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Mitochondria are indispensable for cell viability; however, major mitochondrial functions including citric acid cycle and oxidative phosphorylation are dispensable. Most known essential mitochondrial proteins are involved in preprotein import and assembly, while the only known essential biosynthetic process performed by mitochondria is the biogenesis of iron–sulfur clusters (ISC). The components of the mitochondrial ISC-assembly machinery are derived from the prokaryotic ISC-assembly machinery. We have identified an essential mitochondrial matrix protein, Isd11 (YER048w-a), that is found in eukaryotes only. Isd11 is required for biogenesis of cellular Fe/S proteins and thus is a novel subunit of the mitochondrial ISC-assembly machinery. It forms a complex with the cysteine desulfurase Nfs1 and is required for formation of an Fe/S cluster on the Isu scaffold proteins. We conclude that Isd11 is an indispensable eukaryotic component of the mitochondrial machinery for biogenesis of Fe/S proteins.

BACKGROUND: Activating mutations of the FLT3 gene are common in acute myeloid leukemia (AML), with approximately 25-30% of cases containing an internal tandem duplication (ITD) in the juxtamembrane domain. The presence of a FLT3-ITD mutation is associated with a poor prognosis, the severity of which, can be modulated by the combination of co-occuring mutations. In animal models, expression of Flt3-ITD by transgenesis, bone marrow transplantation or gene knock-in does not lead to an acute leukemia but a myeloproliferative disease, resembling CMML, suggesting a requirement for additional co-operating mutations (Lee et al, Cancer Cell. 2007, 12: 367). This is supported by in vivo models which demonstrate that the combination of the Flt3-ITD mutation with other genetic lesions leads to the development of an acute leukemia in mice (Chen et al, Genes Dev. 2013, 27: 1974). AIMS: To identify and characterise novel genes that alter Flt3-ITD induced MPN by using an N-ethyl-N-Nitroso-urea (ENU) mutagenesis strategy in mice with an Flt3-ITD homozygous knock-in background. METHODS: An autosomal dominant screen for Flt3-ENU co-operating mutations was carried out at the Australian Phenomics Facility by mating ENU-mutagenised male mice homozygous for the Flt3-ITD knock-in allele to homozygous Flt3-ITD females. G1 mice were screened for changes in blood cell parameters indicative of an altered disease state (compared to that induced by Flt3-ITD alone). Mice with blood cell parameters outside two standard deviations of the relevant G1 mean were identified as potential mutation carriers and bred to Flt3-ITD homozygous knock-in mice to test for heritability of the phenotype. Where pedigrees were generated demonstrating heritable phenotypes, multiple affected and unaffected littermates were subject to exome sequencing and analysis to identify a list of candidate gene mutations segregating with the disease phenotype. RESULTS: 150 G1 mice were screened, leading to the identification of four pedigrees with heritable phenotypes marked by an exacerbated MPN. Exome sequencing has identified a short list of 3 genes for one pedigree (pedigree 37) that includes a mutation in Neurofibromatosis 1 (Nf1), a gene known to be involved in the induction of juvenile myelomonocytic leukemia and frequently lost in AML (Parkin et al,Clin Cancer Res 2010, 16:4135). In another pedigree (pedigree 24) we identified a mutation in Ndufa10 as the single candidate segregating with the phenotype (Figure 1A-B). Ndufa10 encodes a subunit of the mitochondrial respiratory complex I, which is the first and largest complex in the mitochondrial electron transport chain. Importantly, germline mutations in this gene lead to a complex I deficiency syndrome in humans, indicating that it is a critical subunit of this complex. We hypothesise that mutation of Ndufa10 leads to altered cellular metabolism in hematopoietic stem and progenitor cells which contributes to exacerbation of the MPN, possibly through an alteration in production of reactive oxygen species and a shift in the balance between glycolysis and oxidative phosphorylation. Breeding of the Ndufa10 mutation onto a non Flt3-ITD background shows that action of the mutation is not dependent on the presence of Flt3-ITD, as these mice also have altered blood counts, including increased WBC (Figure 1C). CONCLUSIONS: It is possible to identify mutations that exacerbate Flt3-ITD induced MPN through mutagenesis and an efficient blood screening strategy. In addition, using this strategy, we have identified novel mutations that act independently of Flt3-ITD to induce changes in the haematological compartment. Translation of these findings to human AML may indicate pathways that will be targets for new and complementary treatments in AML. Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Figure 1. A. Flt3-ITD Pedigree 24 indicating affected mice and genotyping for the Ndufa10 mutation. +/+=Ndufa10 wt, m/+=Ndufa10 heterozygous mutant. B. WBC counts for male mice from Pedigree 24 at 15-17 weeks (+/+, n=3; m/+, n=8). WBC from ENU G1 mice are shown as a comparison (G1, n=75). C. WBC counts for male mice on a wildtype C57 background at 16-18 weeks. +/+=Ndufa10 wt (n=7), m/+=Ndufa10 heterozygous mutant (n=8), m/m=Ndufa10 homozygous mutant (7). Disclosures No relevant conflicts of interest to declare.

