Methods in molecular biology vol 1595 peroxisomes methods and protocols

1 Andreas Manner and Markus Islinger 2 Isolation of Peroxisomes from Mouse Brain Using a Continuous Nycodenz Gradient: A Comparison to the Isolation of Liver and Kidney Peroxisomes.. Pi

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Michael Schrader Editor

Methods and Protocols

Methods in

Molecular Biology 1595

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Me t h o d s i n Mo l e c u l a r Bi o l o g y

Series Editor

John M Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651

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Peroxisomes Methods and Protocols

Edited by

Michael Schrader

College of Life and Enivornmental Sciences, Biosciences, University of Exeter, Exeter, UK

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ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology

ISBN 978-1-4939-6935-7 ISBN 978-1-4939-6937-1 (eBook)

DOI 10.1007/978-1-4939-6937-1

Library of Congress Control Number: 2017937360

This work is subject to copyright All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction

on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed.

The use of general descriptive names, registered names, trademarks, service marks, etc in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use.

The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to

be true and accurate at the date of publication Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Printed on acid-free paper

This Humana Press imprint is published by Springer Nature

The registered company is Springer Science+Business Media LLC

The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.

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This book is dedicated to Tina, Anna and Paula—the

“lighthouses” in my life—who make it all possible and to my parents for their ongoing support

and interest in peroxisomes.

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This edition of Peroxisomes: Methods and Protocols assembles a volume of easily accessible

protocols particularly useful for those already working on peroxisomes (and other brane-bound organelles) as well as for those who would like to start working on this fasci-nating organelle Due to their growing importance in health and development, there is increasing interest in the study of peroxisomes Furthermore, peroxisomes combine prop-erties which render them suitable model organelles to study diverse molecular processes in eukaryotic cells

mem-This edition assembles a comprehensive collection of methods, techniques and gies to investigate the molecular and cellular biology of peroxisomes in different organisms

strate-It aims to provide valuable instructions, guidelines and protocols for molecular cell gists, biochemists and biomedical researchers with an interest in peroxisome biology.Protocols addressing peroxisomes in humans, yeast, fungi and plants are covered Chapters illustrating the isolation of peroxisomes, investigation of properties of membrane proteins, biochemical assays to measure peroxisome metabolic function or protocols to investigate and manipulate peroxisomes in cellular systems have been included Other chap-ters address the detection of peroxisomes, including immunofluorescence, cytochemistry, cryo-immuno-electron microscopy and live cell imaging approaches More specialised chapters deal with peroxisomal redox measurements, determination of pH, peroxisome biogenesis, import of peroxisomal proteins, protein modification or pexophagy, to name a few Finally, the clinical and laboratory diagnosis of peroxisomal disorders and the use of patient fibroblasts are addressed

biolo-I would like to express my sincerest appreciation to all of the authors who contributed chapters to this volume They were a pleasure to work with, providing state-of-the-art pro-tocols (and one review) in a timely fashion, while cheerfully responding to all of my queries

I would also like to thank Professor John Walker, editor of the Methods in Molecular Biology

series, for his invaluable advice and input in all aspects of the formulation of this book.This is truly an exciting time to be involved in peroxisome research, as vital functions

of this dynamic organelle in humans, plants and fungi are being discovered I hope you will get excited about peroxisome biology, that you will take advantage of the methods, tech-niques and strategies provided and that this volume of protocols will serve you well to tackle peroxisome- and organelle-based research questions

Preface

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Preface vii Contributors xiii

1 Isolation of Peroxisomes from Rat Liver and Cultured Hepatoma

Cells by Density Gradient Centrifugation 1

Andreas Manner and Markus Islinger

2 Isolation of Peroxisomes from Mouse Brain Using a Continuous

Nycodenz Gradient: A Comparison to the Isolation of Liver

and Kidney Peroxisomes 13

Miriam J Schönenberger and Werner J Kovacs

3 Determining the Topology of Peroxisomal Proteins Using Protease

Protection Assays 27

Tânia Francisco, Ana F Dias, Ana G Pedrosa, Cláudia P Grou,

Tony A Rodrigues, and Jorge E Azevedo

4 Isolation of Native Soluble and Membrane-Bound Protein Complexes

from Yeast Saccharomyces cerevisiae 37

Tobias Hansen, Anna Chan, Thomas Schröter, Daniel Schwerter,

Wolfgang Girzalsky, and Ralf Erdmann

5 Method for Measurement of Peroxisomal Very Long-Chain Fatty

Acid Beta-Oxidation and De Novo C26:0 Synthesis Activity in Living

Cells Using Stable-Isotope Labeled Docosanoic Acid 45

Malu-Clair van de Beek, Inge M.E Dijkstra, and Stephan Kemp

6 Analysis of Plasmalogen Synthesis in Cultured Cells 55

Masanori Honsho and Yukio Fujiki

7 Transfection of Primary Human Skin Fibroblasts for Peroxisomal Studies 63

Janet Koster and Hans R Waterham

8 siRNA-mediated Silencing of Peroxisomal Genes in Mammalian Cells 69

Tina A Schrader and Michael Schrader

9 Dual Reporter Systems for the Analysis of Translational Readthrough

in Mammals 81

Julia Hofhuis, Severin Dieterle, Rosemol George, Fabian Schueren,

and Sven Thoms

10 Cytochemical Detection of Peroxisomes in Light and Electron

H Dariush Fahimi

11 Cryo-Immuno Electron Microscopy of Peroxisomal Marker Proteins 101

Karina Mildner and Dagmar Zeuschner

12 Detection and Immunolabeling of Peroxisomal Proteins 113

Tina A Schrader, Markus Islinger, and Michael Schrader

Contents

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13 Labeling of Peroxisomes for Live Cell Imaging in the Filamentous

Fungus Ustilago maydis 131

Sofia C Guimarães, Sreedhar Kilaru, Michael Schrader,

and Martin Schuster

14 Quantitative Monitoring of Subcellular Redox Dynamics in Living

Mammalian Cells Using RoGFP2-Based Probes 151

Celien Lismont, Paul A Walton, and Marc Fransen

15 KillerRed as a Tool to Study the Cellular Responses

to Peroxisome-Derived Oxidative Stress 165

Marc Fransen and Chantal Brees

16 Determination of Peroxisomal pH in Living Mammalian Cells

Using pHRed 181

Luis F Godinho and Michael Schrader

17 In Cellulo Approaches to Study Peroxisomal Protein Import – Yeast

Immunofluorescence Microscopy 191

Tobias Hansen, Wolfgang Girzalsky, and Ralf Erdmann

18 Blue Native PAGE: Applications to Study Peroxisome Biogenesis 197

Kanji Okumoto, Shigehiko Tamura, and Yukio Fujiki

19 In Vitro PMP Import Analysis Using Cell-Free Synthesized PMP

and Isolated Peroxisomes 207

Yuqiong Liu, Masanori Honsho, and Yukio Fujiki

20 Peroxisomal Membrane and Matrix Protein Import Using a Semi-Intact

Mammalian Cell System 213

Kanji Okumoto, Masanori Honsho, Yuqiong Liu, and Yukio Fujiki

21 The Use of Glycosylation Tags as Reporters for Protein Entry

into the Endoplasmic Reticulum in Yeast and Mammalian Cells 221

Judith Buentzel and Sven Thoms

22 Detection of Ubiquitinated Peroxisomal Proteins in Yeast 233

Natasha Danda and Chris Williams

23 Assessing Pexophagy in Mammalian Cells 243

Shun-ichi Yamashita and Yukio Fujiki

24 Experimental Systems to Study Yeast Pexophagy 249

Shun-ichi Yamashita, Masahide Oku, Yasuyoshi Sakai,

and Yukio Fujiki

25 Flow Cytometric Analysis of the Expression Pattern of Peroxisomal

Proteins, Abcd1, Abcd2, and Abcd3 in BV-2 Murine Microglial Cells 257

Meryam Debbabi, Thomas Nury, Imen Helali, El Mostafa Karym,

Flore Geillon, Catherine Gondcaille, Doriane Trompier, Amina Najid,

Sébastien Terreau, Maryem Bezine, Amira Zarrouk, Anne Vejux,

Pierre Andreoletti, Mustapha Cherkaoui-Malki, Stéphane Savary,

and Gérard Lizard

26 Study of Peroxisomal Protein Phosphorylation by Functional Proteomics 267

Andreas Schummer, Sven Fischer, Silke Oeljeklaus,

and Bettina Warscheid

Contents

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Oil Mobilization in Arabidopsis thaliana Seedlings 291

