Publications by authors named "Robert E Jensen"

40 Publications

TbKAP6, a mitochondrial HMG box-containing protein in Trypanosoma brucei, is the first trypanosomatid kinetoplast-associated protein essential for kinetoplast DNA replication and maintenance.

Eukaryot Cell 2014 Jul 30;13(7):919-32. Epub 2014 May 30.

Department of Cell Biology, Johns Hopkins Medical School, Baltimore, Maryland, USA.

Kinetoplast DNA (kDNA), the mitochondrial genome of trypanosomatids, is a giant planar network of catenated minicircles and maxicircles. In vivo kDNA is organized as a highly condensed nucleoprotein disk. So far, in Trypanosoma brucei, proteins involved in the maintenance of the kDNA condensed structure remain poorly characterized. In Crithidia fasciculata, some small basic histone H1-like kinetoplast-associated proteins (CfKAP) have been shown to condense isolated kDNA networks in vitro. High-mobility group (HMG) box-containing proteins, such as mitochondrial transcription factor A (TFAM) in mammalian cells and Abf2 in the budding yeast, have been shown essential for the packaging of mitochondrial DNA (mtDNA) into mitochondrial nucleoids, remodeling of mitochondrial nucleoids, gene expression, and maintenance of mtDNA. Here, we report that TbKAP6, a mitochondrial HMG box-containing protein, is essential for parasite cell viability and involved in kDNA replication and maintenance. The RNA interference (RNAi) depletion of TbKAP6 stopped cell growth. Replication of both minicircles and maxicircles was inhibited. RNAi or overexpression of TbKAP6 resulted in the disorganization, shrinkage, and loss of kDNA. Minicircle release, the first step in kDNA replication, was inhibited immediately after induction of RNAi, but it quickly increased 3-fold upon overexpression of TbKAP6. Since the release of covalently closed minicircles is mediated by a type II topoisomerase (topo II), we examined the potential interactions between TbKAP6 and topo II. Recombinant TbKAP6 (rTbKAP6) promotes the topo II-mediated decatenation of kDNA. rTbKAP6 can condense isolated kDNA networks in vitro. These results indicate that TbKAP6 is involved in the replication and maintenance of kDNA.
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http://dx.doi.org/10.1128/EC.00260-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135736PMC
July 2014

Mitochondrial shape and function in trypanosomes requires the outer membrane protein, TbLOK1.

Mol Microbiol 2013 Feb 21;87(4):713-29. Epub 2013 Jan 21.

Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.

In an RNAi library screen for loss of kinetoplast DNA (kDNA), we identified an uncharacterized Trypanosoma brucei protein, named TbLOK1, required for maintenance of mitochondrial shape and function. We found the TbLOK1 protein located in discrete patches in the mitochondrial outer membrane. Knock-down of TbLOK1 in procyclic trypanosomes caused the highly interconnected mitochondrial structure to collapse, forming an unbranched tubule remarkably similar to the streamlined organelle seen in the bloodstream form. Following RNAi, defects in mitochondrial respiration, inner membrane potential and mitochondrial transcription were observed. At later times following TbLOK1 depletion, kDNA was lost and a more drastic alteration in mitochondrial structure was found. Our results demonstrate the close relationship between organelle structure and function in trypanosomes.
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http://dx.doi.org/10.1111/mmi.12089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4715881PMC
February 2013

Kinetic considerations for strength recovery at the fiber-matrix interface based on the Diels-Alder reaction.

ACS Appl Mater Interfaces 2013 Feb 25;5(3):815-21. Epub 2013 Jan 25.

Department of Chemical & Biological Engineering, Drexel University, Philadelphia, Pennsylvania 19104, United States.

The Diels-Alder reaction was used to yield thermal reversibility of the bonding between a partially furan-functionalized epoxy thermosetting matrix and a maleimide-treated glass fiber. Under ambient temperature conditions, the covalent bond forming product reaction dominates, but this reaction reverses at elevated temperatures to allow for interfacial healing. Single-fiber microdroplet pull-out testing was used to characterize the coupled effects of healing temperature and the glass transition temperature (T(g)) of the epoxy on interfacial strength recovery. In particular, the roles of mobility and reaction kinetics were independently varied to understand the individual effects of both.
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http://dx.doi.org/10.1021/am302383vDOI Listing
February 2013

Synthesis and characterization of ionic polymer networks in a room-temperature ionic liquid.