Import of proteins into mitochondria occurs by coordinated actions of preprotein translocases in the outer and inner membranes. Tim9 and Tim10 are translocase components of the intermembrane space, related to deafness-dystonia peptide 1 (DDP1). They coassemble into a hexamer, TIM9.10, which captures and chaperones precursors of inner membrane metabolite carriers as they exit the TOM channel in the outer membrane. The crystal structure of TIM9.10 reveals a previously undescribed alpha-propeller topology in which helical "blades" radiate from a narrow central pore lined with polar residues. The propeller blades are reminiscent of "tentacles" in chaperones Skp and prefoldin. In each TIM9.10 subunit, a signature "twin CX3C" motif forms two intramolecular disulfides. There is no obvious binding pocket for precursors, which we suggest employ the chaperone-like tentacles of TIM9.10 as surrogate lipid contacts. The first reported crystal structure of a mitochondrial translocase assembly provides insights into selectivity and regulation of precursor import.

The purpose of this study was to evaluate and provide insights related to the influence of the intake of stable isotopes of carbon and iodine on the committed doses due to the ingestion of 1 4 C and 1 2 9 I. This was accomplished through the application of two different computational approaches. The first was based on the assumption that ground (drinking) water was the only source of intake of 1 4 C and 1 2 9 I, as well as stable carbon and stable iodine. In the second, the intake of 1 4 C and 1 2 9 I was still assumed to be restricted to that in the ground (drinking) water, but the intake of stable carbon and stable iodine was expanded to include that in other components of the diet. The doses were estimated using either a conversion formula or the applicable dose coefficients in Federal Guidance Reports No. 11 and No. 13. Serving as input for the analyses was the estimated maximum concentrations of 1 4 C or 1 2 9 I that would be present in the ground water due to potential releases from the proposed Yucca Mountain high-level radioactive waste repository during the first 10,000 y after closure. The estimated contributions of stable carbon and iodine through the consumption of ground water were based on analyses of samples collected in the Amargosa Valley, NV. The contributions through dietary intake were based on surveys conducted in the United States. Based on the accompanying analyses, it was noted that stable isotope intake has a significant effect on the estimated doses due to the intake of radioactive isotopes of the same element. While this is a well-known fact, this observation has international implications in terms of dose estimates for key radionuclides, such as 1 4 C and 1 2 9 I, a primary reason being the wide variations in the intakes of stable carbon and iodine in various countries. For this reason, analysts planning to apply the dose coefficients developed by the International Commission on Radiological Protection (ICRP) should either confirm that the average total intake in their country of stable isotope(s) of the radioactive isotope being evaluated is in reasonable agreement with the value assumed by the ICRP or suitably modify the ICRP dose coefficients to account for any differences. If such a procedure is to be implemented, there is a need for periodic updates of the dietary intakes of various stable elements in countries throughout the world. The Importance of this is documented by recent surveys in Asia that revealed that their average total daily intake of stable iodine was less than half of the ICRP value for Reference Man. In this case, application of the ICRP dose coefficients, without modification, would underestimate the dose due to ingested 1 2 9 I by a factor of more than two. A related situation exists in the United States where the latest surveys indicate that the daily intake of stable iodine is 75% of the ICRP value.

Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane. By examining the import pathway of human Tom40, we have been able to elucidate additional features in its import. We identify that Hsp90 is involved in delivery of the Tom40 precursor to mitochondria in an ATP-dependent manner. The precursor then forms its first stable intermediate with the outer face of the TOM complex before its membrane integration and assembly. Deletion of an evolutionary conserved region within Tom40 disrupts the TOM complex intermediate and causes it to stall at a new complex in the intermembrane space that we identify to be the mammalian SAM. Unlike its fungal counterparts, the human Tom40 precursor is not found stably arrested at a SAM intermediate. Nevertheless, we show that Tom40 assembly is reduced in mitochondria depleted of human Sam50. These findings are discussed in context with current models from fungal studies.

Complex I is the largest of the mitochondrial respiratory chain enzyme complexes, consisting of at least 46 subunits, seven of which are encoded by mtDNA. Deficiency of complex I is the most common respiratory chain defect, and can be caused by mutations in both nuclear and mtDNA encoded genes. It has a wide range of clinical presentations, from lethal infantile mitochondrial disease to isolated myopathy.1–3 Mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) is one of the syndromes associated with complex I deficiency and in approximately 80% of cases is caused by a mutation, 3243A→G, in the mitochondrial tRNALeu(UUR) gene ( MTTL1 ). Other mutations in MTTL1 and other transfer RNA genes ( MTTF , MTTV , MTTQ ) account for most of the remainder of cases.4 However, a number of mutations in the mitochondrial MTND subunit genes of complex I have also been reported to cause MELAS, most notably in MTND5 5 and to a lesser extent in MTND6 .6 In stark contrast, there are presently no mutations in the MTND1 subunit gene associated with MELAS. There are several mutations in MTND1 associated with Leber’s hereditary optic neuropathy (LHON) which may be pathogenic, but only one, the 3460G→A mutation, that has robust evidence, including cell biology studies, for pathogenicity.7–9 Here we report three unrelated patients with MELAS and isolated complex I deficiency in skeletal muscle and cultured fibroblasts due to previously unreported mutations in the MTND1 gene. Evidence confirming the pathogenic nature of these mutations includes data from cell fusion experiments and blue native polyacrylamide gel electrophoresis (BN-PAGE), the latter confirming a crucial role for the ND1 subunit in the assembly of complex I holoenzyme.10 ### Patient 1 Patient 1, a white male, presented at 4 years of age with a 3 month history of increasing tiredness, clumsiness, …

The presenilin proteins are required for intramembrane cleavage of a subset of type 1 membrane proteins including the Alzheimer's disease amyloid precursor protein. Previous studies indicate presenilin proteins form enzymatically active high molecular mass complexes consisting of heterodimers of N- and C-terminal fragments in association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1 proteins. Using Blue Native gel electrophoresis (BN/PAGE) we have studied endogenous presenilin 1 complex mass, stability and association with nicastrin, presenilin enhancer-2 and anterior pharynx defective-1. Solubilization of mouse or human brain membranes with dodecyl-d-maltoside produced a 360-kDa species reactive with antibodies to presenilin 1. Presenilin 1 complex levels were high in embryonic brain. Complex integrity was sensitive to Triton X-100 and SDS, but stable to reducing agent. Addition of 5 m urea caused complex dissolution and nicastrin to migrate as a subcomplex. Nicastrin and presenilin enhancer-2 were detected in the presenilin 1 complex following BN/PAGE, electroelution and second-dimension analysis. Anterior pharynx defective-1 was detected as an 18-kDa form and 9-kDa C-terminal fragment by standard SDS/PAGE of mouse tissues, and as a predominant 36-kDa band after presenilin 1 complex second-dimension analysis. Membranes from brain cortex of Alzheimer's disease patients, or from cases with presenilin 1 missense mutations, indicated no change in presenilin 1 complex mobility. Higher molecular mass presenilin 1-reactive species were detected in brain containing presenilin 1 exon 9 deletion mutation. This abnormality was confirmed using cells transfected with the same presenilin deletion mutation.

