DOCSLIB.ORG

Isolation and Characterisation of the Intermembrane Space Components of the Mitochondrial TIM22 Protein Import Machinery of Neurospora Crassa

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Isolation and Characterisation of the Intermembrane Space Components of the Mitochondrial TIM22 Protein Import Machinery of Neurospora Crassa Isolation and characterisation of the intermembrane space components of the mitochondrial TIM22 protein import machinery of Neurospora crassa Dissertation zur Erlangung des doktorgrades der Fakultät für Biologie der Ludwig-Maximilians-Universität München von Andreja Vasiljev aus Subotica, Serbien und Montenegro München, 2004 Mündliche Prüfung am: 15.11.2004 Sondergutachter: Herr Prof. Dr.Dr. Walter Neupert 1. Gutachter: Herr Prof. Dr. Reinhold Herrmann 2. Gutachter: Herr PD Dr. Enrico Schleiff To my grandmothers, Jelena and Marija Table of Contents 1. Introduction ....................................................................................1 1.1. Mitochondrial protein translocation machineries........................................................ 1 1.1.1. Mitochondrial structure and function................................................................. 1 1.1.2. Protein translocation in mitochondria of N. crassa and S. cerevisiae................ 4 1.2.2.1. Targeting of preproteins to mitochondria.......................................................... 4 1.2.2.2. Translocases of the outer mitochondrial membrane ......................................... 6 1.2.2.3. Translocases of the inner mitochondrial membrane ......................................... 8 1.2. Zinc fingers............................................................................................................... 13 1.3. Aims of the present study.......................................................................................... 17 2. Material and methods ...................................................................18 2.1. Molecular biology methods....................................................................................... 18 2.1.1. PCR (polymerase chain reaction)..................................................................... 18 2.1.2. DNA purification and analysis......................................................................... 19 2.1.2.1. Analytical and preparative gel electrophoresis ............................................... 19 2.1.2.2. DNA concentration measurement ................................................................... 19 2.1.3. Cloning of DNA fragments.............................................................................. 20 2.1.3.1. Enzymatic manipulation of DNA: restriction and ligation reactions.............. 20 2.1.3.2. Preparation and transformation of E. coli competent cells ............................. 20 2.1.4. E. coli strains used............................................................................................ 21 2.1.5. Small and large scale isolation of plasmid DNA from E. coli ......................... 21 2.1.6. Plasmids and genomic library clones used....................................................... 22 2.1.7. Cloning strategies............................................................................................. 23 2.1.8. S. cerevisiae strains used.................................................................................. 26 2.1.9. Preparation of yeast DNA ................................................................................ 26 2.1.10. N. crassa strains used....................................................................................... 27 2.1.11. Screening of N. crassa cosmid libraries........................................................... 27 2.1.12. Southern blot.................................................................................................... 29 2.1.13. Screening of clones through in situ colony-blotting ........................................ 29 2.2. Cell biology methods ................................................................................................ 29 2.2.1. E. coli: Media and culture ................................................................................ 29 2.2.2. N. crassa: Media and culture............................................................................ 30 2.2.2.1. Media and solutions for N. crassa................................................................... 30 2.2.2.2. Growth of N. crassa hyphae............................................................................ 32 2.2.2.3. Transformation of N. crassa............................................................................ 34 2.2.3. Isolation of mitochondria from N. crassa hyphae............................................ 35 2.2.4. Crude isolation of mitochondria from N. crassa (“mini” prep) ....................... 36 2.2.5. S. cerevisiae: Culture and Media...................................................................... 37 2.2.5.1. Media for S.cerevisiae..................................................................................... 37 2.2.5.2. S. cerevisiae growth ........................................................................................ 37 2.2.5.3. Transformation of S. cerevisiae (lithium acetate method) .............................. 37 2.2.6. Dilution assay................................................................................................... 38 2.2.7. Isolation of mitochondria from S. cerevisiae ................................................... 38 2.2.8. Isolation of crude mitochondria from S. cerevisiae ......................................... 39 2.3. Biochemical methods ................................................................................................ 39 2.3.1. Electrophoretic methods................................................................................... 