ACS Appl Mater Interfaces 2012 Nov 5;4(11):6142-50. Epub 2012 Nov 5.

Department of Chemical & Biological Engineering, Drexel University, Philadelphia, Pennsylvania 19104, USA.

Ionic liquid gels (ILGs) for potential use in ion transport and separation applications were generated via a free radical copolymerization of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and N,N'-methylene(bis)acrylamide (MBA) using 1-ethyl-3-methylimidazolium ethylsulfate (IL) as a room temperature ionic liquid solvent medium. The AMPS and MBA monomer solubility window in the IL in the temperature range of 25 to 65 °C was determined. In situ ATR-FTIR showed near complete conversion of monomers to a cross-linked polymer network. ILGs with glass transition temperatures (T(g)s) near -50 °C were generated with T(g) decreasing with increasing IL content. The elastic moduli in compression (200 to 6600 kPa) decreased with increasing IL content and increasing AMPS content while the conductivities (0.35 to 2.14 mS cm⁻¹) increased with increasing IL content and decreasing MBA content. The polymer-IL interaction parameter (χ) (0.48 to 0.55) was determined via a modified version of the Bray and Merrill equation.
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http://dx.doi.org/10.1021/am301777hDOI Listing
November 2012

Network news: the replication of kinetoplast DNA.

Annu Rev Microbiol 2012 ;66:473-91

Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

One of the most fascinating and unusual features of trypanosomatids, parasites that cause disease in many tropical countries, is their mitochondrial DNA. This genome, known as kinetoplast DNA (kDNA), is organized as a single, massive DNA network formed of interlocked DNA rings. In this review, we discuss recent studies on kDNA structure and replication, emphasizing recent developments on replication enzymes, how the timing of kDNA synthesis is controlled during the cell cycle, and the machinery for segregating daughter networks after replication.
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http://dx.doi.org/10.1146/annurev-micro-092611-150057DOI Listing
January 2013

Role for two conserved intermembrane space proteins, Ups1p and Ups2p, [corrected] in intra-mitochondrial phospholipid trafficking.

J Biol Chem 2012 May 7;287(19):15205-18. Epub 2012 Mar 7.

Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

Mitochondrial membranes maintain a specific phospholipid composition. Most phospholipids are synthesized in the endoplasmic reticulum (ER) and transported to mitochondria, but cardiolipin and phosphatidylethanolamine are produced in mitochondria. In the yeast Saccharomyces cerevisiae, phospholipid exchange between the ER and mitochondria relies on the ER-mitochondria encounter structure (ERMES) complex, which physically connects the ER and mitochondrial outer membrane. However, the proteins and mechanisms involved in phospholipid transport within mitochondria remain elusive. Here, we investigated the role of the conserved intermembrane space proteins, Ups1p and Ups2p, and an inner membrane protein, Mdm31p, in phospholipid metabolism. Our data show that loss of the ERMES complex, Ups1p, and Mdm31p causes similar defects in mitochondrial phospholipid metabolism, mitochondrial morphology, and cell growth. Defects in cells lacking the ERMES complex or Ups1p are suppressed by Mdm31p overexpression as well as additional loss of Ups2p, which antagonizes Ups1p. Combined loss of the ERMES complex and Ups1p exacerbates phospholipid defects. Finally, pulse-chase experiments using [(14)C]serine revealed that Ups1p and Ups2p antagonistically regulate conversion of phosphatidylethanolamine to phosphatidylcholine. Our results suggest that Ups proteins and Mdm31p play important roles in phospholipid biosynthesis in mitochondria. Ups proteins may function in phospholipid trafficking between the outer and inner mitochondrial membranes.
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http://dx.doi.org/10.1074/jbc.M111.338665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3346110PMC
May 2012

TbPIF8, a Trypanosoma brucei protein related to the yeast Pif1 helicase, is essential for cell viability and mitochondrial genome maintenance.

Mol Microbiol 2012 Feb 4;83(3):471-85. Epub 2012 Jan 4.

Departments of Biological Chemistry Cell Biology, Johns Hopkins Medical School, Baltimore, MD 21205, USA.