Björn Hielscher, Lennart Charton, Tabea Mettler-Altmann,

and Nicole Linka

28 Peroxisome Mini-Libraries: Systematic Approaches to Study

Peroxisomes Made Easy 305

Noa Dahan, Maya Schuldiner, and Einat Zalckvar

29 Generation of Peroxisome-Deficient Somatic Animal Cell Mutants 319

Kanji Okumoto and Yukio Fujiki

30 Clinical and Laboratory Diagnosis of Peroxisomal Disorders 329

Ronald J.A Wanders, Femke C.C Klouwer, Sacha Ferdinandusse,

Hans R Waterham, and Bwee Tien Poll-Thé

Index 343

Contents

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Pierre Andreoletti • Laboratoire ‘Biochimie du peroxysome, inflammation et

métabo-lisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne Franche Comté, Dijon, France

Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de

Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal; Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal

MAryeM Bezine • Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme

lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne Franche Comté, Dijon, France; Laboratoire de Venins et Biomolécules Thérapeutiques (LVMT), Université de Tunis El Manar-Institut Pasteur, Tunis, Tunisia

ChAntAl Brees • Laboratory of Lipid Biochemistry and Protein Interactions,

Department of Cellular and Molecular Medicine, KU Leuven – University of

Leuven, Leuven, Belgium

Judith Buentzel • Department of Pediatrics and Adolescent Health, University

Medical Center, University of Göttingen, Göttingen, Germany

Pathobiochemie, Medizinische Fakultät der Ruhr- Universität Bochum,

Bochum, Germany

lennArt ChArton • Institute for Plant Biochemistry and Cluster of Excellence on

Plant Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany

MustAPhA CherkAoui-MAlki • Laboratoire ‘Biochimie du peroxysome, inflammation

et métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université

de Bourgogne Franche Comté, Dijon, France

Rehovot, Israel

nAtAshA dAndA • Molecular Cell Biology, Groningen Biomolecular Sciences and

Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands

MeryAM deBBABi • Laboratoire ‘Biochimie du peroxysome, inflammation et

métabo-lisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne Franche Comté, Dijon, France; Faculté de Médecine, Laboratoire de Nutrition—Aliments Fonctionnels et Santé Vasculaire (LR12ES05), Monastir & Faculté de Médecine, Université de Monastir, Sousse, Tunisia

Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal;

Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, Porto, Portugal

Contributors

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severin dieterle • Department of Pediatrics and Adolescent Health, University

Medical Center Göttingen, University of Göttingen, Göttingen, Germany

Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

rAlF erdMAnn • Abteilung für Systembiochemie, Institut für Biochemie und

Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum,

Ruhr-Universität Bochum, Bochum, Germany

h dAriush FAhiMi • Division of Medical Cell Biology, Department of Anatomy

and Cell Biology, University of Heidelberg, Heidelberg, Germany

sAChA FerdinAndusse • Laboratory Genetic Metabolic Diseases, Departments of

Paediatrics and Clinical Chemistry, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

sven FisCher • Department of Biochemistry and Functional Proteomics, Institute of

Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany

tâniA FrAnCisCo • Instituto de Investigação e Inovação em Saúde, Universidade do

Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal

MArC FrAnsen • Laboratory of Lipid Biochemistry and Protein Interactions,

Department of Cellular and Molecular Medicine, University of Leuven - KU Leuven, Leuven, Belgium

yukio FuJiki • Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

Flore geillon • Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme

lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne Franche Comté, Dijon, France

roseMol george • Department of Pediatrics and Adolescent Health, University

Medical Center Göttingen, Georg August University Göttingen, Göttingen, Germany

WolFgAng girzAlsky • Abteilung für Systembiochemie, Institut für Biochemie und

Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Bochum, Germany

(iBiMED), University of Aveiro, Aveiro, Portugal

CAtherine gondCAille • Laboratoire ‘Biochimie du peroxysome, inflammation et

métabolisme lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université

CláudiA P grou • Instituto de Investigação e Inovação em Saúde, Universidade do

Porto, Porto, Portugal; Organelle Biogenesis and Function Group, Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal

University of Exeter, Exeter, UK

toBiAs hAnsen • Abteilung für Systembiochemie, Institut für Biochemie und

iMen helAli • Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme

lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne Franche Comté, Dijon, France; Faculté de Pharmacie, Laboratoire des Maladies

Contributors

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Transmissibles et Substances Biologiquement Actives (LR99ES27), Université de Monastir, Monastir, Tunisia

BJörn hielsCher • Institute for Plant Biochemistry and Cluster of Excellence on Plant

Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany

JuliA hoFhuis • Department of Pediatrics and Adolescent Health, University Medical

Center Göttingen, University of Göttingen, Göttingen, Germany

MAsAnori honsho • Medical Institute of Bioregulation, Kyushu University, Fukuoka,

Japan

MArkus islinger • Center for Biomedicine and Medical Technology Mannheim,

Institute of Neuroanatomy, University of Heidelberg, Mannheim, Germany

el MostAFA kAryM • Laboratoire ‘Biochimie du peroxysome, inflammation et

de Bourgogne Franche Comté, Dijon, France; Laboratoire de Biochimie et

Neuroscience, Faculté de Sciences et Techniques, Université Hassan 1er, Settat,

Morocco

stePhAn keMP • Laboratory Genetic Metabolic Diseases (F0-226), Departments of

Pediatrics and Clinical Chemistry, Academic Medical Center, University of

Amsterdam, Amsterdam, The Netherlands

sreedhAr kilAru • College of Life and Environmental Sciences, Biosciences, University

of Exeter, Exeter, UK

Paediatrics and Clinical Chemistry, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

JAnet koster • Laboratory Genetic Metabolic Diseases, Department of Clinical

Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Werner J kovACs • Institute of Molecular Health Sciences, ETH Zurich, Zurich,

Switzerland

niCole linkA • Institute for Plant Biochemistry and Cluster of Excellence on Plant

Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany

Celien lisMont • Laboratory of Lipid Biochemistry and Protein Interactions,

Department of Cellular and Molecular Medicine, University of Leuven - KU Leuven, Leuven, Belgium

yuqiong liu • Graduate School of Systems Life Sciences, Kyushu University Graduate

School, Fukuoka, Japan

gérArd lizArd • Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme

AndreAs MAnner • Institute of Neuroanatomy, Center for Biomedicine and Medical

Technology Mannheim, University of Heidelberg, Mannheim, Germany

tABeA Mettler-AltMAnn • Institute for Plant Biochemistry and Cluster of Excellence

on Plant Sciences (CEPLAS), Heinrich Heine University, Düsseldorf, Germany

kArinA Mildner • Max-Planck-Institute for Molecular Biomedicine, Electron

Microscopy, Muenster, Germany

Contributors

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thoMAs nury • Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme

silke oelJeklAus • Department of Biochemistry and Functional Proteomics, Institute

of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany

MAsAhide oku • Division of Applied Life Sciences, Graduate School of Agriculture,

Kyoto University, Kyoto, Japan

kAnJi okuMoto • Department of Biology, Faculty of Sciences, Kyushu University,

Fukuoka, Japan

Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

yAsuyoshi sAkAi • Division of Applied Life Sciences, Graduate School of Agriculture,

Kyoto University, Kyoto, Japan

stePhAne sAvAry • Laboratoire ‘Biochimie du peroxysome, inflammation et

MiriAM J sChönenBerger • Institute of Physiology, University of Zurich, Zurich,

Switzerland

MiChAel sChrAder • College of Life and Environmental Sciences, Biosciences,

thoMAs sChröter • Abteilung für Systembiochemie, Institut für Biochemie und

FABiAn sChueren • Department of Pediatrics and Adolescent Health, University

Medical Center Göttingen, Georg August University Göttingen, Göttingen, Germany

MAyA sChuldiner • Department of Molecular Genetics, Weizmann Institute of Science,