39 2.3.1.1. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) ................................. 39 2.3.1.2. High Tris-urea SDS-Polyacrylamide gel electrophoresis............................... 40 2.3.1.3. Blue-Native gel electrophoresis (BNGE)........................................................ 40 I 2.3.1.4. 2D Blue-Native gel electrophoresis (BNGE).................................................. 41 2.3.1.5. Coomassie blue staining of SDS gels.............................................................. 42 2.3.1.6. Silver staining of SDS gels.............................................................................. 42 2.3.1.7. Transfer of proteins to nitrocellulose/PVDF membrane (Western-Blot)........ 43 2.3.2. Protein concentration determination ................................................................ 43 2.3.3. Protein quantification by autoradiography and phosphorimaging ................... 44 2.3.4. Synthesis of radioactively labelled proteins in vitro ........................................ 44 2.3.5. Import of preproteins into isolated mitochondria............................................. 45 2.3.6. Generation of mitoplasts (“swelling”).............................................................. 46 2.3.7. Trichloroacetic acid (TCA) precipitation of proteins....................................... 46 2.3.8. Ammonium sulphate precipitation of proteins................................................. 46 2.3.9. Carbonate extraction........................................................................................ 47 2.3.10. Expression and purification of proteins ........................................................... 47 2.3.10.1 Purification of recombinant proteins expressed in E. coli ............................. 47 2.3.10.2 Purification of immunoglobulin G (IgG) ....................................................... 48 2.3.10.3 Purification of Tim9·Tim10 complex from N. crassa mitochondria ............. 48 2.3.11. Gel filtration ..................................................................................................... 49 2.3.12. Digitonin fractionation..................................................................................... 50 2.3.13. Thin layer chromatography (TLC) for determination of detergent traces in protein preparations.......................................................................................... 50 2.3.14. Chemical cross-linking..................................................................................... 51 2.3.15. Screening of peptide libraries with the purified Tim9·Tim10 complex........... 51 2.3.16. Pull-down assay................................................................................................ 52 2.3.17. In-gel digestion of proteins for sequencing...................................................... 53 2.4. Immunological methods............................................................................................ 54 2.4.1. Generation of specific antibodies against N. crassa Tim9 and Tim10 proteins in rabbits........................................................................................................... 54 2.4.2. Affinity purification of antibodies against Tim9 and Tim10 proteins ............. 55 2.4.3. Immunodecoration ........................................................................................... 56 2.4.4. Immunoprecipitation and co-immunoprecipitation.......................................... 56 3. Results ..........................................................................................58 3.1. Identification of the N. crassa tim9 and tim10 genes................................................ 58 3.1.1. Identification of the N. crassa tim10 gene ....................................................... 58 3.1.2. Identification of the N. crassa tim9 gene ......................................................... 59 3.1.3. Tim9 is an essential protein in N. crassa ........................................................
Load more

Recommended publications
  • Electron Transport Generates a Proton Gradient Across the Membrane
    Electron Transport Generates a Proton Gradient Across the Membrane Each of respiratory enzyme complexes couples the energy released by electron transfer across it to an uptake of protons from water in the mitochondrial matrix, accompanied by the release of protons on the other side of the membrane into the intramembrane space. As result, the energetically favorable flow of electrons along the electron- transport chain pumps protons across the membrane out of the matrix. This event creates electrochemical protons across the inner membrane. The Proton Gradient Drives ATP Synthesis The electrochemical proton gradient across the inner mitochondrial membrane is used to drive ATP synthesis in the process of oxidative Phosphorylation. The device that makes this possible is a large membrane-bound enzyme called ATP synthase. This enzymes creates a hydrophilic pathway across the inner mitochondrial membrane that allows protons to follow down their electrochemical gradient. As these ions thread their way through the ATP synthase, they are used to drive the energetically unfavorable reaction between ADP and Pi. 1 Proton Gradients Produce Most of the Cell’s ATP Glycolysis alone produces a net yield of two molecules of ATP for every molecule of glucose, which is the total energy yield for the fermentation process that occur in the absence of oxygen. In contrast, during the oxidative Phosphorylation each pair of electrons donated by NADH produced mitochondria is thought to provide energy for the formation of the about 2.5 molecules of ATP, once one includes the energy needed for transporting this ATP to cytosol. Oxidative Phosphorylation also produces 1.5 ATP molecules per electron pair of FADH2, or from the NADH molecules produced by glycolysis in the cytosol.