The trypanosome mitochondrial genome, kinetoplast DNA (kDNA), is a massive network of interlocked DNA rings, including several thousand minicircles and dozens of maxicircles. The unusual complexity of kDNA would indicate that numerous proteins must be involved in its condensation, replication, segregation and gene expression. During our investigation of trypanosome mitochondrial PIF1-like helicases, we found that TbPIF8 is the smallest and most divergent. It lacks some conserved helicase domains, thus implying that unlike other mitochondrial PIF1-like helicases, this protein may have no enzymatic activity. TbPIF8 is positioned on the distal face of kDNA disk and its localization patterns vary with different kDNA replication stages. Stem-loop RNAi of TbPIF8 arrests cell growth and causes defects in kDNA segregation. RNAi of TbPIF8 causes only limited kDNA shrinkage but the networks become disorganized. Electron microcopy of thin sections of TbPIF8-depleted cells shows heterogeneous electron densities in the kinetoplast disk. Although we do not yet know its exact function, we conclude that TbPIF8 is essential for cell viability and is important for maintenance of kDNA.
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http://dx.doi.org/10.1111/j.1365-2958.2011.07938.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3262056PMC
February 2012

Depletion of mitochondrial acyl carrier protein in bloodstream-form Trypanosoma brucei causes a kinetoplast segregation defect.

Eukaryot Cell 2011 Mar 14;10(3):286-92. Epub 2011 Jan 14.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205, USA.

Like other eukaryotes, trypanosomes have an essential type II fatty acid synthase in their mitochondrion. We have investigated the function of this synthase in bloodstream-form parasites by studying the effect of a conditional knockout of acyl carrier protein (ACP), a key player in this fatty acid synthase pathway. We found that ACP depletion not only caused small changes in cellular phospholipids but also, surprisingly, caused changes in the kinetoplast. This structure, which contains the mitochondrial genome in the form of a giant network of several thousand interlocked DNA rings (kinetoplast DNA [kDNA]), became larger in some cells and smaller or absent in others. We observed the same pattern in isolated networks viewed by either fluorescence or electron microscopy. We found that the changes in kDNA size were not due to the disruption of replication but, instead, to a defect in segregation. kDNA segregation is mediated by the tripartite attachment complex (TAC), and we hypothesize that one of the TAC components, a differentiated region of the mitochondrial double membrane, has an altered phospholipid composition when ACP is depleted. We further speculate that this compositional change affects TAC function, and thus kDNA segregation.
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http://dx.doi.org/10.1128/EC.00290-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067480PMC
March 2011

The killing of African trypanosomes by ethidium bromide.

PLoS Pathog 2010 Dec 16;6(12):e1001226. Epub 2010 Dec 16.

Department of Biological Chemistry, Johns Hopkins Medical School, Baltimore, Maryland, United States of America.

Introduced in the 1950s, ethidium bromide (EB) is still used as an anti-trypanosomal drug for African cattle although its mechanism of killing has been unclear and controversial. EB has long been known to cause loss of the mitochondrial genome, named kinetoplast DNA (kDNA), a giant network of interlocked minicircles and maxicircles. However, the existence of viable parasites lacking kDNA (dyskinetoplastic) led many to think that kDNA loss could not be the mechanism of killing. When recent studies indicated that kDNA is indeed essential in bloodstream trypanosomes and that dyskinetoplastic cells survive only if they have a compensating mutation in the nuclear genome, we investigated the effect of EB on kDNA and its replication. We here report some remarkable effects of EB. Using EM and other techniques, we found that binding of EB to network minicircles is low, probably because of their association with proteins that prevent helix unwinding. In contrast, covalently-closed minicircles that had been released from the network for replication bind EB extensively, causing them, after isolation, to become highly supertwisted and to develop regions of left-handed Z-DNA (without EB, these circles are fully relaxed). In vivo, EB causes helix distortion of free minicircles, preventing replication initiation and resulting in kDNA loss and cell death. Unexpectedly, EB also kills dyskinetoplastic trypanosomes, lacking kDNA, by inhibiting nuclear replication. Since the effect on kDNA occurs at a >10-fold lower EB concentration than that on nuclear DNA, we conclude that minicircle replication initiation is likely EB's most vulnerable target, but the effect on nuclear replication may also contribute to cell killing.
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http://dx.doi.org/10.1371/journal.ppat.1001226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3002999PMC
December 2010

Dual role of the receptor Tom20 in specificity and efficiency of protein import into mitochondria.

Proc Natl Acad Sci U S A 2011 Jan 20;108(1):91-6. Epub 2010 Dec 20.

Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

Mitochondria import most of their resident proteins from the cytosol, and the import receptor Tom20 of the outer-membrane translocator TOM40 complex plays an essential role in specificity of mitochondrial protein import. Here we analyzed the effects of Tom20 binding on NMR spectra of a long mitochondrial presequence and found that it contains two distinct Tom20-binding elements. In vitro import and cross-linking experiments revealed that, although the N-terminal Tom20-binding element is essential for targeting to mitochondria, the C-terminal element increases efficiency of protein import in the step prior to translocation across the inner membrane. Therefore Tom20 has a dual role in protein import into mitochondria: recognition of the targeting signal in the presequence and tethering the presequence to the TOM40 complex to increase import efficiency.
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http://dx.doi.org/10.1073/pnas.1014918108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3017135PMC
January 2011

Room-temperature healing of a thermosetting polymer using the Diels-Alder reaction.

ACS Appl Mater Interfaces 2010 Apr;2(4):1141-9

Department of Chemical & Biological Engineering, Drexel University, Philadelphia, PA19104, USA.

Self-healing materials are particularly desirable for load-bearing applications because they offer the potential for increased safety and material lifetimes. A furan-functionalized polymer network was designed that can heal via covalent bonding across the crack surface with the use of a healing agent consisting of a bismaleimide in solution. Average healing efficiencies of approximately 70% were observed. The healing ability of fiber-reinforced composite specimens was investigated with flexural, short beam shear, and double cantilever beam specimens. It was found that solvent amount and maleimide concentration play key roles in determining healing efficiency.
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http://dx.doi.org/10.1021/am9009378DOI Listing
April 2010

Reversibly cross-linked polymer gels as healing agents for epoxy-amine thermosets.

ACS Appl Mater Interfaces 2009 May;1(5):992-5

The Diels-Alder reaction was used to develop a reversibly cross-linking gel as a healing agent for traditional epoxy-amine thermosets. Direct application of the reversibly cross-linking network to a crack surface in an epoxy-amine thermoset resulted in the recovery of 37% of the initial epoxy-amine network's strength. Composites in which the reversibly cross-linking gel was incorporated as a secondary particulate phase recovered 21% of the initial composite strength after the first healing cycle, with healing possible up to five times.
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http://dx.doi.org/10.1021/am900104wDOI Listing
May 2009

Phosphorylation of the F(1)F(o) ATP synthase beta subunit: functional and structural consequences assessed in a model system.

Circ Res 2010 Feb 24;106(3):504-13. Epub 2009 Dec 24.

Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA.

Rationale: We previously discovered several phosphorylations to the beta subunit of the mitochondrial F(1)F(o) ATP synthase complex in isolated rabbit myocytes on adenosine treatment, an agent that induces cardioprotection. The role of these phosphorylations is unknown.

Objective: The present study focuses on the functional consequences of phosphorylation of the ATP synthase complex beta subunit by generating nonphosphorylatable and phosphomimetic analogs in a model system, Saccharomyces cerevisiae.

Methods And Results: The 4 amino acid residues with homology in yeast (T58, S213, T262, and T318) were studied with respect to growth, complex and supercomplex formation, and enzymatic activity (ATPase rate). The most striking mutant was the T262 site, for which the phosphomimetic (T262E) abolished activity, whereas the nonphosphorylatable strain (T262A) had an ATPase rate equivalent to wild type. Although T262E, like all of the beta subunit mutants, was able to form the intact complex (F(1)F(o)), this strain lacked a free F(1) component found in wild-type and had a corresponding increase of lower-molecular-weight forms of the protein, indicating an assembly/stability defect. In addition, the ATPase activity was reduced but not abolished with the phosphomimetic mutation at T58, a site that altered the formation/maintenance of dimers of the F(1)F(o) ATP synthase complex.

Conclusions: Taken together, these data show that pseudophosphorylation of specific amino acid residues can have separate and distinctive effects on the F(1)F(o) ATP synthase complex, suggesting the possibility that several of the phosphorylations observed in the rabbit heart can have structural and functional consequences to the F(1)F(o) ATP synthase complex.
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http://dx.doi.org/10.1161/CIRCRESAHA.109.214155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2835499PMC
February 2010

Mgr3p and Mgr1p are adaptors for the mitochondrial i-AAA protease complex.

Mol Biol Cell 2008 Dec 8;19(12):5387-97. Epub 2008 Oct 8.

Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

By screening yeast knockouts for their dependence upon the mitochondrial genome, we identified Mgr3p, a protein that associates with the i-AAA protease complex in the mitochondrial inner membrane. Mgr3p and Mgr1p, another i-AAA-interacting protein, form a subcomplex that bind to the i-AAA subunit Yme1p. We find that loss of Mgr3p, like the lack of Mgr1p, reduces proteolysis by Yme1p. Mgr3p and Mgr1p can bind substrate even in the absence of Yme1p, and both proteins are needed for maximal binding of an unfolded substrate by the i-AAA complex. We speculate that Mgr3p and Mgr1p function in an adaptor complex that targets substrates to the i-AAA protease for degradation.
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http://dx.doi.org/10.1091/mbc.e08-01-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2592643PMC
December 2008

What happens when Trypanosoma brucei leaves Africa.

Trends Parasitol 2008 Oct 18;24(10):428-31. Epub 2008 Aug 18.

Department of Cell Biology, Johns Hopkins Medical School, Baltimore, MD 21205, USA.

Julius Lukes and co-workers evaluated the evolutionary origin of Trypanosoma equiperdum and Trypanosoma evansi, parasites that cause horse and camel diseases. Although similar to T. brucei, the sleeping-sickness parasite, these trypanosomes do not cycle through the tsetse fly and have been able to spread beyond Africa. Transmission occurs sexually, or via blood-sucking flies or vampire bats. They concluded that these parasites, which resemble yeast petite mutants, are T. brucei sub-species, which have evolved recently through changes in mitochondrial DNA.
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http://dx.doi.org/10.1016/j.pt.2008.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2725760PMC
October 2008

Fluorescence mapping of mitochondrial TIM23 complex reveals a water-facing, substrate-interacting helix surface.

Cell 2008 Aug;134(3):439-50

Department of Molecular and Cellular Medicine, Texas A&M Health Science Center, College Station, TX 77843-1114, USA.

Protein translocation across the mitochondrial inner membrane is mediated by the TIM23 complex. While its central component, Tim23, is believed to form a protein-conducting channel, the regions of this subunit that face the imported protein are unknown. To examine Tim23 structure and environment in intact membranes at high resolution, various derivatives, each with a single, environment-sensitive fluorescent probe positioned at a specific site, were assembled into functional TIM23 complexes in active mitochondria and analyzed by multiple spectral techniques. Probes placed sequentially throughout a transmembrane region that was identified by crosslinking as part of the protein-conducting channel revealed an alpha helix in an amphipathic environment. Probes on the aqueous-facing helical surface specifically underwent spectral changes during protein import, and their accessibility to hydrophilic quenching agents is considered in terms of channel gating. This approach has therefore provided an unprecedented view of a translocon channel structure in an intact, fully operational, membrane-embedded complex.
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http://dx.doi.org/10.1016/j.cell.2008.06.007DOI Listing
August 2008

Tom20 and Tom22 share the common signal recognition pathway in mitochondrial protein import.

J Biol Chem 2008 Feb 6;283(7):3799-807. Epub 2007 Dec 6.

Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan.

Precise targeting of mitochondrial precursor proteins to mitochondria requires receptor functions of Tom20, Tom22, and Tom70 on the mitochondrial surface. Tom20 is a major import receptor that recognizes preferentially mitochondrial presequences, and Tom70 is a specialized receptor that recognizes presequence-less inner membrane proteins. The cytosolic domain of Tom22 appears to function as a receptor in cooperation with Tom20, but how its substrate specificity differs from that of Tom20 remains unclear. To reveal possible differences in substrate specificities between Tom20 and Tom22, if any, we deleted the receptor domain of Tom20 or Tom22 in mitochondria in vitro by introducing cleavage sites for a tobacco etch virus protease between the receptor domains and transmembrane segments of Tom20 and Tom22. Then mitochondria without the receptor domain of Tom20 or Tom22 were analyzed for their abilities to import various mitochondrial precursor proteins targeted to different mitochondrial subcompartments in vitro. The effects of deletion of the receptor domains on the import of different mitochondrial proteins for different import pathways were quite similar between Tom20 and Tom22. Therefore Tom20 and Tom22 are apparently involved in the same step or sequential steps along the same pathway of targeting signal recognition in import.
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http://dx.doi.org/10.1074/jbc.M708339200DOI Listing
February 2008

Quaternary structure of the mitochondrial TIM23 complex reveals dynamic association between Tim23p and other subunits.

Mol Biol Cell 2008 Jan 24;19(1):159-70. Epub 2007 Oct 24.

Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.