Both nuclear and mitochondrial DNA mutations can cause energy generation disorders. Respiratory chain complex I deficiency is the most common energy generation disorder and a frequent cause of infantile mitochondrial encephalopathies such as Leigh's disease and lethal infantile mitochondrial disease. Most such cases have been assumed to be caused by nuclear gene defects, but recently an increasing number have been shown to be caused by mutations in the mitochondrially encoded complex I subunit genes ND4, ND5, and ND6. We report the first four cases of infantile mitochondrial encephalopathies caused by mutations in the ND3 subunit gene. Three unrelated children have the same novel heteroplasmic mutation (T10158C), only the second mutation reported in ND3, and one has the previously identified T10191C mutation. Both mutations cause disproportionately greater reductions in enzyme activity than in the amount of fully assembled complex I, suggesting the ND3 subunit plays an unknown but important role in electron transport, proton pumping, or ubiquinone binding. Three cases appear to have a de novo mutation, with no mutation detected in maternal relatives. Mitochondrial DNA disease may be considerably more prevalent in the pediatric population than currently predicted and should be considered in patients with infantile mitochondrial encephalopathies and complex I deficiency.

The new antigen receptor (IgNAR) from sharks is a disulphide bonded dimer of two protein chains, each containing one variable and five constant domains, and functions as an antibody. In order to assess the antigen-binding capabilities of isolated IgNAR variable domains (VNAR), we have constructed an in vitro library incorporating synthetic CDR3 regions of 15-18 residues in length. Screening of this library against the 60 kDa cytosolic domain of the 70 kDa outer membrane translocase receptor from human mitochondria (Tom70) resulted in one dominant antigen-specific clone (VNAR 12F-11) after four rounds of in vitro selection. VNAR 12F-11 was expressed into the Escherichia coli periplasm and purified by anti-FLAG affinity chromatography at yields of 3 mg x L(-1). Purified protein eluted from gel filtration columns as a single monomeric protein and CD spectrum analysis indicated correct folding into the expected beta-sheet conformation. Specific binding to Tom70 was demonstrated by ELISA and BIAcore (Kd = 2.2 +/- 0.31 x 10(-9) m-1) indicating that these VNAR domains can be efficiently displayed as bacteriophage libraries, and selected against target antigens with an affinity and stability equivalent to that obtained for other single domain antibodies. As an initial step in producing 'intrabody' variants of 12F-11, the impact of modifying or removing the conserved immunoglobulin intradomain disulphide bond was assessed. High affinity binding was only retained in the wild-type protein, which combined with our inability to affinity mature 12F-11, suggests that this particular VNAR is critically dependent upon precise CDR loop conformations for its binding affinity.

Because current methods of tracking hypertension are time-consuming and expensive, the prevalence of hypertension in retired military populations is often unknown. Tracking military retiree hypertension is important because it can help to conserve scarce resources and save lives by preventing strokes and other diseases. Intended as a case study, an alternative method of tracking hypertension using pharmacy records and ambulatory data system records to confirm diagnosis of hypertension was used to determine the prevalence of hypertension in a retired military population (ages 40-85 years). The results indicated that matching pharmacy data with ICD9 401 diagnosis data and medical records is a more efficient method of tracking hypertension.

Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes1,2,3,4,5. It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination1,2,5,6,7,8,9,10. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11–15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.

The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytosol and subsequently imported into mitochondria through protein translocation machineries of the outer and inner membranes. In this review, we discuss the arrangement of the various translocation complexes and the function of individual import components. We also outline the various targeting pathways that preproteins can take in order to reach their appropriate sub-mitochondrial compartment.