    [Show full text]
  • Protein Translocation Pathways of the Mitochondrion
    FEBS 23788 FEBS Letters 476 (2000) 27^31 View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Minireview Protein translocation pathways of the mitochondrion Carla M. Koehler* Department of Chemistry and Biochemistry, Box 951569, University of California, Los Angeles, CA 90095-1569, USA Received 5 May 2000 Edited by Gunnar von Heijne membrane (reviewed by [2^6]). The translocase of the outer Abstract The biogenesis of mitochondria depends on the coordinated import of precursor proteins from the cytosol membrane (TOM) consists of protein import receptors and coupled with the export of mitochondrially coded proteins from the import channel. The receptors (Tom20, 22, 37 and 70, the matrix to the inner membrane. The mitochondria contain an with the number indicating molecular weight) on the mito- elaborate network of protein translocases in the outer and inner chondrial surface recognize targeting information on mito- membrane along with a battery of chaperones and processing chondrial precursors, while components Tom40 and the small enzymes in the matrix and intermembrane space to mediate Tom proteins 5, 6 and 7 form the channel through which the protein translocation. A mitochondrial protein, often with an translocating precursor passes [6]. After passage through the amino-terminal targeting sequence, is escorted through the TOM complex, proteins are sorted via a number of mecha- cytosol by chaperones to the TOM complex (translocase of the nisms either directly to the outer membrane, the intermem- outer membrane). After crossing the outer membrane, the import brane space, or the translocase of the inner membrane (TIM).
    [Show full text]
  • Ionic Strength of the Intermembrane Space of Intact Mitochondria As Estimated with Fluorescein-BSA Delivered by Low Ph Fusion Jorge D
    Ionic Strength of the Intermembrane Space of Intact Mitochondria as Estimated with Fluorescein-BSA Delivered by Low pH Fusion Jorge D. Cortese, A. Laura Voglino, and Charles R. Hackenbrock The Department of Cell Biology and Anatomy, Laboratories for Cell Biology, University of North Carolina, The School of Medicine, Chapel Hill, North Carolina 27599-7090 Abstract. The electrostatic interactions of cytochrome c (octadecylrhodamine B) and the soluble FITC-BSA with its redox partners and membrane lipids, as well fluorescent probe, which report membrane and contents as other protein interactions and biochemical reactions, mixing, respectively. Assays were consistent with a Downloaded from http://rupress.org/jcb/article-pdf/113/6/1331/1061492/1331.pdf by guest on 27 September 2021 may be modulated by the ionic strength of the inter- rapid, low pH-induced vesicle-outer membrane fusion membrane space of the mitochondrion. FITC-BSA was and delivery of FITC-BSA into the intermembrane used to determine the relative value of the mitochon- space. Similar affinities for the ionic strength-depen- drial intermembrane ionic strength with respect to bulk dent change in fluorescence were found for bulk medium, medium external to the mitochondrial outer mem- soluble (9.8 ± 0.8 mM) and intermembrane space- brane. FITC-BSA exhibited an ionic strength-depen- entrapped FITC-BSA (10.2 + 0.6 mM). FITC-BSA con- dent fluorescence change with an affinity in the mM sistently reported an ionic strength in the intermem- range as opposed to its pH sensitivity in the #M range. brane space of the functionally and structurally intact A controlled, low pH-induced membrane fusion pro- mitochondria within ±20% of the external bulk solu- cedure was developed to transfer FITC-BSA encapsu- tion.