Tim23p is an essential channel-forming component of the multisubunit TIM23 complex of the mitochondrial inner membrane that mediates protein import. Radiolabeled Tim23p monocysteine mutants were imported in vitro, incorporated into functional TIM23 complexes, and subjected to chemical cross-linking. Three regions of proximity between Tim23p and other subunits of the TIM23 complex were identified: Tim17p and the first transmembrane segment of Tim23p; Tim50p and the C-terminal end of the Tim23p hydrophilic region; and the entire hydrophilic domains of Tim23p molecules. These regions of proximity reversibly change in response to changes in membrane potential across the inner membrane and also when a translocating substrate is trapped in the TIM23 complex. These structural changes reveal that the macromolecular arrangement within the TIM23 complex is dynamic and varies with the physiological state of the mitochondrion.
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http://dx.doi.org/10.1091/mbc.e07-07-0669DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2174187PMC
January 2008

Regulation of mitochondrial fusion and division.

Trends Cell Biol 2007 Nov 23;17(11):563-9. Epub 2007 Oct 23.

Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK.

In many organisms, ranging from yeast to humans, mitochondria fuse and divide to change their morphology in response to a multitude of signals. During the past decade, work using yeast and mammalian cells has identified much of the machinery required for fusion and division, including the dynamin-related GTPases--mitofusins (Fzo1p in yeast) and OPA1 (Mgm1p in yeast) for fusion and Drp1 (Dnm1p) for division. However, the mechanisms by which cells regulate these dynamic processes have remained largely unknown. Recent studies have uncovered regulatory mechanisms that control the activity, assembly, distribution and stability of the key components for mitochondrial fusion and division. In this review, we discuss how mitochondrial dynamics are controlled and how these events are coordinated with cell growth, mitosis, apoptosis and human diseases.
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http://dx.doi.org/10.1016/j.tcb.2007.08.006DOI Listing
November 2007

Awaking TIM22, a dynamic ligand-gated channel for protein insertion in the mitochondrial inner membrane.

J Biol Chem 2007 Jun 26;282(26):18694-701. Epub 2007 Apr 26.

Department of Biochemistry and Molecular Biology, University of Extremadura, 10071 Cáceres, Spain.

Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.
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http://dx.doi.org/10.1074/jbc.M700775200DOI Listing
June 2007

The morphology proteins Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria.

EMBO J 2007 May 5;26(9):2229-39. Epub 2007 Apr 5.

Institut für Biochemie und Molekularbiologie, Zentrum für Biochemie und Molekulare Zellforschung, Universität Freiburg, Freiburg, Germany.

The beta-barrel proteins of mitochondria are synthesized on cytosolic ribosomes. The proteins are imported by the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been assumed that the SAM(core) complex with the subunits Sam35, Sam37 and Sam50 represents the last import stage common to all beta-barrel proteins, followed by splitting in a Tom40-specific route and a route for other beta-barrel proteins. We have identified new components of the beta-barrel assembly machinery and show that the major beta-barrel pathway extends beyond SAM(core). Mdm12/Mmm1 function after SAM(core) yet before splitting of the major pathway. Mdm12/Mmm1 have been known for their role in maintenance of mitochondrial morphology but we reveal assembly of beta-barrel proteins as their primary function. Moreover, Mdm10, which functions in the Tom40-specific route, can associate with SAM(core) as well as Mdm12/Mmm1 to form distinct assembly complexes, indicating a dynamic exchange between the machineries governing mitochondrial beta-barrel assembly. We conclude that assembly of mitochondrial beta-barrel proteins represents a major function of the morphology proteins Mdm12/Mmm1.
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http://dx.doi.org/10.1038/sj.emboj.7601673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864972PMC
May 2007

Yeast mitochondrial division and distribution require the cortical num1 protein.

Dev Cell 2007 Mar;12(3):363-75

Department of Cell Biology, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA.

Yeast mitochondrial division requires the dynamin-related Dnm1 protein. By isolating high-copy suppressors of a dominant-negative Dnm1p mutant, we uncovered an unexpected role in mitochondrial division and inheritance for Num1p, a protein previously shown to facilitate nuclear migration. num1 mutants contain an interconnected network of mitochondrial tubules, remarkably similar to cells lacking Dnm1p, and time-lapse microscopy confirms that mitochondrial fission is greatly reduced in num1Delta cells. We also find that Num1p assembles into punctate structures, which often colocalize with mitochondrial-bound Dnm1p particles. Suggesting a role for both Num1p and Dnm1p in mitochondrial inheritance, we find that num1 dnm1 double mutants accumulate mitochondria in daughter buds and that mother cells are frequently devoid of all mitochondria. Thus, our studies have revealed an additional role for Dnm1p in mitochondrial transmission through its interaction with Num1p, thereby providing a link between mitochondrial division and inheritance.
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http://dx.doi.org/10.1016/j.devcel.2007.01.017DOI Listing
March 2007

The Tim9p/10p and Tim8p/13p complexes bind to specific sites on Tim23p during mitochondrial protein import.