Rehovot, Israel

AndreAs sChuMMer • Department of Biochemistry and Functional Proteomics,

Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany

MArtin sChuster • College of Life and Environmental Sciences, Biosciences,

Contributors

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dAniel sChWerter • Abteilung für Systembiochemie, Institut für Biochemie und

shigehiko tAMurA • Division for Experimental Natural Science, Faculty of Arts

and Science, Kyushu University, Fukuoka, Japan

seBAstien terreAu • Laboratoire ‘Biochimie du peroxysome, inflammation et

Center, University of Göttingen, Göttingen, Germany

doriAne troMPier • Laboratoire ‘Biochimie du peroxysome, inflammation et

Departments of Pediatrics and Clinical Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Department of Cellular and Molecular Medicine, University of Leuven - KU Leuven, Leuven, Belgium; Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada

ronAld J.A WAnders • Laboratory Genetic Metabolic Diseases, Departments of

Paediatric and Clinical Chemistry, Emma Children’s Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

BettinA WArsCheid • Department of Biochemistry and Functional Proteomics,

Institute of Biology II, Faculty of Biology, University of Freiburg, Freiburg, Germany; BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany

Chemistry, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Chris WilliAMs • Molecular Cell Biology, Groningen Biomolecular Sciences and

Biotechnology Institute (GBB), University of Groningen, Groningen, The Netherlands

shun-iChi yAMAshitA • Department of Cellular Physiology, Graduate School of

Medical and Dental Sciences, Niigata University, Niigata, Japan

einAt zAlCkvAr • Department of Molecular Genetics, Weizmann Institute of Science,

Rehovot, Israel

AMirA zArrouk • Laboratoire ‘Biochimie du peroxysome, inflammation et métabolisme

lipidique’, EA7270/INSERM, Faculté des Sciences Gabriel, Université de Bourgogne Franche Comté, Dijon, France; Faculté de Médecine, Laboratoire de Nutrition – Aliments Fonctionnels et Santé Vasculaire, Monastir & Faculté de Médecine,

Université de Monastir, Sousse, Tunisia

dAgMAr zeusChner • Max-Planck-Institute for Molecular Biomedicine, Electron

Microscopy, Muenster, Germany

Contributors

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Michael Schrader (ed.), Peroxisomes: Methods and Protocols, Methods in Molecular Biology, vol 1595,

DOI 10.1007/978-1-4939-6937-1_1, © Springer Science+Business Media LLC 2017

Chapter 1

Isolation of Peroxisomes from Rat Liver and Cultured

Hepatoma Cells by Density Gradient Centrifugation

Andreas Manner and Markus Islinger

Abstract

Subcellular fractionation is still a valuable technique to unravel organelle-specific proteomes, validate the location of uncharacterized proteins, or to functionally analyze import and metabolism in individual subcellular compartments In this respect, density gradient centrifugation still represents a very classic, indis- pensable technique to isolate and analyze peroxisomes Here, we present two independent protocols for the purification of peroxisomes from either liver tissue or the HepG2 hepatoma cell line While the former permits the isolation of highly pure peroxisomes suitable for, e.g., subcellular proteomics experiments, the latter protocol yields peroxisomal fractions from considerably less purity but allows to easily modify metabolic conditions in the culture medium or to genetically manipulate the peroxisomal compartment In this respect, both purification methods represent alternative tools to be applied in experiments investigating peroxisome physiology.

Key words Peroxisomes, Liver, Density gradient centrifugation, Organelle purification, ACAD11

1 Introduction

Peroxisomes are ubiquitous organelles, which can be found in fering amounts in all eukaryotic cells Morphologically, peroxi-somes appear in varying sizes and shapes in different tissues and contain a differing subset of proteins Accordingly, there is no stan-dard procedure for peroxisome isolation and protocols have to be adapted to the tissue of interest In mammals, highest amounts of peroxisomes can be found in hepatocytes and the kidney cells of the proximal tubule, where they can comprise up to 2% of the total cellular content In these organs peroxisomes do not only stand out by their sheer amount but also organelle size and can reach

kidney possess unique physical features that enable the isolation of highly pure peroxisome fractions that can still not be obtained in this quality in other tissues Also historically, the liver of rats were the source of the fractions used for the initial biochemical

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This initial protocol, as well as most of the methods applied to date, is based on a three-step isolation procedure consisting of (1)

a mild homogenization of the tissue using a Potter-Elvenheijm homogenizer at low velocities, (2) a series of differential centrifu-gations leading to a peroxisome-enriched fraction, and (3) a final centrifugation step applying a density gradient For the fundamen-tal experiments performed by the De Duve group, linear sucrose density gradients were used for isopycnic centrifugation; however, they required the intravenous injection of Triton WR-1339 prior

to sacrificing the animals In addition, the isolation of peroxisomes

of high purity required the use of a special Beaufay-type rotor

Metrizamide, a tri-iodinated benzamido-derivative of glucose is—compared to sucrose—a considerably less viscous gradient medium showing lower osmolality and is not able to penetrate biological

of peroxisomes with purities >90% from rodent liver and kidney

Metrizamide was subsequently replaced by the iodinated cose-based benzamido-derivatives Nycodenz and Optiprep, which are more stable, less toxic, and show significantly less interference

been frequently used to isolate peroxisomes from rodent liver or kidney and have been shown to yield peroxisome fractions of high purity suitable for subsequent proteome analysis without further

isoosmotic solutions at velocities overlapping with small dria, lysosomes, and partially microsomes, they reach unexpectedly high densities, when centrifuged in gradients from the iodinated media mentioned above This remarkable behavior may be due to the selective permeability of peroxisomes to the gradient media Interestingly, peroxisomes possess comparably large protein pores allowing the free diffusion of molecules up to 600 Da across their

and Optiprep range between 821 and 1550 Da, respectively, and should not allow those molecules to enter peroxisomes through these pores A more likely explanation may lie in the high fragility

damage during the isolation may result in a transient disruption of the peroxisomal membrane enabling the exchange of soluble com-pounds Thus, the lower osmotic pressure in gradient media from higher molecular weight compounds would lead to a better preser-vation of and reduced uptake of separation medium by the organ-elles Indeed, liver peroxisomes sediment in Metrizamide at a mean

that the bulkier Optiprep penetrates the organelle membrane less efficiently

Andreas Manner and Markus Islinger

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Since peroxisomes from liver and kidney have been thoroughly characterized in numerous publications one may ask if a chapter describing the isolation of peroxisomes from these tissues is still required in a current method compilation However, as the purities

of these peroxisome fractions still exceed those from other tissues and cell culture, they still remain the gold-standard for the localiza-tion of newly identified, ubiquitously expressed peroxisomal pro-teins Still, some peroxisomal constituents may only localize at the organelle under specific physiological conditions or show tissue-specific expression In this respect, cell cultures represent ideal models to manipulate metabolic conditions by administration of selected compounds or to modify peroxisomal functions by knock-down or overexpression of distinct peroxisomal proteins Hence, peroxisomes isolated from cell lines may be used to allocate novel endogenous or overexpressed peroxisomal proteins that do not per-manently associate with the organelle or which under standard con-ditions are only present at very low concentrations HepG2 cells possess a considerable number of peroxisomes and according to their liver origin express the proteins of the classic peroxisomal met-abolic pathways Thus, they can be easily analyzed using commer-cially available antibodies against peroxisomal marker proteins Peroxisomes in this cell line are on average considerably smaller than those from rodent liver or kidney and the majority of peroxi-somes sediments at densities that are closer to the endoplasmic

frac-tions from HepG2 cells do not reach comparable purities but are nevertheless suitable to allocate individual proteins to distinct organelle fractions To this end, we established a protocol to sepa-rate peroxisomes from HepG2 cells from the remaining organelles using a flat, linear density gradient As both isolation schemes (from liver and HepG2 cells) represent multi-step procedures, representa-

compare the methods used for isolating peroxisomes from cells and tissues As an example for a localization experiment, we show here the distribution of one of the more recently identified peroxisomal