    [Show full text]
  • Lecture 37 & 38: Electron Transport Chain and Oxidative Phosphorylation
    CHM333 LECTURES 37 & 38: 4/27 – 29/13 SPRING 2013 Professor Christine Hrycyna Electron Transport and Oxidative Phosphorylation • Final stages of aerobic Mitochondrial Electron Transport oxidation of biomolecules in • How did we get here? eukaryotes occur in the mitochondrion • Summary of glycolysis glucose + 2 NAD+ + 2P + 2ADP 2 pyruvate + 2ATP + 2NADH + 2H+ • Reduced coenzymes NADH i → and FADH2 from: • Summary of the citric acid cycle (including pyruvate dehydrogenase) pyruvate + 4 NAD+ + FAD + GDP + 2 H 0 3 CO + 4NADH + 4H+ + P + GTP + FADH (1) Aerobic oxidation of 2 → 2 i 2 pyruvate by the citric acid cycle (2) Oxidation of fatty acids and amino acids Electron Transport Chain is the process by which NADH and FADH2 are oxidized and a proton gradient is formed. Electron Transport and Oxidative Phosphorylation Oxidative phosphorylation is the process of making ATP by using the proton gradient generated by the ETC. Respiration by mitochondria • Oxidation of substrates is coupled to the phosphorylation of ADP • Respiration (consumption of oxygen) proceeds only when ADP is present • The amount of O2 consumed depends upon the amount of ADP added Location of mitochondrial complexes • Inner mitochondrial membrane: a. Electron transport chain: oxidizes reduced coenzymes b. ATP synthase: machinery to synthesize ATP 280 CHM333 LECTURES 37 & 38: 4/27 – 29/13 SPRING 2013 Professor Christine Hrycyna Electron transport and oxidative phosphorylation capture the energy in the redox potential of NADH and FADH2 – 2 separate processes that are COUPLED to result in ATP production Extensive folding of IMM provides a large surface area on the matrix side to form lots of assemblies of proteins to maximize ATP production (1) Respiratory electron-transport chain (ETC) Series of enzyme complexes embedded in the inner mitochondrial membrane, which oxidize NADH and FADH2.
    [Show full text]
  • Sperm-Specific COX6B2 Enhances Oxidative Phosphorylation
    RESEARCH ARTICLE Sperm-specific COX6B2 enhances oxidative phosphorylation, proliferation, and survival in human lung adenocarcinoma Chun-Chun Cheng1, Joshua Wooten2, Zane A Gibbs1, Kathleen McGlynn1, Prashant Mishra3, Angelique W Whitehurst1* 1Department of Pharmacology, Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, Dallas, United States; 2Nuventra, Durham, United States; 3Children’s Research Institute, UT Southwestern Medical Center, Dallas, United States Abstract Cancer testis antigens (CTAs) are proteins whose expression is normally restricted to the testis but anomalously activated in human cancer. In sperm, a number of CTAs support energy generation, however, whether they contribute to tumor metabolism is not understood. We describe human COX6B2, a component of cytochrome c oxidase (complex IV). COX6B2 is expressed in human lung adenocarcinoma (LUAD) and expression correlates with reduced survival time. COX6B2, but not its somatic isoform COX6B1, enhances activity of complex IV, increasing oxidative phosphorylation (OXPHOS) and NAD+ generation. Consequently, COX6B2-expressing cancer cells display a proliferative advantage, particularly in low oxygen. Conversely, depletion of COX6B2 attenuates OXPHOS and collapses mitochondrial membrane potential leading to cell death or senescence. COX6B2 is both necessary and sufficient for growth of human tumor xenografts in mice. Our findings reveal a previously unappreciated, tumor-specific metabolic pathway hijacked from one of the most ATP-intensive processes in the animal
    [Show full text]
  • Mitochondria and Chloroplasts
    ANATOMICAL, PHYSIOLOGICAL AND BIOCHEMICAL SIMILARITIES OF MITOCHONDRIA AND CHLOROPLASTS Mitochondria are membranous organelles found in association with almost all types of eukaryotic microorganisms, while chloroplasts are found only in association with certain forms, e.g., algae and euglenoids. Although these organelles vary considerable in size, shape and number in different species of organisms, they tend to follow some basic patterns, and have many similarities in their structure, function and biochemistry. Both mitochondria and chloroplasts are surrounded by outer membranes typical of eukaryotic cells (50:50 lipid protein ratio plus ring form lipids), and have inner membranes typical of prokaryotic cells (40:60 lipid protein ratio and no ring-form lipids). These inner membranes are highly folded (forming cristae or thylakoids), and contain ATP-synthase enzymes. ATP synthesis involves photophosphorylation in chloroplasts, and oxidative phosphorylation in both mitochondria and chloroplasts. Like bacteria, mitochondria and chloroplasts contain ccc-DNA and 70S ribosomes, they reproduce by means of binary fission, and are sensitive to antimicrobial drugs. Basic Organizational Pattern of a Typical Mitochondrion Basic Organizational Pattern of a Typical Chloroplast EVIDENCE FOR THE ENDOSYMBIOTIC THEORY Mitochondria evolved from Proteobacteria and Chloroplasts evolved from cyanobacteria; both organelles were initially prokaryotic organisms that were ingested by eukaryotic cells. Anatomical Features: Significant Details: Outer membrane Encloses organelle, composition typical of eukaryotic plasma membrane with 50:50 lipid to protein ratio plus ring-form lipids. Intermembrane space Potential space between membranes (spatially equivalent to the periplasmic space typical of prokaryotic cells). Inner membrane Composition typical of prokaryotic plasma membrane with 40:60 lipid to protein ratio and no ring-form lipids.
