Brett A. Kaufman

MCUR1 is an essential component of mitochondrial Ca2+ uptake that regulates cellular metabolism

The mitochondrial calcium uniporter (MCU) mediates calcium uptake by mitochondria and thus regulates cellular bioenergetics, but how MCU activity is modulated is not fully understood. Madesh, Foskett and colleagues report that the integral mitochondrial membrane protein MCUR1 (mitochondrial calcium uniporter regulator 1) binds to the MCU and promotes MCU-dependent calcium uptake to control ATP production and autophagy.

Mutation in TACO1, encoding a translational activator of COX I, results in cytochrome c oxidase deficiency and late-onset Leigh syndrome

Defects in mitochondrial translation are among the most common causes of mitochondrial disease, but the mechanisms that regulate mitochondrial translation remain largely unknown. In the yeast Saccharomyces cerevisiae, all mitochondrial mRNAs require specific translational activators, which recognize sequences in 5′ UTRs and mediate translation. As mammalian mitochondrial mRNAs do not have significant 5′ UTRs, alternate mechanisms must exist to promote translation. We identified a specific defect in the synthesis of the mitochondrial DNA (mtDNA)-encoded COX I subunit in a pedigree segregating late-onset Leigh syndrome and cytochrome c oxidase (COX) deficiency. We mapped the defect to chromosome 17q by functional complementation and identified a homozygous single-base-pair insertion in CCDC44, encoding a member of a large family of hypothetical proteins containing a conserved DUF28 domain. CCDC44, renamed TACO1 for translational activator of COX I, shares a notable degree of structural similarity with bacterial homologs, and our findings suggest that it is one of a family of specific mammalian mitochondrial translational activators.

Mitochondrial transcription factor A regulates mitochondrial transcription initiation, DNA packaging, and genome copy number.

Mitochondrial transcription factor A (mtTFA, mtTF1, TFAM) is an essential protein that binds mitochondrial DNA (mtDNA) with and without sequence specificity to regulate both mitochondrial transcription initiation and mtDNA copy number. The abundance of mtDNA generally reflects TFAM protein levels; however, the precise mechanism(s) by which this occurs remains a matter of debate. Data suggest that the usage of mitochondrial promoters is regulated by TFAM dosage, allowing TFAM to affect both gene expression and RNA priming for first strand mtDNA replication. Additionally, TFAM has a non-specific DNA binding activity that is both cooperative and high affinity. TFAM can compact plasmid DNA in vitro, suggesting a structural role for the non-specific DNA binding activity in genome packaging. This review summarizes TFAM-mtDNA interactions and describes an emerging view of TFAM as a multipurpose coordinator of mtDNA transactions, with direct consequences for the maintenance of gene expression and genome copy number. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.

Reconstitution of Mitochondria Derived Vesicle Formation Demonstrates Selective Enrichment of Oxidized Cargo

The mechanisms that ensure the removal of damaged mitochondrial proteins and lipids are critical for the health of the cell, and errors in these pathways are implicated in numerous degenerative diseases. We recently uncovered a new pathway for the selective removal of proteins mediated by mitochondrial derived vesicular carriers (MDVs) that transit to the lysosome. However, it was not determined whether these vesicles were selectively enriched for oxidized, or damaged proteins, and the extent to which the complexes of the electron transport chain and the mtDNA-containing nucloids may have been incorporated. In this study, we have developed a cell-free mitochondrial budding reaction in vitro in order to better dissect the pathway. Our data confirm that MDVs are stimulated upon various forms of mitochondrial stress, and the vesicles incorporated quantitative amounts of cargo, whose identity depended upon the nature of the stress. Under the conditions examined, MDVs did not incorporate complexes I and V, nor were any nucleoids present, demonstrating the specificity of cargo incorporation. Stress-induced MDVs are selectively enriched for oxidized proteins, suggesting that conformational changes induced by oxidation may initiate their incorporation into the vesicles. Ultrastructural analyses of MDVs isolated on sucrose flotation gradients revealed the formation of both single and double membranes vesicles of unique densities and uniform diameter. This work provides a framework for a reductionist approach towards a detailed examination of the mechanisms of MDV formation and cargo incorporation, and supports the emerging concept that MDVs are critical contributors to mitochondrial quality control.

A function for the mitochondrial chaperonin Hsp60 in the structure and transmission of mitochondrial DNA nucleoids in Saccharomyces cerevisiae.

The yeast mitochondrial chaperonin Hsp60 has previously been implicated in mitochondrial DNA (mtDNA) transactions: it is found in mtDNA nucleoids associated with single-stranded DNA; it binds preferentially to the template strand of active mtDNA ori sequences in vitro; and wild-type (ρ+) mtDNA is unstable in hsp60 temperature-sensitive (ts) mutants grown at the permissive temperature. Here we show that the mtDNA instability is caused by a defect in mtDNA transmission to daughter cells. Using high resolution, fluorescence deconvolution microscopy, we observe a striking alteration in the morphology of mtDNA nucleoids in ρ+ cells of an hsp60-t s mutant that suggests a defect in nucleoid division. We show that ρ− petite mtDNA consisting of active ori repeats is uniquely unstable in the hsp60-t s mutant. This instability of ori ρ− mtDNA requires transcription from the canonical promoter within the ori element. Our data suggest that the nucleoid dynamics underlying mtDNA transmission are regulated by the interaction between Hsp60 and mtDNA ori sequences.

