redox-regulated protein required for mitochondrial fusion and normal cristae morphology. We showed that oxidative stress promoted the formation of high–molecular weight ROMO1 complexes and that knockdown of ROMO1 promoted mitochondrial fission. ROMO1 was essential for the oligomerization of the inner membrane guanosine triphosphatase (GTPase) OPA1, which is required to maintain the integrity of cristae junctions. As a consequence, cells lacking ROMO1 displayed fragmented mitochondria and loss of cristae, causing impaired mitochondrial respiration and increased sensitivity to cell death stimuli. Together, our data identify ROMO1 as a critical molecular switch that couples metabolic stress and mitochondrial morphology, linking mitochondrial fusion to cell survival.

Mitochondrial Ribosomal Protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription

Basal transcription of human mitochondrial DNA (mtDNA) in vitro requires the single-subunit, bacteriophage-related RNA polymerase, POLRMT, and transcription factor h-mtTFB2. This two-component system is activated differentially at mtDNA promoters by human mitochondrial transcription factor A (h-mtTFA). Mitochondrial ribosomal protein L7/L12 (MRPL12) binds directly to POLRMT, but whether it does so in the context of the ribosome or as a “free” protein in the matrix is unknown. Furthermore, existing evidence that MRPL12 activates mitochondrial transcription derives from overexpression studies in cultured cells and transcription experiments using crude mitochondrial lysates, precluding direct effects of MRPL12 on transcription to be assigned. Here, we report that depletion of MRPL12 from HeLa cells by shRNA results in decreased steady-state levels of mitochondrial transcripts, which are not accounted for by changes in RNA stability. We also show that a significant “free” pool of MRPL12 exists in human mitochondria not associated with ribosomes. “Free” MRPL12 binds selectively to POLRMT in vivo in a complex distinct from those containing h-mtTFB2. Finally, using a fully recombinant mitochondrial transcription system, we demonstrate that MRPL12 stimulates promoter-dependent and promoter-independent transcription directly in vitro. Based on these results, we propose that, when not associated with ribosomes, MRPL12 has a second function in transcription, perhaps acting to facilitate the transition from initiation to elongation. We speculate that this is one mechanism to coordinate mitochondrial ribosome biogenesis and transcription in human mitochondria, where transcription of rRNAs from the mtDNA presumably needs to be adjusted in accordance with the rate of import and assembly of the nucleus-encoded MRPs into ribosomes.

The core human mitochondrial transcription initiation complex: It only takes two to tango.

We recently demonstrated that the core transcription initiation complex in human mitochondria is two-component system (POLRMT and h-mtFB2). Human mtTFA/TFAM, previously proposed to be a requisite initiation complex member, is dispensable for promoter-specific initiation in vitro and we propose instead regulates relative promoter activity and/or overall nucleoid transcription and replication potential.