    [Show full text]
  • The Role of the Bcl-2 Family in the Regulation of Outer Mitochondrial Membrane Permeability
    Cell Death and Differentiation (2000) 7, 1182 ± 1191 ã 2000 Macmillan Publishers Ltd All rights reserved 1350-9047/00 $15.00 www.nature.com/cdd Review The role of the Bcl-2 family in the regulation of outer mitochondrial membrane permeability 1 ,1 MH Harris and CB Thompson* Introduction 1 Abramson Family Cancer Research Institute, University of Pennsylvania, Mitochondria became the subject of intensive scientific Philadelphia, PA 19104, USA investigation in the mid-twentieth century when their role as * Corresponding author: CB Thompson, Abramson Family Cancer Research cellular energy producers was discovered. This culminated in Institute, 450 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160, USA. the discovery and characterization of oxidative phosphoryla- E-mail: [email protected] tion as the mechanism by which most cellular ATP is Received 29.6.00; accepted 14.9.00 produced. Free exchange of substrate and ATP/ADP Edited by G Kroemer between mitochondria and the cytosol was proposed to provide the homeostatic mechanism by which glycolysis and oxidative phosphorylation are coupled to maintain the Abstract intracellular ATP/ADP ratio.1,2 In the last few years, there has been a resurgence of interest in mitochondria following Mitochondria are well known as sites of electron transport and the discovery that mitochondria play a crucial role in the generators of cellular ATP. Mitochondria also appear to be regulation of programmed cell death, or apoptosis.3±9 A sites of cell survival regulation. In the process of programmed number of molecules involved in the execution of apoptosis cell death, mediators of apoptosis can be released from normally reside in mitochondria, safely sequestered from their mitochondria through disruptions in the outer mitochondrial targets and co-factors.10 ± 16 Following an apoptotic stimulus, membrane; these mediators then participate in the activation these proteins can be released from mitochondria and initiate of caspases and of DNA degradation.
    [Show full text]
  • Energetics at the Molecular Level Douglas R Moellering, Ph.D
    Energetics: Scientific Foundations of Obesity and Other Health Aspects Energetics at the Molecular Level Douglas R Moellering, Ph.D. UAB Nutrition Sciences UAB COMPREHENSIVE CENTER FOR HEALTHY AGING UAB COMPREHENSIVE CARDIOVASCULAR CENTER PUH 690 Energetics 1.24.2013 ‐ Moellering 1 2 Overview of Metabolism Organic Water Compounds: Vitamins Hydrogen: H Minerals Oxygen: O Carbon: C Nitrogen: N Phosphorous: P Sulfur: S http://www.elmhurst.edu/~chm/vchembook/600glycolysis.html 3 Energy Metabolism Overview (a) Pyruvate produced in glycolysis is oxidized in (b) the tricarboxylic acid (TCA) cycle. (c) Electrons liberated in this oxidation flow through the electron-transport chain and drive the synthesis of ATP in oxidative phosphorylation. In 8/25/1900 – 11/22/1981 eukaryotic cells, this overall process occurs in mitochondria. ‘1937’ Krebs cycle = TCA cycle = Citric acid cycle 4 Overview: Harnessing Energy – Chemical Bonds Complete oxidation of glucose: o -1 C6H12O6 + 6O2 --> 6CO2 + 6H2O G ’ = -2823 kJ mol Electron transfer: 1. Oxidation of glucose carbon atoms + - C6H12O6 + 6H2O --> 6CO2 + 24H + 24e cytosol 2. Reduction of molecular oxygen + - 6O2 + 24H + 24e --> 12H2O mito Oxidation Reduction Is e- Is glucose Loss Gain Glycolysis 10 NADH Electron transport & ATP TCA 2 FADH2 Oxidative Phosphorylation Figure 22-1 The sites of electron transfer that form NADH and FADH2 in glycolysis and the citric acid cycle. Page 798 5 Vo e t Biochemistry 3e. © 2004 John Wiley & Sons, Inc.; Ch 22, pgs 797-840 Where does Metabolism Start? 6 http://www.medbio.info/Horn/Body%20Energy/body_energy.htm 7 Cellular Metabolism Cellular metabolism refers to all of the chemical processes that occur inside living cells.