2 Materials

1 Refractometer for the preparation of density gradients

2 Ultracentrifuge and a fixed angle rotor (e.g., VTi 50, VTi 65.1 type vertical rotor, Beckman Coulter, Brea, USA)

3 Homogenization buffer (HB): 250 mM sucrose, 5 mM MOPS, 1 mM EDTA, 2 mM PMSF, 1 mM DTT, 1 mM ɛ-aminocaproic acid, pH 7.4 adjusted with KOH

2.1 General

Materials

Peroxisome Isolation from Liver and Hepatoma Cells

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4 Gradient buffer (GB): 5 mM MOPS, 1 mM EDTA, 2 mM

adjusted with KOH

1 Motor-driven Potter-Elvehjem tissue grinder with loose fitting pestle (clearance 0.1–0.15 mm, vol 30 mL)

2 Optiprep: 60% (w/v) iodixanol solution in water (Axis Shield, Rodeløkka, Sweden)

3 Quick-seal polyallomer tubes 25 × 89 mm (39 mL, Beckman Coulter)

4 0.9% (w/v) NaCl solution

1 Gradient mixer (10–25 mL volume per chamber)

2 Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM

3 Cell scrapers to remove HepG2 cells from culture dishes

4 Syringe (5 mL) with a 27G needle

5 Quick-seal polyallomer tubes 16 × 76 mm (13.5 mL, Beckman Coulter)

2.2 Isolation of Rat

Liver Peroxisomes

2.3 Separation

of Peroxisomes

from HepG2 Cells

Fig 1 Schematic overview of peroxisome isolation from rodent liver (a) and HepG2 cells (b) PNS post nuclear

supernatant, HM heavy mitochondrial fraction, LM light mitochondrial fraction, MIC microsomal fraction, CYT

cytosolic fraction

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3 Methods

1 To produce the sigmoid-shaped density gradient prepare Optiprep solutions of 1.12, 1.15, 1.19, 1.22, and 1.26 g/mL

by diluting the 60% Optiprep stock solution (1.32 g/mL) with

GB To adjust the correct density use a refractometer and the formula:

ρ = 3.350 × refractive index − 3.462

2 Using the 39 mL Quick-Seal tubes (Beckman Coulter Inc.), layer sequentially 4, 3, 6, 7, and 10 mL of the Optiprep dilutions

in a decreasing order of concentration (1.26–1.12 g/mL)

3 Freeze the discontinuous gradient rapidly in liquid nitrogen

4 Precool all solutions and vessels used on ice; all subsequent purification steps are carried out at a temperature of 4 °C

5 After anesthesia open the body cavity of the animals and excise

the liver, rinse with 0.9% NaCl and determine its weight (see

Note 2) Cut the liver into small pieces and wash away drained

blood with HB Finally suspend liver pieces in ice-cold HB at a

6 Homogenize the liver pieces with the Potter-Elvehjem tissue

pellet cellular debris and nuclei

8 Keep the supernatant on ice, resuspend the pellet in approx

10 mL and re-homogenize the pellet for a second time at

centrifugation step

9 Pool the supernatants from both homogenization steps and

mitochondrial fraction, which mainly contains large mitochondria

10 Carefully drain the pellet from the supernatant and manually suspend the pellet in HB using a glass rod Make sure not to disturb the blood pellet at the bottom of the tube

12 Combine the supernatants from both runs and centrifuge at

pellet of light mitochondria, which contains peroxisomes They are enriched by the factor of 3–4 if compared to the PNS, combined with microsomes, mitochondria, and lysosomes

aspi-rate the gel-like, reddish “fluffy” layer positioned at the top of the pellet, which mainly contains microsomes

3.1 Isolation

of Peroxisomes

from Rat Liver

Trang 20

14 Stir the remaining dry pellet with a glass rod until no clumps are visible and subsequently add drop-wise HB Continue stir-ring until you obtain a homogenous organelle suspension Adjust volume of HB to at least 2 mL/g of pellet

15 Wash the light mitochondrial pellet using another

centrifuga-tion step at 37,000 × g, 15 min, 4 °C.

16 Again remove the remaining fluffy-layer and resuspend the residual pellet in 1–2 mL HB/g as described above This sus-pension comprises the final light mitochondrial fraction, which

is further separated by the density gradient centrifugation

17 Slowly defrost the Optiprep gradient described above at room temperature in a metal stand This takes around 30 min and can be initiated in parallel to the last steps of the differential centrifugation procedure

18 Layer 5 mL of the light mitochondrial fraction on the top of

the Optiprep gradient Overlay with GB and seal the tubes (see

Note 6).

19 Centrifuge in a vertical rotor (e.g., VTi50) at an integrated

accel-eration/deceleration, 4 °C

20 After centrifugation, three narrow but clearly detectable bands will be visible near the bottom of the tube The lowermost is com-posed of crystalloid cores from disrupted peroxisomes Somewhat above you will detect two bands containing intact peroxisomes The lower band at the higher density of 1.20 g/mL is the purest fraction in the gradient containing more than 95% of peroxi-somes The one above at 1.18 g/mL shows a higher contamina-tion with mitochondria but still contains usually above 90% of peroxisomes To collect the individual fractions, puncture the tubes with a syringe and aspirate band by band

21 To concentrate the samples and wash out the Optiprep, dilute the sample at least 3:1 in HB Pellet the organelles by centrifu-

of HB

22 Determine enzyme activities of organelle marker enzymes as

1 Remove HepG2 cells from minimum five 80% confluent

analysis 10–12 flasks are recommended) Clear cells from

10 mL PBS by repeating the centrifugation procedure

3.2 Purification

of Peroxisomes

from HepG2 Cells

Trang 21

Fig 2 Peroxisome isolation from rodent liver (a) Sketch of a typical sigmoid Optiprep-gradient after centrifugation

Organelles enrich in characteristic bands at densities given to the right, arrow—location where fraction LM is

marker proteins Note that peroxisomes enrich in two individual bands, LM1 and LM2 While the LM1 fraction usually possesses a purity above 95%, LM2 show a higher contamination with mitochondria, which can, however,

PNS post nuclear supernatant, HM heavy mitochondrial fraction, LM light mitochondrial fraction, MIC microsomal

(1:10,000, gift from D Crane, Griffith University, Brisbane), rabbit α Catalase (1:10,000, gift from A Völkl, University

Trang 22

2 Resuspend the cell pellet in 2 mL HB/tissue culture flask and disrupt the cells by shearing through a syringe with a 27G nee-dle for seven times

3 Collect undisrupted cells, cellular debris, and nuclei by

on ice until further use Homogenize the pellet for a second

time as described above and centrifuge again at 600 × g,

10 min, 4 °C

4 Pool both post nuclear supernatants (PNS) and centrifuge at

pro-duce the light mitochondrial pellet (LM) Resuspend the pellet

in 1 mL of HB using a glass rod

6 For the next centrifugation step, pour a linear Nykodenz- gradient ranging from 1.14 to 1.20 g/mL immediately before

gradient, prepare two stock solutions of Nycodenz of 1.14 and 1.19 g/mL in GB, pH 7.4 You will require 6 mL of each Nykodenz solution per 16 × 76 mm tube (e.g., 13.5 mL Quick-Seal tubes, Beckman Coulter Inc.)

7 Layer the pellet suspended in 1 mL HB on the top of the Nycodenz gradient Overlay with GB and seal the tubes

Centrifuge at 100,000 × g for 3 h, 4 °C.