Mol Biol Cell 2007 Feb 22;18(2):475-86. Epub 2006 Nov 22.

Department of Molecular and Cellular Medicine, Texas A&M University System Health Science Center, College Station, TX 77843-1114, USA.

The import of polytopic membrane proteins into the mitochondrial inner membrane (IM) is facilitated by Tim9p/Tim10p and Tim8p/Tim13p protein complexes in the intermembrane space (IMS). These complexes are proposed to act as chaperones by transporting the hydrophobic IM proteins through the aqueous IMS and preventing their aggregation. To examine the nature of this interaction, Tim23p molecules containing a single photoreactive cross-linking probe were imported into mitochondria in the absence of an IM potential where they associated with small Tim complexes in the IMS. On photolysis and immunoprecipitation, a probe located at a particular Tim23p site (27 different locations were examined) was found to react covalently with, in most cases, only one of the small Tim proteins. Tim8p, Tim9p, Tim10p, and Tim13p were therefore positioned adjacent to specific sites in the Tim23p substrate before its integration into the IM. This specificity of binding to Tim23p strongly suggests that small Tim proteins do not function solely as general chaperones by minimizing the exposure of nonpolar Tim23p surfaces to the aqueous medium, but may also align a folded Tim23p substrate in the proper orientation for delivery and integration into the IM at the TIM22 translocon.
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http://dx.doi.org/10.1091/mbc.e06-06-0546DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1783793PMC
February 2007

Ups1p, a conserved intermembrane space protein, regulates mitochondrial shape and alternative topogenesis of Mgm1p.

J Cell Biol 2006 Jun;173(5):651-8

Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Mgm1p is a conserved dynamin-related GTPase required for fusion, morphology, inheritance, and the genome maintenance of mitochondria in Saccharomyces cerevisiae. Mgm1p undergoes unconventional processing to produce two functional isoforms by alternative topogenesis. Alternative topogenesis involves bifurcate sorting in the inner membrane and intramembrane proteolysis by the rhomboid protease Pcp1p. Here, we identify Ups1p, a novel mitochondrial protein required for the unique processing of Mgm1p and for normal mitochondrial shape. Our results demonstrate that Ups1p regulates the sorting of Mgm1p in the inner membrane. Consistent with its function, Ups1p is peripherally associated with the inner membrane in the intermembrane space. Moreover, the human homologue of Ups1p, PRELI, can fully replace Ups1p in yeast cells. Together, our findings provide a conserved mechanism for the alternative topogenesis of Mgm1p and control of mitochondrial morphology.
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http://dx.doi.org/10.1083/jcb.200603092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2063882PMC
June 2006

A genomewide screen for petite-negative yeast strains yields a new subunit of the i-AAA protease complex.

Mol Biol Cell 2006 Jan 2;17(1):213-26. Epub 2005 Nov 2.

Department of Cell Biology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Unlike many other organisms, the yeast Saccharomyces cerevisiae can tolerate the loss of mitochondrial DNA (mtDNA). Although a few proteins have been identified that are required for yeast cell viability without mtDNA, the mechanism of mtDNA-independent growth is not completely understood. To probe the relationship between the mitochondrial genome and cell viability, we conducted a microarray-based, genomewide screen for mitochondrial DNA-dependent yeast mutants. Among the several genes that we discovered is MGR1, which encodes a novel subunit of the i-AAA protease complex located in the mitochondrial inner membrane. mgr1Delta mutants retain some i-AAA protease activity, yet mitochondria lacking Mgr1p contain a misassembled i-AAA protease and are defective for turnover of mitochondrial inner membrane proteins. Our results highlight the importance of the i-AAA complex and proteolysis at the inner membrane in cells lacking mitochondrial DNA.
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http://dx.doi.org/10.1091/mbc.e05-06-0585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1345660PMC
January 2006

Control of mitochondrial shape.

Authors:
Robert E Jensen

Curr Opin Cell Biol 2005 Aug;17(4):384-8

Department of Cell Biology, Johns Hopkins Medical School, 725 N. Wolfe Street, Baltimore, MD 21205, USA.