Rationale Ischaemic heart disease is the leading cause of death and disability worldwide. Modulating mitochondrial morphology by inhibiting mitochondrial fission during acute ischaemia-reperfusion injury (IRI) has been reported to protect the heart against cell death. Here, we investigate the newly discovered mitochondrial fission proteins, MiD49 and MiD51, as novel targets for cardioprotection. Methods and Results Genetic ablation of both MiD49 and MiD51 in HL-1 cardiac cell using shRNAs had the following effects: A significant increase in the proportion of cells expressing predominantly elongated mitochondria (81.5±2.9% MiD49+MiD51 knockdown vs. 49.4±4.9% vector control: n=120 cells/group; P A delay in time taken to induce mitochondrial permeability transition pore (MPTP) opening (485±61 seconds MiD49+MiD51 knockdown vs. 233±20 seconds vector control: n=120 cells/group; P Less cell death following simulated IRI (27.9±3.0% MiD49+MiD51 knockdown vs. 56.7±2.4% vector control: n=300 cells/group; P Ablation of MiD49 or MiD51 individually had no beneficial effects. Unexpectedly, the over-expression of either MiD49 or MiD51 also had beneficial effects: The formation of abnormally elongated (hyperfused) mitochondria with a peri-nuclear distribution; It delayed the induction of MPTP opening; and It reduced cell death following simulated IRI. The explanation for this apparent beneficial effect with MiD49 or MiD51 over-expression, may be due to Drp1 sequestration to mitochondria, leading to unopposed abnormal mitochondrial elongation (hyperfusion). Conclusions Here, we show that combined knockdown of the newly discovered mitochondrial fission proteins, MiD49 and MiD51, induced mitochondrial elongation, delayed MPTP opening, and protected cells against simulated acute IRI, positioning these proteins as novel targets for cardioprotection.

One of the most challenging aspects of regulatory compliance can be demonstrating compliance with internal dosimetry requirements. For long-lived alpha-emitting radionuclides in particular, the sensitivity and accuracy of bioassay analysis and whole body counting may not allow for adequate assessment of intakes. Simple and effective measures can be used to control the workplace for the internal hazards associated with long-lived radioactive material using methods that measure directly the air to which workers are exposed. This paper provides an easy assessment tool that uses direct measurement of the specific activity of dusts in breathing zone air to evaluate internal exposures. Using this method, sensitive assessments can be made to determine if intakes are likely to have occurred and, if so, at what magnitude. It is not a substitute for confirmatory bioassay or whole body counting but a simple method to evaluate expectations for internal exposures. Health Phys.

Genes found in the nucleus encode almost all enzymes. Enzymes, along with proteins involved in their maintenance, must therefore be targeted to their correct destination. Proteins are synthesized with signal sequences that guide them to receptor proteins that in turn transfer them to large transmembrane protein channels of organelles. Following their translocation across membranes, the newly inserted proteins are often folded with the assistance of molecular chaperones. Keywords: organelles; protein import; protein export; receptors; targeting