The diabetes susceptibility gene Clec16a regulates mitophagy

Clec16a has been identified as a disease susceptibility gene for type 1 diabetes, multiple sclerosis, and adrenal dysfunction, but its function is unknown. Here we report that Clec16a is a membrane-associated endosomal protein that interacts with E3 ubiquitin ligase Nrdp1. Loss of Clec16a leads to an increase in the Nrdp1 target Parkin, a master regulator of mitophagy. Islets from mice with pancreas-specific deletion of Clec16a have abnormal mitochondria with reduced oxygen consumption and ATP concentration, both of which are required for normal β cell function. Indeed, pancreatic Clec16a is required for normal glucose-stimulated insulin release. Moreover, patients harboring a diabetogenic SNP in the Clec16a gene have reduced islet Clec16a expression and reduced insulin secretion. Thus, Clec16a controls β cell function and prevents diabetes by controlling mitophagy. This pathway could be targeted for prevention and control of diabetes and may extend to the pathogenesis of other Clec16a- and Parkin-associated diseases.

Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU

Calcium signaling stimulates the accumulation of the mitochondrial calcium uniporter to regulate mitochondrial metabolism. Maintaining mitochondrial calcium uptake The calcium uniporter complex, which includes the protein MCU, mediates mitochondrial calcium uptake, a process that buffers excess cytosolic calcium and regulates mitochondrial metabolism. Shanmughapriya et al. examined mitochondrial calcium uptake and function in a B lymphocyte cell line deficient in one or more proteins necessary for mediating two types of calcium signals—IICR, calcium released from the endoplasmic reticulum through the calcium-permeable IP3 receptors, and SOCE, calcium influx through store-operated calcium channels. Without IICR or SOCE, the activity of the transcription factor CREB, which bound to the MCU promoter, and the expression and abundance of MCU were reduced, mitochondrial calcium uptake was compromised, and mitochondrial metabolism was altered. Cells deficient in IICR or SOCE lacked an oscillating basal calcium signal. Thus, IICR and SOCE control the capacity of mitochondria to uptake calcium and therefore regulate mitochondrial metabolism. Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+-permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+-regulated transcription factor CREB (cyclic adenosine monophosphate response element–binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+-dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

The mitochondrial genome exists in numerous structural conformations, complicating the study of mitochondrial DNA (mtDNA) metabolism. Here, we describe the development of 2D intact mtDNA agarose gel electrophoresis (2D-IMAGE) for the separation and detection of approximately two-dozen distinct topoisomers. Although the major topoisomers were well conserved across many cell and tissue types, unique differences in certain cells and tissues were also observed. RNase treatment revealed that partially hybridized RNAs associated primarily with covalently closed circular DNA, consistent with this structure being the template for transcription. Circular structures composed of RNA:DNA hybrids contained only heavy-strand DNA sequences, implicating them as lagging-strand replication intermediates. During recovery from replicative arrest, 2D-IMAGE showed changes in both template selection and replication products. These studies suggest that discrete topoisomers are associated with specific mtDNA-directed processes. Because of the increased resolution, 2D-IMAGE has the potential to identify novel mtDNA intermediates involved in replication or transcription, or pathology including oxidative linearization, deletions or depletion.

Diabetes susceptibility genes Pdx1 and Clec16a function in a pathway regulating mitophagy in β-cells

Mitophagy is a critical regulator of mitochondrial quality control and is necessary for elimination of dysfunctional mitochondria to maintain cellular respiration. Here, we report that the homeodomain transcription factor Pdx1, a gene associated with both type 2 diabetes and monogenic diabetes of the young, regulates mitophagy in pancreatic β-cells. Loss of Pdx1 leads to abnormal mitochondrial morphology and function as well as impaired mitochondrial turnover. High-throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates the expression of Clec16a, a type 1 diabetes gene and itself a key mediator of mitophagy through regulation of the E3 ubiquitin ligase Nrdp1. Indeed, expression of Clec16a and Nrdp1 are both reduced in Pdx1 haploinsufficient islets, and reduction of Pdx1 impairs fusion of autophagosomes containing mitochondria to lysosomes during mitophagy. Importantly, restoration of Clec16a expression after Pdx1 loss of function restores mitochondrial trafficking during mitophagy and improves mitochondrial respiration and glucose-stimulated insulin release. Thus, Pdx1 orchestrates nuclear control of mitochondrial function in part by controlling mitophagy through Clec16a. The novel Pdx1-Clec16a-Nrdp1 pathway we describe provides a genetic basis for the pathogenesis of mitochondrial dysfunction in multiple forms of diabetes that could be targeted for future therapies to improve β-cell function.

Mitochondrial regulation of β-cell function: maintaining the momentum for insulin release

All forms of diabetes share the common etiology of insufficient pancreatic β-cell function to meet peripheral insulin demand. In pancreatic β-cells, mitochondria serve to integrate the metabolism of exogenous nutrients into energy output, which ultimately leads to insulin release. As such, mitochondrial dysfunction underlies β-cell failure and the development of diabetes. Mitochondrial regulation of β-cell function occurs through many diverse pathways, including metabolic coupling, generation of reactive oxygen species, maintenance of mitochondrial mass, and through interaction with other cellular organelles. In this chapter, we will focus on the importance of enzymatic regulators of mitochondrial fuel metabolism and control of mitochondrial mass to pancreatic β-cell function, describing how defects in these pathways ultimately lead to diabetes. Furthermore, we will examine the factors responsible for mitochondrial biogenesis and degradation and their roles in the balance of mitochondrial mass in β-cells. Clarifying the causes of β-cell mitochondrial dysfunction may inform new approaches to treat the underlying etiologies of diabetes.