Although mitochondria are known to exhibit a wide variety of morphologies in different cells, the mechanism by which these shapes are established and regulated are largely unknown. Several potential shape-forming proteins have been recently identified. Some studies suggest that these proteins control shape by mediating attachment of mitochondria to the cytoskeleton, while other studies indicate that these proteins form part of a connection between the mitochondrial outer and inner membranes. Complicating matters, a recent study raises the possibility that one or more of these shape-forming proteins plays a direct role in the import and assembly of mitochondrial proteins synthesized in the cytosol.
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http://dx.doi.org/10.1016/j.ceb.2005.06.011DOI Listing
August 2005

Functional analysis of subunit e of the F1Fo-ATP synthase of the yeast Saccharomyces cerevisiae: importance of the N-terminal membrane anchor region.

Eukaryot Cell 2005 Feb;4(2):346-55

Department of Biological Sciences, Marquette University, Milwaukee, WI 53233, USA.

Mitochondrial F1Fo-ATP synthase complexes do not exist as physically independent entities but rather form dimeric and possibly oligomeric complexes in the inner mitochondrial membrane. Stable dimerization of two F1Fo-monomeric complexes involves the physical association of two membrane-embedded Fo-sectors. Previously, formation of the ATP synthase dimeric-oligomeric network was demonstrated to play a critical role in modulating the morphology of the mitochondrial inner membrane. In Saccharomyces cerevisiae, subunit e (Su e) of the Fo-sector plays a central role in supporting ATP synthase dimerization. The Su e protein is anchored to the inner membrane via a hydrophobic region located at its N-terminal end. The hydrophilic C-terminal region of Su e resides in the intermembrane space and contains a conserved coiled-coil motif. In the present study, we focused on characterizing the importance of these regions for the function of Su e. We created a number of C-terminal-truncated derivatives of the Su e protein and expressed them in the Su e null yeast mutant. Mitochondria were isolated from the resulting transformant strains, and a number of functions of Su e were analyzed. Our results indicate that the N-terminal hydrophobic region plays important roles in the Su e-dependent processes of mitochondrial DNA maintenance, modulation of mitochondrial morphology, and stabilization of the dimer-specific Fo subunits, subunits g and k. Furthermore, we show that the C-terminal coiled-coil region of Su e functions to stabilize the dimeric form of detergent-solubilized ATP synthase complexes. Finally, we propose a model to explain how Su e supports the assembly of the ATP synthase dimers-oligomers in the mitochondrial membrane.
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http://dx.doi.org/10.1128/EC.4.2.346-355.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC549337PMC
February 2005

Prediction of the formulation dependence of the glass transition temperatures of amine-epoxy copolymers using a QSPR based on the AM1 method.

J Chem Inf Comput Sci 2004 May-Jun;44(3):912-20

United States Army Research Laboratory, Weapons and Materials Research Directorate, AMSRL-WM-BD, Aberdeen Proving Ground, MD 21005-5066, USA.

A designer Quantitative Structure-Property Relationship, based upon molecular properties calculated using the AM1 semiempirical quantum mechanical method, was developed to predict the glass transition temperature of amine-cured epoxy resins based on the diglycidyl ether of bisphenol A. The QSPR (R2 = 0.9977) was generated using the regression analysis program, COmprehensive DEscriptors for Structural and Statistical Analysis. By applying an ad hoc treatment based on the elementary probability theory to the quantitative structure-property relationship analysis a method was developed for computing bulk polymer glass transition temperatures for stoichiometric and nonstoichiometric monomeric formulations. A model polymer was synthesized and found to validate our model predictions.
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http://dx.doi.org/10.1021/ci030290dDOI Listing
March 2005

Mitochondrial building blocks.

Trends Cell Biol 2004 May;14(5):215-8

Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Despite many genomic and proteomic attempts, approximately half of all mitochondrial proteins remain unidentified. Moreover, the composition of mitochondria varies in different mammalian cell types and the details of this tissue specificity are unclear. Two recent reports provide a major advance in our understanding of mitochondrial function. Sickmann et al. used an exhaustive proteomic approach and came very close to identifying the complete set of yeast mitochondrial proteins. Mootha et al. examined mitochondria from mouse brain, heart, kidney and liver cells, finding that a surprising fraction of the proteins are expressed in only a subset of tissues.
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http://dx.doi.org/10.1016/j.tcb.2004.03.006DOI Listing
May 2004