    [Show full text]
  • Hypoxia Promotes Mitochondrial Complex I Abundance Via HIF-1Α in Complex III and Complex IV Deficient Cells
    cells Article Hypoxia Promotes Mitochondrial Complex I Abundance via HIF-1α in Complex III and Complex IV Deficient Cells Amy Saldana-Caboverde 1 , Nadee Nissanka 1 , Sofia Garcia 1, Anne Lombès 2 and Francisca Diaz 1,* 1 Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA; asaldana@fiu.edu (A.S.-C.); [email protected] (N.N.); sofi[email protected] (S.G.) 2 Institut Cochin, Unité U1016, INSERM, UMR 8104, CNRS, Université Paris 5, F-75014 Paris, France; [email protected] * Correspondence: [email protected]; Tel.: +1-305-243-7489 Received: 4 September 2020; Accepted: 21 September 2020; Published: 29 September 2020 Abstract: Murine fibroblasts deficient in mitochondria respiratory complexes III (CIII) and IV (CIV) produced by either the ablation of Uqcrfs1 (encoding for Rieske iron sulfur protein, RISP) or Cox10 (encoding for protoheme IX farnesyltransferase, COX10) genes, respectively, showed a pleiotropic effect in complex I (CI). Exposure to 1–5% oxygen increased the levels of CI in both RISP and COX10 KO fibroblasts. De novo assembly of the respiratory complexes occurred at a faster rate and to higher levels in 1% oxygen compared to normoxia in both RISP and COX10 KO fibroblasts. Hypoxia did not affect the levels of assembly of CIII in the COX10 KO fibroblasts nor abrogated the genetic defect impairing CIV assembly. Mitochondrial signaling involving reactive oxygen species (ROS) has been implicated as necessary for HIF-1α stabilization in hypoxia. We did not observe increased ROS production in hypoxia. Exposure to low oxygen levels stabilized HIF-1α and increased CI levels in RISP and COX10 KO fibroblasts.
    [Show full text]
  • Chapter 14 Slides 2017
    12/1/17 CHAPTER 14: Metabolism and Bioenergetics SUMMARY OF CATABOLISM • Your body is capable of “burning” many sorts of fuel to produce ATP Ø Carbohydrates Ø Lipids Ø Proteins • All three “fuels” feed into the citric acid cycle to complete catabolism: Ø Begins with acetyl-CoA Ø Requires O2 Ø Occurs in the mitochondria CHAPTER 14: Metabolism and Bioenergetics OUTLINE • 14.1 Acetyl CoA and the Citric Acid Cycle • 14.2 Oxidative Phosphorylation • 14.3 Entropy and Bioenergetics (not covered) CHAPTER 14: Metabolism and Bioenergetics ACETYL-CoA PRODUCTION FROM PYRUVATE • Acetyl CoA is produced between the second & third stages of carbohydrate catabolism: Ø Both proteins & fat can also can be converted into acetyl-CoA to produce ATP • Further catabolism of acetyl-CoA forms NADH & FADH2 during the citric acid cycle. Ø NADH & FADH2 are re-oxidized to make ATP 1 12/1/17 CHAPTER 14: Metabolism and Bioenergetics ACETYL CoA • Acetyl CoA is a complex molecule consisting of pantothenic acid (vitamin B5), a thiol amine, and ADP, and the acetyl group • Coenzyme A can carry other groups besides the acetyl in other metabolic pathways (ie. acyl chains) CHAPTER 14: Metabolism and Bioenergetics THE CHEMISTRY OF ACETYL CoA • The key to Coenzyme-A chemistry is the thiol group located at the very end of the molecule: The thiol can react with a variety of carbonyl-containing molecules to form a high- energy thioester bond CHAPTER 12: Carbohydrates: Structure and Function OXIDATION OF PYRUVATE TO ACETYL-CoA • If oxygen is present, pyruvate is oxidized to acetyl-
    [Show full text]
  • Mitochondrial Markers 1