8 As the individual bands in the gradient are usually very faint, puncture the tube at the bottom with a syringe and retrieve equal-sized samples (e.g., 1 mL)

9 To remove the Nycodenz and enrich organelles, dilute the

samples at minimum 3:1 in HB and centrifuge at >30,000 × g

for 20 min, 4 °C Suspend the resulting pellets in a small

10 Perform immunoblots or enzyme assays (see above) to

4 Notes

1 Freezing of the density step-gradient used in the liver isolation protocol is a prerequisite for a successful separation The char-acteristic sigmoid density distribution is generated during the thawing process

2 Glycogen deposits in the liver will disturb the separation in the density gradient Thus, to obtain highly pure peroxisome frac-tions the animals have to be fasted overnight

3 The protocol for liver peroxisomes can also be applied for the isolation of peroxisomes from kidney If comparable amounts

Trang 23

of starting material are used, however, the peroxisome yield will be lower than in liver As kidney peroxisomes do not con-tain uricase (UOX), the separation will produce no core frac-tion, which is a characteristic for liver peroxisomes

4 Peroxisomes are particularly fragile and leaky organelles; thus vigorous homogenization using multiple pestle strokes should

be avoided to maximize organelle integrity

5 Optionally the supernatant produced while pelleting the light mitochondrial fraction can be further separated into a microsome- enriched pellet and a supernatant representing the cytosol These fractions can be used to evaluate the subcellular location of a protein of interest To obtain both fractions, add

6 As the thumb-rule for efficient peroxisome purification, the maximum amount of LM fraction, which can be applied on one density gradient, equals approximately 10 g of liver tissue used as a starting material

7 Adaptors to fit the small 16 mm diameter tubes into a VTi 50

or comparable rotor with a larger cavity are commercially available

Fig 3 Peroxisome isolation from HepG2 cells (a) Sketch of the linear gradient (1.14–1.19 g/mL) used for the

organelle separation from HepG2 cells Positions of the individual fractions analyzed in the neighboring

immu-noblot and their correspondent densities after the centrifugation step are depicted The arrow represents the

mitochondria, endoplasmic reticulum, and peroxisomes in the gradient Maxima of the individual organelle

peaks are marked by arrowheads Note that ACAD11 is not associated with mitochondria in HepG2 cells (see

differs from the maxima of the peroxisome markers ACBD3, ACOX1, and Pex14 Since ACAD11 expression is highly dynamic in HepG2 cells, this observation may point to a specific peroxisome subfraction containing

ACAD11 PNS post nuclear supernatant, HM heavy mitochondrial fraction, LM light mitochondrial fraction

COX4 (1:2000, Abcam)

Trang 24

8 Compared to the situation in the density gradients from liver, microsomes migrate to a significantly higher density in the lin-ear Nykodenz-gradients, switching the position with mito-chondria This might be due to the different methods used for homogenization, which may fractionate the tubular ER cister-nae to a different degree

Acknowledgments

We thank all colleagues, who donated antibodies used in this work

We would further like to thank D Türker and Dr S Kühl for nical assistance

tech-References

1 Baudhuin P, Beaufay H, De Duve C (1965)

Combined biochemical and morphological

study of particulate fractions from rat liver

Analysis of preparations enriched in lysosomes

or in particles containing urate oxidase,

d-amino acid oxidase, and catalase J Cell Biol

26:219–243

2 De Duve C, Baudhuin P (1966) Peroxisomes

(microbodies and related particles) Physiol

Rev 46:323–357

3 Leighton F et al (1968) The large-scale

separa-tion of peroxisomes, mitochondria, and

lyso-somes from the livers of rats injected with

triton WR-1339 Improved isolation

proce-dures, automated analysis, biochemical and

morphological properties of fractions J Cell

Biol 37:482–513

4 Rickwood D, Birnie GD (1975) Metrizamide,

a new density-gradient medium FEBS Lett

50:102–110

5 Volkl A, Fahimi HD (1985) Isolation and

characterization of peroxisomes from the liver

of normal untreated rats Eur J Biochem

149:257–265

6 Hajra AK, Wu D (1985) Preparative isolation

of peroxisomes from liver and kidney using

metrizamide density gradient centrifugation in

a vertical rotor Anal Biochem 148:233–244

7 Crane DI, Hemsley AC, Masters CJ (1985)

Purification of peroxisomes from livers of

nor-mal and clofibrate-treated mice Anal Biochem

148:436–445

8 Rickwood D, Ford T, Graham J (1982)

Nycodenz: a new nonionic iodinated gradient

medium Anal Biochem 123:23–31

9 Ford T, Graham J, Rickwood D (1994)

Iodixanol: a nonionic iso-osmotic

centrifuga-tion medium for the formacentrifuga-tion of self- generated gradients Anal Biochem 220:360–366

10 Islinger M, Luers GH, Li KW, Loos M, Volkl

A (2007) Rat liver peroxisomes after fibrate treatment A survey using quantitative mass spectrometry J Biol Chem 282:23055–23069

11 Wiese S et al (2007) Proteomics tion of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling Mol Cell Proteomics 6:2045–2057

12 Rokka A et al (2009) Pxmp2 is a channel- forming protein in Mammalian peroxisomal membrane PLoS One 4:e5090

13 Antonenkov VD, Sormunen RT, Hiltunen JK (2004) The behavior of peroxisomes in vitro: mammalian peroxisomes are osmotically sensi- tive particles Am J Physiol Cell Physiol 287:C1623–C1635

14 Islinger M, Luers GH, Zischka H, Ueffing M, Volkl A (2006) Insights into the membrane proteome of rat liver peroxisomes: microsomal glutathione-S-transferase is shared by both subcellular compartments Proteomics 6:804–816

15 Schrader M, Baumgart E, Volkl A, Fahimi HD (1994) Heterogeneity of peroxisomes in human hepatoblastoma cell line HepG2 Evidence of distinct subpopulations Eur J Cell Biol 64:281–294

16 Kikuchi M et al (2004) Proteomic analysis of rat liver peroxisome: presence of peroxisome- specific isozyme of Lon protease J Biol Chem 279:421–428

17 Camoes F et al (2014) New insights into the peroxisomal protein inventory: Acyl-CoA oxi- dases and -dehydrogenases are an ancient fea- ture of peroxisomes Biochim Biophys Acta 1853:111–125

Trang 25

18 Islinger M, Abdolzade-Bavil A, Liebler S, Weber

G, Volkl A (2012) Assessing heterogeneity of

peroxisomes: isolation of two subpopulations

from rat liver Methods Mol Biol 909:83–96

19 He M et al (2011) Identification and acterization of new long chain acyl-CoA dehydrogenases Mol Genet Metab 102: 418–429

char-Peroxisome Isolation from Liver and Hepatoma Cells

Trang 26

Chapter 2

Isolation of Peroxisomes from Mouse Brain Using

a Continuous Nycodenz Gradient: A Comparison

to the Isolation of Liver and Kidney Peroxisomes

Miriam J Schönenberger and Werner J Kovacs

Abstract

In the central nervous system (CNS) peroxisomes are present in all cell types, namely neurons, drocytes, astrocytes, microglia, and endothelial cells Brain peroxisomes are smaller in size compared to peroxisomes from other tissues and are therefore referred to as microperoxisomes We have established a purification procedure to isolate highly purified peroxisomes from the central nervous system that are well separated from the endoplasmic reticulum and mitochondria and are free of myelin contamination The major difficulty in purification of brain peroxisomes compared to peroxisomes from liver or kidney is the presence of large amounts of myelin in the CNS, which results in contamination of the subcellular fractions Hence, the crucial step of the isolation procedure is the elimination of myelin by the use of a sucrose gradient, since without the elimination of myelin no significant enrichment of purified peroxisomes can be achieved Another difficulty is that in brain tissue the abundance of peroxisomes decreases significantly during postnatal development We provide a detailed protocol for the isolation of peroxisomes from mouse central nervous system as well as a protocol for the isolation of peroxisomes from the liver and kidney using

oligoden-a continuous Nycodenz groligoden-adient.