Mitochondria import several hundred different proteins from the cytosol. Upon synthesis on cytosolic polysomes, targeting signals of the preproteins are recognized by receptors on the mitochondrial surface. The preproteins are then translocated across the outer mitochondrial membrane through a general import pore (GIP) and are transferred to the further mitochondrial subcompartments (32, 35, 37, 45). Three proteins of the translocase of the outer membrane (TOM) have been identified as receptors for preproteins. Tom20 and Tom70 are the initial receptors for preproteins with N-terminal targeting signals and internal targeting signals, respectively. Tom22 functions as central receptor and is associated with the channel-forming subunit Tom40. Coimmunoprecipitation and blue native polyacrylamide gel electrophoresis (BN-PAGE) of mitochondria from the yeast Saccharomyces cerevisiae led to the identification of a ∼400-kDa (400K) complex, termed the GIP complex, that contains Tom22, Tom40, and three small Tom proteins, Tom5, Tom6, and Tom7 (9, 10, 52). Tom5 mediates transfer of preproteins from Tom22 to Tom40 (10), while Tom6 and Tom7 modulate assembly steps of the GIP complex (3, 9, 19, 40). The TOM complex was also isolated from Neurospora crassa mitochondria, and its holo form was shown to contain not only Tom40, Tom22, Tom7, and Tom6 but also Tom20 and some Tom70; however, no Neurospora homolog of Tom5 has been found (1, 27). Electron microscopic analysis and assessment of the stoichiometry of TOM complexes have suggested that the TOM complex contains two or three translocation channels (1, 9, 27, 52). Electrophysiological studies with the Neurospora TOM complex resolved conductance states suggesting the presence of two independent channels (27, 28). Little is known about the functional architecture of the TOM machinery and the interactions of its subunits. It is unknown which Tom proteins and type of interactions are responsible for keeping a preprotein in the import channel. Moreover, it is an open question whether translocase components of the intermembrane space or inner membrane (TIM) are needed for stable accumulation of preproteins in the GIP (30, 31) or whether the TOM complex is functional by itself. Possible candidates are the TIM23 complex for preproteins containing N-terminal targeting signals or the small Tim proteins of the intermembrane space and inner membrane, such as Tim10 and Tim12, for preproteins containing internal targeting signals (4, 22, 26, 56). In this study we attempted to address these questions regarding the functional architecture of the mitochondrial TOM machinery. We found that the GIP complex is of unusual high stability, with a Tom40-Tom22 core structure responsible for stably holding a preprotein by nonionic forces. Tim proteins are not needed for accumulation of a preprotein in the GIP complex, and evidence for a coupled function of two channel pores in a GIP complex is presented.

The ADP/ATP carrier (AAC) is a major representative of mitochondrial preproteins lacking an N-terminal presequence. AAC contains targeting information in each of its three modules, which has led to a search for the dominant targeting region. An alternative, not yet tested model would be that several distinct targeting signals function simultaneously in import of the preprotein. We report that the three AAC modules cooperate in binding to the receptor Tom70 such that three Tom70 dimers are recruited to one preprotein. The modules are transferred to the import pore in a stepwise manner and cooperate again in the accumulation of AAC in the general import pore complex. AAC can cross the outer membrane with an internal segment first, i.e. in a loop formation. Each module of AAC is required for dimerization in the inner membrane. We propose a new concept for import of the hydrophobic carrier proteins into mitochondria where multiple signals cooperate in receptor recruitment, outer membrane translocation via loop formation and assembly in the inner membrane.

Publisher Summary Hsp70 proteins are essential components of the protein translocation systems of the endoplasmic reticulum (ER) and mitochondria. The role of Hsp70s in protein translocation is not confined to the ER and mitochondria. For most proteins of the ER, translocation is coupled with protein synthesis. The synthesis of the growing polypeptide from the ribosome docked onto the ER is sufficient to drive the forward movement of the preprotein into the organelle. However, it was found that some ER-targeted proteins, especially those found in yeast, could be imported into this organelle in a posttranslational fashion. Polypeptide binding and release by Hsp70 is coupled to its nucleotide binding state. Furthermore, Hsp70 proteins often act in concert with cofactors involved in the regulation of its ATPase cycle. These cofactors thereby indirectly contribute to the process of polypeptide binding and release. In contrast to the ER, a mitochondrial homolog of the co-chaperone and nucleotide exchange factor GrpE, termed Mgel, is present in the matrix and is involved in the cycling of the mtHsp70 ATPase by releasing ADP from mtHsp70.

Publisher Summary Most proteins found in the mitochondria originate in the cytosol and therefore must be imported into this organelle following their synthesis. Most of the translocase components of the outer and inner mitochondrial membranes (TOM and TIM, respectively) have been identified in Saccharomyces cerevisiae and Neurospora crassa . A preprotein is directed to the mitochondria by virtue of its targeting signal, which is most often situated at the N terminus. Preproteins are first bound to receptors of the mitochondrial membrane—Tom70, Tom22, and Tom20—before being translocated across the outer membrane channel, which is formed by Tom40. Many preproteins destined to reside in the outer membrane transverse laterally out of the channel upon contact of their hydrophobic anchors with the TOM machinery. Further, another group of inner membrane proteins contain internal targeting signals that direct them from the inner face of the TOM machinery to a different TIM complex (the TIM22 complex) via their association with small intermembrane space TIM proteins. The characterization of these different import pathways has been achieved mainly through the use of mitochondrial in vitro import studies. In such cases, isolated mitochondria are incubated with in vitro -translated 35 S-labeled preproteins under varying conditions, and their import characteristics and requirements are analyzed. This chapter describes various methods employed to characterize both the mitochondrial import of preproteins and the protein import machinery in vitro .