    Mitochondrial Markers 1 MITOCHONDRIAL MARKERS www.ptglab.com Mitochondrial Markers 2 INTRODUCTION Mitochondria are important cellular organelles that maintain cellular energy balance, contain key regulators of cell death processes, and play a significant role in cellular oxidative stress generation and maintenance of calcium homeostasis. Links to cancer, apoptosis, autophagy, and hypoxia have brought mitochondria to the forefront of scientific studies in recent years. Knowledge of the subcellular location of a protein may reveal the potential role it plays in a variety of cellular processes. Proteintech offers approximately all the antibodies needed for mitochondria research. What’s Inside 3–4 Mitochondrial Markers 5 Citric Acid Cycle 6–7 Mitochondrial Respiratory Complexes 8 Mitochondrial Fission Mitochondrial Fusion 9 Mitochondrial Mediated Apoptosis 10 Mitochondrial Autophagy 11 Mitochondrial Translation Mitochondrial Protein Import 12 Contact Us Mitochondrial Markers 3 Mitochondrial Mitochondria are composed of the inner and outer membranes, the intermembrane space, the cristae, and the matrix, and they contain Markers their own DNA separated from the nucleus. Knowledge of the subcellular location of a protein may reveal the potential role it plays in a variety of cellular processes. Colocalization with one of the organelle-specific markers listed here can confirm the subcellular location of a mitochondrial protein of interest. Related Antibodies Antibody Name Catalog Number Type Applications AIF 7 17984-1-AP Rabbit Poly ELISA, IF,
    [Show full text]
  • HIGD-Driven Regulation of Cytochrome C Oxidase Biogenesis and Function
    cells Review HIGD-Driven Regulation of Cytochrome c Oxidase Biogenesis and Function Alba Timón-Gómez 1 , Emma L. Bartley-Dier 2, Flavia Fontanesi 2 and Antoni Barrientos 1,2,* 1 Department of Neurology, University of Miami Miller School of Medicine, Miami, FL 33136, USA; [email protected] 2 Department of Biochemistry and Molecular Biology, University of Miami Miller School of Medicine, Miami, FL 33136, USA; [email protected] (E.L.B.-D.); ff[email protected] (F.F.) * Correspondence: [email protected] Received: 23 November 2020; Accepted: 4 December 2020; Published: 6 December 2020 Abstract: The biogenesis and function of eukaryotic cytochrome c oxidase or mitochondrial respiratory chain complex IV (CIV) undergo several levels of regulation to adapt to changing environmental conditions. Adaptation to hypoxia and oxidative stress involves CIV subunit isoform switch, changes in phosphorylation status, and modulation of CIV assembly and enzymatic activity by interacting factors. The latter include the Hypoxia Inducible Gene Domain (HIGD) family yeast respiratory supercomplex factors 1 and 2 (Rcf1 and Rcf2) and two mammalian homologs of Rcf1, the proteins HIGD1A and HIGD2A. Whereas Rcf1 and Rcf2 are expressed constitutively, expression of HIGD1A and HIGD2A is induced under stress conditions, such as hypoxia and/or low glucose levels. In both systems, the HIGD proteins localize in the mitochondrial inner membrane and play a role in the biogenesis of CIV as a free unit or as part as respiratory supercomplexes. Notably, they remain bound to assembled CIV and, by modulating its activity, regulate cellular respiration. Here, we will describe the current knowledge regarding the specific and overlapping roles of the several HIGD proteins in physiological and stress conditions.
    [Show full text]