Key words Peroxisomes, Microperoxisomes, Brain, Liver, Kidney, Central nervous system, Myelin,

Fractionation, Nycodenz gradient

1 Introduction

Peroxisomes are ubiquitous and highly dynamic organelles whose number, size, and function are dependent on cell type and metabolic needs They play essential roles in reactive oxy-gen species and lipid metabolism The importance of peroxisomal metabolism for mammalian physiology is illustrated by peroxi-

some biogenesis disorders (i.e., Zellweger spectrum diseases) in

which functional peroxisomes are absent or disorders caused by single peroxisomal enzyme and membrane transporter deficien-

Trang 27

associated with abnormal CNS neuronal migration, abnormal white matter (demyelination, dysmyelination, hypomyelination), abnormal Purkinje cell dendritic arborization, loss of axonal integ-rity, neuroinflammation, and other neurodegenerative processes

addressing the cause of Zellweger syndrome neuropathology

In particular, studies on mice with either ubiquitous or

brain-specific deletion of Pex2, Pex5, and Pex13 have demonstrated a

range of neuropathological changes similar to those of Zellweger syndrome patients

In the brain, peroxisomes have been detected in all neural cell types, namely in neurons, oligodendrocytes, and astrocytes and in

microperoxi-somes Several studies showed that peroxisome abundance in the

brain development, peroxisomal activity remains constant in the cerebral cortex (a typical gray matter region), whereas microper-oxisomes are especially abundant in myelin-forming oligodendro-cytes prior to the appearance of myelin sheaths and for several days

is a decrease in the overall frequency of microperoxisomes and in

myelin formation is most pronounced during the first 3 weeks after birth Cholesterol, the main lipid component of myelin, is rapidly synthesized during this period We found that activities for cata-

enzyme, and the cholesterol biosynthetic enzymes isopentenyl diphosphate:dimethylallyl diphosphate isomerase and 3-hydroxy- 3-methylglutaryl coenzyme A reductase showed a similar postnatal development with high activities in brain stem, cerebellum, and

system-atic comparison by immunohistochemistry, western blot analysis, and catalase activity measurements found the maximum level two days after birth, and subsequently the abundance of peroxisomes

enzyme/pro-tein composition of brain peroxisomes during postnatal ment In summary, these data suggest that the high peroxisomal activity in the first weeks of postnatal life might relate to lipid syn-thesis accompanying rapid myelin formation and to the elaboration

develop-of plasma membranes for growing neurons in early postnatal brain

As with most subcellular organelles, methods for the isolation

of peroxisomes have come largely from work with rat and mouse liver The major difficulty in purification of brain peroxisomes

Miriam J Schönenberger and Werner J Kovacs

Trang 28

3 OptiSeal polyallomer centrifuge tubes (e.g., Beckman; Cat

No 362183; 25 × 86 mm or 1 × 3.5 in.; 36.2 mL capacity)

To avoid damage of peroxisomes, clean all glassware, fuge tubes, and equipment without detergents, and prepare buf-fers in detergent-free glassware

1 Homogenization buffer (HB): 5 mM MOPS (pH 7.4),

250 mM sucrose, 1 mM EDTA, 0.1% (v/v) ethanol Adjust

pH to 7.4 with NaOH Store at 4 °C Add protease inhibitors prior to usage We used the cOmplete EDTA-free protease inhibitor cocktail from Roche Diagnostics

eth-anol, 0.01% (v/v) Triton X-100 Adjust the pH of the cold buffer (4 °C) to 7.6 with HCl Store at 4 °C

1 Gradient buffer: Homogenization buffer without protease inhibitors

2 Prepare 25 mL of a 50% Nycodenz solution in HB to prepare one Nycodenz gradient Dissolve 12.5 g of Nycodenz in

15 mL of HB by shaking at 37 °C Once it is dissolved, adjust the volume to 25 mL

3 Nycodenz gradient for the isolation of brain peroxisomes: pare 4 mL each of 5, 10, 15, 20, 25, 30, 35, and 40% Nycodenz solutions Nycodenz gradient for the isolation of liver and kid-ney peroxisomes: prepare 5 mL each of 10, 15, 20, 25, 30, 35% Nycodenz solutions and 2 mL of a 40% Nycodenz solu-tion for the cushion Pour the gradient as a step gradient one day in advance of the isolation in an OptiSeal polyallomer cen-

Pour the gradient slowly with a long glass Pasteur pipette and avoid air bubbles

Trang 29

1 Catalase substrate: 10 mL 0.2 M imidazole buffer (pH 7.0),

2 Substrate solution: Dissolve 32.6 mg o-nitrophenyl acetate in

1 mL methanol (prepare the substrate solution fresh; keep on ice)

1 Reaction buffer: 50 mM potassium phosphate buffer (pH 7.4),

Fig 1 Scheme of the preparation of Nycodenz gradients for the isolation of

gradient one day in advance of the organelle isolation in an OptiSeal polyallomer centrifuge tube and allow linearizing overnight at 4 °C

Trang 30

1 0.05 M Tris–HCl (pH 7.5)

2 1% (v/v) Triton X-100

3 Reaction buffer: 0.2 M imidazole (pH 6.2)

frozen in small aliquots

5 Stop solution: glacial acetic acid

9 Cellulose MN 300 plates (Macherey-Nagel, Düren, Germany) for thin-layer chromatography

3 Methods

The protocol for the isolation of CNS peroxisomes described here

point of active myelination and when peroxisomes are still abundant

in the brain We used CNS regions that are rich in myelin and fore lipid metabolism plays an important role Note that the amount

there-of myelin in the brain there-of adult mice is much higher, whereas some abundance is lower Hence, elimination of myelin will be cru-cial to isolate highly purified peroxisomes from adult brain

peroxi-All steps should be performed at a temperature of 4 °C unless

1 For the isolation of peroxisomes, euthanize 15-day-old mouse

cerebel-lum, and spinal cord After determining its weight, mince the

cold homogenization buffer (HB) at a ratio of 3 mL/g tissue in the precooled glass vessel of the Potter-Elvehjem homogenizer

2 Homogenize the tissue slowly with three up and down strokes using a motor-driven Potter-Elvehjem tissue grinder and a loose-fitting Teflon pestle rotating at 1000 rpm

3 Centrifuge the homogenate at 1000 × g for 10 min at 4 °C to

sediment nuclei and cellular debris

4 Store the supernatant on ice and resuspend the pellet in

HB Rehomogenize the pellet by three strokes of 1000 rpm

and centrifuge at 600 × g for 10 min.

from the Central

Nervous System Using

a Continuous

Nycodenz Gradient

Isolation of Mouse Brain Peroxisomes

Trang 31

Brain stem, cerebellum

& spinal cord

Postnuclear supernatant

Light mitochondrial pellet 1

Heavy mitochondrial pellet

Microsomal pellet

Liver or kidney

Whole homogenate

100 g, 10 min, 2x + Homogenization buffer, 1 Stroke

Nuclear fraction

Postmitochondrial supernatant

Cytosol Supernatant

on 0-35% (w/v) Nycodenz gradient

Liver/kidney peroxisomes

Fig 2 (a) Fractionation scheme for the isolation of peroxisomes from brain Myelin-free light mitochondrial

fractions prepared by differential centrifugation are further purified by equilibrium density centrifugation on a

isolation of peroxisomes from liver and kidneys Light mitochondrial fractions prepared by differential gation are further purified by equilibrium density centrifugation on a linear Nycodenz gradient, and fractions

organelles on the Nycodenz gradient for the isolation of liver and kidney peroxisomes after centrifugation Note that the ER distributes also over the gradient fractions containing mitochondria

Trang 32

6 Combine the supernatants (postnuclear supernatant; PNS) and dilute it to 10% (w/v) with HB Store a small sample of

Discard the final pellet that consists mainly of nuclei, large myelin fragments, and tissue debris

7 Centrifuge the PNS at 5500 × g for 10 min at 4 °C to obtain a

heavy mitochondrial pellet (M) and a postmitochondrial natant (PMS) Resuspend the M pellet manually with a glass

super-rod in 1 mL/g HB and spin again at 5500 × g for 10 min The

combined supernatants represent the PMS Resuspend the M pellet manually in 1 mL/g HB

8 Centrifuge the PMS at 18,000 × g for 30 min at 4 °C to obtain

a light mitochondrial pellet (L1) Remove the supernatant

care-fully and centrifuge at 105,000 × g for 1 h at 4 °C to obtain a

microsomal pellet (P) and a final supernatant (contains cytosolic proteins) Resuspend the microsomal pellet in 1 mL/g HB

9 Resuspend the pellet L1 in 0.85 M sucrose in 5 mM Mops,

pH 7.4, 1 mM EDTA, and 0.1% (v/v) ethanol Resuspend the pellet L1 carefully with a glass rod, add the buffer drop-wise until a homogenous suspension is gained, and then adjust the volume to 19 mL and transfer to an open-top poly-propylene or Ultra-Clear centrifuge tube (tubes for SW27 rotor or similar) Overlay with an equal volume of 0.25 M sucrose in 5 mM Mops, pH 7.4, 1 mM EDTA, and 0.1%