In order for proteins to be imported into subcellular compartments, they must first traverse the organellar membranes. In mitochondria, hydrophilic protein channels in both the outer and inner membranes serve such a purpose. Recently, the channel protein of the outer mitochondrial membrane was identified to be Tom40. Tom40 is found in a high molecular weight complex termed the general import pore (GIP) complex where it is tightly associated with the receptor protein Tom22 along with Tom7, Tom6 and Tom5. Tom7 and Tom6 seem to modulate the dynamics of the GIP complex while Tom5 is involved in preprotein transfer from receptors to Tom40. The receptor proteins Tom70 and Tom20 associate with this complex in a weaker manner where they are involved in the initial recognition of preproteins. This review focuses on the identification and characterisation of the transport machinery of the outer mitochondrial membrane and how they are involved in the co-ordination and regulation of events required for the translocation of preproteins into mitochondria.

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23–Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22–Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23–Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

Planning, preparing, submitting and tracking license and permit applications through the regulatory process is complex and time consuming. A disciplined approach can make the process easier. This paper discusses practical steps to take and some guidelines to follow in making the licensing or permitting process easier.

Chaperonins are a class of stress-inducible molecular chaperones involved in protein folding. We report the cloning, sequencing and characterisation of the rat mitochondrial chaperonin 60 and chaperonin 10 genes. The two genes are arranged in a head-to-head configuration and together comprise 14 kb and contain 14 introns. The genes are linked together by a region of approximately 280 bp, which constitutes a bidirectional promoter and includes a common heat-shock element. Insertion of the shared promoter region between two reporter genes is sufficient to drive their expression under both constitutive and heat-shock conditions. The arrangement of the mammalian chaperonin genes suggests the potential to provide the coordinated regulation of their products in a manner that is mechanistically distinct from, yet conceptually similar to, that employed by the bacterial chaperonin (groE) operon.

Molecular chaperones play a critical role in many cellular processes. This review concentrates on their role in targeting of proteins to the mitochondria and the subsequent folding of the imported protein. It also reviews the role of molecular chaperones in protein degradation, a process that not only regulates the turnover of proteins but also eliminates proteins that have folded incorrectly or have aggregated as a result of cell stress. Finally, the role of molecular chaperones, in particular the mitochondrial chaperonins, in disease is reviewed. In support of the endosymbiont theory on the origin of mitochondria, the chaperones of the mitochondrial compartment show a high degree of similarity to bacterial molecular chaperones. Thus, studies of protein folding in bacteria such as Escherichia coli have proved to be instructive in understanding the process in the eukaryotic cell. As in bacteria, the molecular chaperone genes of eukaryotes are activated by a variety of stresses. The regulation of stress genes involved in mitochondrial chaperone function is reviewed and major unsolved questions regarding the regulation, function, and involvement in disease of the molecular chaperones are identified.

Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis., Author Summary Mitochondrial complex III deficiency is a devastating disorder that impairs energy generation, and leads to variable symptoms such as developmental regression, seizures, kidney dysfunction and frequently death. The genetic basis of complex III deficiency is not fully understood, with around half of cases having no known cause. This lack of genetic diagnosis is partly due to an incomplete understanding of the genes required for complex III assembly and function. We have identified two key proteins required for complex III, UQCC1 and UQCC2, and have elucidated the role of these inter-dependent proteins in the biogenesis of cytochrome b, the only complex III subunit that is encoded by mitochondrial DNA. We have shown that mutations in UQCC2 cause human complex III deficiency in a patient with neonatal lactic acidosis and renal tubulopathy. This work contributes to an improved understanding of complex III biogenesis, and will aid future molecular diagnoses of complex III deficiency.

Sparganosis is an infection caused by migrating larvae of the cestode genus Spirometra. There have been approximately 62 cases of sparganosis reported in the United States. Although a subcutaneous mass is the most common manifestation, sparganosis is not well-described in the dermatology literature. We present a case of cutaneous sparganosis in a 52-year-old Filipino American woman. Histologically, the sections showed a granulomatous panniculitis and dermatitis containing a section of a sparganum. A transverse section of an intact sparganum reveals an eosinophilic cuticle, loose stroma, calcareous bodies, and smooth muscle fibers.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI-specific phospholipase C (PI-PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI-PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8-barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI-PLC in complex with its inhibitor, myo-inositol, at 2.6 A resolution (R factor = 19.5%). This substrate-like inhibitor interacts with a number of residues highly conserved among prokaryotic PI-PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI-PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.

We used affinity chromatography on DnaK columns to identify a mitochondrial GrpE homologue from bovine, porcine and rat liver mitochondria. The 24 kDa GrpE homologue bound specifically to the DnaK column and was not eluted with 1 M KCl but readily with 5 mM ATP. Sequence analysis of the bovine homologue (85 residues) revealed 42% positional identity to mitochondrial GrpEp from S. cerevisiae and about 30% identity to the bacterial counterparts. Thus, GrpE homologues from higher and lower eukaryotes are highly conserved.

Because current methods of tracking hypertension are time-consuming and expensive, the prevalence of hypertension in retired military populations is often unknown. Tracking military retiree hypertension is important because it can help to conserve scarce resources and save lives by preventing strokes and other diseases. Intended as a case study, an alternative method of tracking hypertension using pharmacy records and ambulatory data system records to confirm diagnosis of hypertension was used to determine the prevalence of hypertension in a retired military population (ages 40-85 years). The results indicated that matching pharmacy data with ICD9 401 diagnosis data and medical records is a more efficient method of tracking hypertension.

Because Topoisomerase I (TOP1) is critical for the relaxation of DNA supercoils and because it is the target for anticancer activity of camptothecins, we assessed TOP1 transcript levels in the 60 cell line panel (the NCI-60) of the National Cancer Institute's anticancer drug screen. TOP1 expression levels varied over a 5.7-fold range across the NCI-60. HCT116 colon and MCF-7 breast cancer cells were the highest expressers; SK-MEL-28 melanoma and HS578T breast carcinoma cells were the lowest. TOP1 mRNA expression was highly correlated with Top1 protein levels, indicating that TOP1 transcripts could be conveniently used to monitor Top1 protein levels and activity in tissues. Assessment of the TOP1 locus by array comparative genomic hybridization across the NCI-60 showed copy numbers ranging from 1.71 to 4.13 and a statistically significant correlation with TOP1 transcript levels (p Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C149.

By the end of the sixteenth century, the Christian mission overseas had fallen on hard times. With the great geographical discoveries a little more than a century old, the rosy optimism of early missionaries that the new worlds could be converted quickly and easily had begun to sour. Second and third generation missionaries in areas of colonial control had to confront the fact that baptism and conversion were not always identical and that indigenous religions could neither be mandated nor persecuted out of existence. The tenacity of heathenism and the ignorance of earlier missionaries were refrains from an increasingly audible jeremaid.1 In Mexico, the Franciscan Bernardino de Sahagun meticulously compiled his religious ethnography of the Indians to show the manifold ways in which superstitution had survived the arrival of the Gospel.2 To the south, in Peru, the influential Jesuit Jose de Acosta wrote his important missionary manual, De procuranda indorum salute (1588), both to console disappointed missionaries who found their work frustrated at every turn and to suggest ways by which the Indians might be more effectively proselytized.3 In Asia, the situation was, if anything, worse. Christian missions in India remained stalled within the narrow perimeter of Portuguese control. Outside the Iberian orbit it was no different. The death of the sympathetic Akbar in 1605 seemed to have robbed the Jesuits of a Catholic Mughal Empire. Most alarming of all, however, was the sudden reversal of fortunes in Japan.