(v/v) ethanol and centrifuge at 107,960 × g for 45 min at

4 °C (Beckman SW27 rotor or similar) to remove any ing myelin The majority of myelin will be at the interface

10 Remove the upper layer (0.25 M sucrose) and myelin at the interface Resuspend the pellet and combine it with the lower layer (0.85 M sucrose) and dilute the suspension with HB to a

final concentration of 0.25 M sucrose Centrifuge at 18,000 × g

for 30 min at 4 °C to obtain the myelin-free light drial pellet (L2)

11 Resuspend the pellet L2 carefully in HB with a glass rod, add the buffer drop-wise until a homogenous suspension is gained

12 Layer the resuspended L2 dropwise with a plastic Pasteur pipette on the top of a 0–40% (w/v) continuous linear Nycodenz gradient Seal the tube with a cap and make sure to avoid air

bubbles Centrifuge at 142,000 × g for 85 min at 8 °C in a

Beckman VTI50 or a Sorvall TV-850 vertical rotor with slow acceleration/deceleration Remove the cap from the centrifuge tube and collect fractions (1.25 mL) in an Eppendorf tube from the bottom of the tube with a two-way needle The peroxi-

Trang 33

be performed at a temperature of 4 °C unless otherwise specified

1 Starve mice overnight before the isolation experiment (see

Note 3).

2 For the isolation of liver peroxisomes, the mouse is

mouse is completely anesthetized and shows no signs of response Open the abdominal cavity and perfuse the liver with ice-cold PBS or 0.8% saline solution through the portal vein using a pump system generating a constant buffer speed of

13 mL/min Perfusion is carried out until blood is drained away completely For the isolation of kidney peroxisomes euth-

perfusion of the organ

3 After determining its weight, mince the tissue on an ice-cold metal block with razorblades into pieces of approximately

at a ratio of 3 mL/g tissue directly in the precooled glass vessel

4 Homogenize the tissue very slowly with one up and down stroke (each 1 min) using a motor-driven Potter-Elvehjem tis-sue grinder and a loose-fitting Teflon pestle rotating at

5 Centrifuge the homogenate at 100 × g for 10 min at 4 °C to

sediment nuclei and cellular debris

6 Store the supernatant (Postnuclear supernatant 1, PNS1) on ice and resuspend the pellet in 2 g/mL HB Rehomogenize the pellet by one stroke of 1000 rpm and centrifuge again at

100 × g for 10 min at 4 °C Combine the supernatant with

PNS1 and discard the pellet

7 Centrifuge the PNS1 once at 600 × g for 10 min at

4 °C Resuspend the nuclear pellet in 1 mL HB (P1) Combine the PNS1 and dilute it to 10% (w/v) (postnuclear supernatant 2; PNS2) Store a small aliquot of the postnuclear supernatant (PNS2)

8 Centrifuge the PNS2 at 1950 × g for 10 min Store the

supernatant (postmitochondrial supernatant, PMS) on ice Resuspend the pellet in 1 mL HB and centrifuge again at

1950 × g for 10 min Combine both supernatants (PMS) and

save a small aliquot Resuspend the second pellet, which is the heavy mitochondrial fraction (M), manually in 1 mL HB

Trang 34

9 Centrifuge the PMS at 25,500 × g for 20 min at 4 °C to obtain

a light mitochondrial pellet (LM) Remove the supernatant carefully and resuspend the pellet carefully with a glass rod, add the buffer dropwise until a homogenous suspension is gained Start resuspension of the pellet with 1 mL HB and only once a homogenous suspension is gained add 4 mL of HB

10 Centrifuge the supernatant from the 25,500 × g spin at 100,000 × g for 1 h at 4 °C to obtain a microsomal pellet (P)

and a final supernatant (contains cytosolic proteins) Resuspend

11 Add the LM dropwise with a plastic Pasteur pipette on the top of

a 0–35% (w/v) continuous linear Nycodenz gradient Seal the tube with a cap and make sure to avoid air bubbles Centrifuge at

142,000 × g for 85 min at 8 °C in a Beckman VTI50 or a Sorvall

TV-850 vertical rotor with slow acceleration/deceleration Remove the cap from the centrifugation tube and collect frac-tions (1.25 mL) in an Eppendorf tube from the bottom of the

peroxisomes and provides very reliable data The catalase assay is usually carried out at 0 °C However, since catalase activities in the subcellular fractions and gradient fractions from brain tissues are much lower compared to liver and kidney, the assay is carried out

at 37 °C Dilute samples with TVBE buffer Keep the substrate solution at 0 °C

soda-lime culture tubes; 75 × 10 × 0.6) in an ice bath

set up a reagent control using TVBE and Triton X-100 Perform assays in duplicate

3 Mix well and incubate for at least 1 min

4 Add 1 mL of substrate solution (substrate should be chilled to

0 °C), adding the substrate solution to successive tubes at timed 10 s intervals Incubate each sample for exactly the same time at 0 °C

the solution to successive tubes at timed 10 s intervals

6 Vortex immediately and transfer the glass tubes to room temperature

7 Wait for at least 10 min for full color development

absorbance of the samples and the reagent control at 410 nm

Trang 35

around 1–1.3

9 Calculate the activity according to the following formula:

Enzyme activity (BU/mL) = (1 + x mL Sample + x mL Triton

X-100)/50 × 1/(incubation time in min) × 1/(Sample volume in mL) × log (Reagent control OD/Sample OD) × Dilution factor

sub-strate solution The assay is performed at 25 °C

2 Measure the absorption at 420 nm directly after starting the reaction for at least 2 min

3 Calculate the activity according to the following formula:

1/3.06 × Reaction volume (mL)/Sample volume (mL) × Dilution factor

The activity of glutamate dehydrogenase is measured according to [14]

1 Bring the reaction buffer to room temperature (25 °C) and carry out all operations at this temperature

and mix well

3 Blank the spectrophotometer against air Record the bance at 340 nm until the endogenous rate disappears, and

4 Mix well and continue to record the absorbance at 340 nm until a linear increase in value can be measured over a period of 1–2 min

5 Calculate the activity according to the following formula:Enzyme activity (U/mL) = Rate × Reaction volume (mL)/Sample volume (mL) × 1/6.22 × Dilution factor

Phosphoglucose isomerase is used as a marker for the cytosolic

1 Bring the reaction buffer to room temperature (25 °C) and carry out all operations at this temperature Use 0.1 M Tris buffer (pH 8.0) to dilute samples

Isomerase for Cytosol

Trang 36

3 Blank the spectrophotometer against air Record the bance at 340 nm until a linear increase in value can be mea-sured over a period of 1–2 min

4 Calculate the activity according to the following formula:Enzyme activity (U/mL) = Rate × Reaction volume (mL)/

3.1.4.37, CNPase), a marker enzyme for myelin, is assayed

pretreat-ment of samples using detergents resulted in an increase in the measured specific activity of the enzyme and allowed more repro-ducible results to be obtained

1 Activation of samples (subcellular fractions and gradient

Triton X-100 Incubate for 10 min at 0–4 °C Dilute with water so that the enzyme activity can be determined using

4 Centrifuge the reaction mixture at ~16,000 × g for 10 min.

plate and develop for about 4 h in 80:18:2 (v/v/v) saturated

6 Dry the TLC plate and visualize the spots under UV light, and

pen-cil Scrape the circled areas and transfer into small test tubes

7 Dissolve the scraped spots in 1–2 mL of 10 mM HCl by vortex mixing for 5 s, centrifuge to pellet the cellulose, and measure the absorption of the supernatant fractions at 260 nm The absorbance values are corrected for the blank cellulose spot absorbance

pro-tein Calculate the activity according to the following formula:

× 1/t [min] × 1/protein [mg]

3.3.5 2 ′,3′-Cyclic

Nucleotide

3 ′-Phosphodiesterase

(CNPase) Assay for Myelin

Trang 37

To verify the relative purity of the organelles, subject equal umes of the gradient fractions to SDS-polyacrylamide gel electro-phoresis and transfer to nitrocellulose membranes To characterize the subcellular fractions, subject equal amounts of protein to SDS- polyacrylamide gel electrophoresis After blocking for 1 h in a Tris- buffered saline containing 0.05% Tween 20 and 1% bovine serum albumin, probe membranes with indicated antibodies listed in

second-ary antibodies conjugated to horseradish peroxidase and visualize using enhanced chemiluminescence

4 Notes

marker enzyme determinations Determine the volume of each supernatant and resuspended pellet to be able to calculate total enzyme content

3.4 Immunoblotting

for Purity Validation

Table 1

Organelle marker antibodies for western blot analysis of subcellular and Nycodenz gradient fractions

Peroxisomes Pex14p Rabbit 1:1000 10594-1-AP, Proteintech

Peroxisomes Catalase Rabbit 1:8000 219010, Calbiochem

Peroxisomes PMP70 Sheep 1:1000 Gift from S Gould

Peroxisomes/Cytosol Pex5p Rabbit 1:1000 BD 6115941

Mitochondria Vdac Rabbit 1:5000 AB10527, Millipore

Mitochondria Tom20 Rabbit 1:1000 sc11415, Santa Cruz

Mitochondria Trap1 Mouse 1:2000 sc-135944, Santa Cruz

Endoplasmic reticulum Grp78 Goat 1:500 sc-1051, Santa Cruz

Endoplasmic reticulum Grp94 Rat 1:200 RT-102-P1, Neomarkers

Myelin CNPase Mouse 0.5 μg/mL MAB326R, Chemicon

Cytosol Hsp90 Mouse 1:1000 ADI-SPA-830-D, Enzo Life Sciences

Trang 38

4 For an optimal separation of peroxisomes from mitochondria and

ER do not use more than 1 g and not less than 600 mg tissue

5 Try to avoid air bubbles

6 This is a crucial step, since peroxisomes are fragile and if the let is resuspended too harshly the peroxisomes will break up

7 Measure catalase activity in the collected gradient fractions the

8 Dilute the samples in TVBE buffer to a concentration, which avoids a complete discoloring of the reaction solution and to obtain absorbance values between 0.350 and 0.850 The sam-ple volume can be increased if necessary, but always use the same volume of sample and 2% Triton X-100

reaction buffer

10 We recommend performing the assay in duplicate with either two different protein concentrations or two different incuba-tion times

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

References

1 Raymond GV, Watkins P, Steinberg S, Powers

J (2009) Peroxisomal disorders In: Lajtha A,

Tettamanti G, Goracci G (eds) Handbook of

neurochemistry and molecular neurobiology:

neural lipids Springer Science+Business

Media, Berlin, Germany, pp 631–670

2 Berger J, Dorninger F, Forss-Petter S, Kunze M

(2015) Peroxisomes in brain development and

function Biochim Biophys Acta 1863:934–955

3 Adamo AM, Aloise PA, Pasquini JM (1986) A

possible relationship between concentration of

microperoxisomes and myelination Int J Dev

Neurosci 4:513–517

4 Arnold G, Holtzman E (1978) Microperoxisomes

in the central nervous system of the postnatal rat

Brain Res 155:1–17

5 Kovacs WJ, Faust PL, Keller GA, Krisans SK

(2001) Purification of brain peroxisomes and

7 Völkl A, Fahimi HD (1985) Isolation and terization of peroxisomes from the liver of normal untreated rats Eur J Biochem 149:257–265

8 Biardi L, Sreedhar A, Zokaei A, Vartak NB, Bozeat RL, Shackelford JE, Keller GA, Krisans

SK (1994) Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders

J Biol Chem 269:1197–1205

9 Walter KM, Schönenberger MJ, Trötzmüller

M, Horn M, Elsässer H-P, Moser AB, Lucas

MS, Schwarz T, Gerber PA, Faust PL, Moch

H, Köfeler HC, Krek W, Kovacs WJ (2014) Hif-2 α promotes degradation of mammalian peroxisomes by selective autophagy Cell Metab 20:882–897

Trang 39

10 Chantrenne H (1955) Effects of a catalase

inhibitor on the induced formation of catalase

in yeast Biochim Biophys Acta 16:410–417

11 Baudhuin P, Beaufay H, Rahman-Li Y,

Sellinger OZ, Wattiaux R, Jacques P, De Duve

C (1964) Tissue fractionation studies 17

Intracellular distribution of monoamine

oxi-dase, aspartate aminotransferase, alanine

ami-notransferase, d-amino acid oxidase and

catalase in rat-liver tissue Biochem

J 92:179–184

12 Patel CC, Mohan MS (1960) Nature of the

colour-forming species in peroxy titanium

sul-phate Nature 186:803–804

13 Beaufay H, Amar-Costesec A, Feytmans E,

Thinès-Sempoux D, Wibo M, Robbi M,

Berthet J (1974) Analytical study of somes and isolated subcellular membranes from rat liver: I Biochemical methods J Cell Biol 61:188–200

14 Schmidt E (1974) Methods of enzymatic ysis In: Bergmeyer HV(ed), 2nd English edn, vol 2 Verlag-Chemie, Weinheim, Germany,

anal-pp 650–656

15 Noltmann EA (1966) Phosphoglucose erase: I Rabbit muscle (crystalline) Methods Enzymol 9:557–565

16 Sprinkle TJ, McMorris FA, Yoshino J, DeVries

GH (1985) Differential expression of

2 ′:3′-cyclic nucleotide 3′-phosphodiesterase in cultured central, peripheral, and extraneural cells Neurochem Res 10:919–931

Trang 40

Chapter 3

Determining the Topology of Peroxisomal Proteins

Using Protease Protection Assays

Tânia Francisco, Ana F Dias, Ana G Pedrosa, Cláudia P Grou,

Tony A Rodrigues, and Jorge E Azevedo

Abstract

Protease protection assays are powerful tools to determine the topology of organelle proteins Their simplicity, together with the fact that they are particularly suited to characterize endogenous proteins, are their major advantages and the reason why these assays have been in use for so many years Here, we provide a detailed protocol to use with mammalian peroxisomes Suggestions on how these assays can be controlled, and how to identify some technical pitfalls, are also presented.

Key words Peroxisome, Peroxisomal proteins, Protease protection assay, Protein topology,

Proteinase K

1 Introduction

Knowledge on the topology of peroxisomal proteins is crucial to understand their function This is valid not only for enzymes, but also for all proteins involved in the biogenesis/structure of the organelle Only then can one know whether a given enzyme acts

on the cytosolic or organelle pools of its substrate or on which side

of the peroxisomal membrane does a given protein-protein action take place

inter-Many different strategies can be used to define the topology of these proteins For instance, in the case of mammalian peroxisomes immunofluorescence techniques using semi-permeabilized cells

antibody directed to a protein of interest is available, a simple tease protection assay using an organelle suspension can also pro-

are several-fold First, it is very simple Indeed, besides requiring just a few specific reagents (i.e., a protease and a protease inhibitor) and common protein analysis equipment (e.g., SDS-PAGE and

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1. Manning G, Whyte DB, Martinez R et al (2002) The protein kinase complement of the human genome. Science 298(5600):1912–	Khác
4. Hunter T (2012) Why nature chose phosphate to modify proteins. Philos Trans R Soc Lond BBiol Sci 367(1602):2513–2516. doi:10.1098/rstb.2012.0013	Khác
5. Oeljeklaus S, Schummer A, Mastalski T et al (2016) Regulation of peroxisome dynamics by phosphorylation. Biochim Biophys Acta.doi:10.1016/j.bbamcr.2015.12.022	Khác
6. Jung S, Marelli M, Rachubinski RA et al (2010) Dynamic changes in the subcellular distribu- tion of Gpd1p in response to cell stress. J Biol Chem 285(9):6739–6749. doi:10.1074/jbc.M109.058552	Khác
7. Knoblach B, Rachubinski RA (2010) Phosphorylation-dependent activation of per- oxisome proliferator protein PEX11 controls Peroxisomal Protein Phosphorylation	Khác