Functional characterization of the human peroxins PEX3 and PEX19, proteins essential for early peroxisomal membrane biogenesis [Elektronische Ressource] / Peter Uli Mayerhofer

Peter Mayerhofer

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2003
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

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Institut für Experimentelle Genetik,
GSF-Forschungszentrum für Umwelt und Gesundheit, Neuherberg
Dr. von Haunersches Kinderspital
Functional Characterization of the Human
Peroxins PEX3 and PEX19, Proteins Essential for
Early Peroxisomal Membrane Biogenesis
Peter Uli Mayerhofer
Vollständiger Abdruck der von der
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
der Technischen Universität München
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
genehmigten Dissertation.
Vorsitzender: Univ.-Prof. Dr.rer.nat. Alfons Gierl
Prüfer der Dissertation: 1. Priv.-Doz. Dr.rer.nat. Dr.rer.biol.hum.habil. Jerzy Adamski
2. Univ.-Prof. Dr.rer.nat. Hans-Werner Mewes
3. Univ.-Prof. Dr.med. Adelbert A. Roscher,
Ludwig-Maximilians-Universität München
Die Dissertation wurde am 11.07.2002 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 01.03.2003 angenommen.TABLE OF CONTENTS i
Table of Contents
Abstract ................................................................................................................................iii
Zusammenfassung .................................................................................................................v
CHAPTER I: Introduction ..................................................................................................... 1
1 Peroxisomes: Morphology and Metabolic Functions ................................................... 2
2 Peroxisomal Diseases.................................................................................................. 5
2.1 X-Linked Adrenoleukodystrophy: A Peroxisomal Single Enzyme deficiency......... 6
2.2 Peroxisomal Biogenesis Disorders............................................................................ 8
3 The Peroxisomal Biogenesis ..................................................................................... 10
3.1 Targeting and Import of Matrix Enzymes ............................................................... 13
3.2 Peroxisomal Membrane Biogenesis........................................................................ 17
3.3 PEX3 and PEX19, Peroxins Expected to be Involved in the Early Steps of
Peroxisomal Biogenesis ......................................................................................... 20
CHAPTER II: Aims of the Thesis....................................................................................... 22
CHAPTER III: Results and Discussion ................................................................................ 24
1 PEX19 Interacts With a Variety of Peroxisomal Membrane Proteins......................... 25
2 Characterization of PEX19 Splice Variants: Functional Diversity
and New Insights in the Role of Posttranslational Farnesylation ............................. 27
3 Proposed Biological Functions of PEX19.................................................................. 30
4 PEX3, a Key Factor in Early Human Peroxisome Synthesis ...................................... 32
5 Two-Path Model for Peroxisome Membrane Synthesis in Mammalian Cells ............. 35
6 Further Perspectives: Identification of the Origin of the Endomembrane Template.... 37
References........................................................................................................................... 38
Appendix 1: Full Length cDNA Cloning, Promoter Sequence, and Genomic Organization
of the Human Adrenoleukodystrophy Related (ALDR) Gene Functionally
Redundant to the Gene Responsible for X-Linked Adrenoleukodystrophy....... 50
Appendix 2: Human Adrenoleukodystrophy Protein and Related Peroxisomal ABC
Transporters Interact with the Peroxisomal Assembly Protein PEX19p ........... 63
Appendix 3: Two Splice Variants of Human PEX19 Exhibit Distinct Functions
in Peroxisomal Assembly ................................................................................ 77
Appendix 4: Genomic Organization, Expression Analysis, and Chromosomal Localization
of the Mouse PEX3 Gene Encoding a Peroxisomal Assembly Protein ............. 90TABLE OF CONTENTS ii
Appendix 5: The Human PEX3 Gene Encoding a Peroxisomal Assembly Protein:
Genomic Organization, Positional Mapping, and Mutation Analysis
in Candidate Phenotypes ............................................................................... 100
Appendix 6: Defective Peroxisome Membrane Synthesis Due to Mutations
in Human PEX3 Causes Zellweger Syndrome, Complementation Group G ... 112
Abbreviations .................................................................................................................... 125
Danksagung....................................................................................................................... 126
List of Publications............................................................................................................ 127
Curriculum Vitae ............................................................................................................... 130ABSTRACT iii
AbstractABSTRACT iv
Abstract
Peroxisomes are single-membrane-bound organelles present in virtually all eukaryotic cells.
Their significance in human metabolism is illustrated by the existence of severe inherited
diseases caused by the failure of peroxisomal biogenesis (peroxisomal biogenesis disorders;
PBDs). Proteins required for peroxisomal assembly are termed “peroxins” and are encoded by
at least 23 PEX-genes. In most cases, defects in PEX genes lead to a disruption of
peroxisomal matrix protein import, whereas various peroxisomal membrane components are
synthesized and accumulate in peroxisomal membrane remnants (“peroxisomal ghosts”).
Since yeast mutants in PEX3 and PEX19 have been shown to lack peroxisomal ghosts, these
peroxins were expected to be involved in the early stages of peroxisomal membrane synthesis.
This work focused on the functional characterization of human PEX3 and PEX19 to gain
insights into their role in human biology and disease. PEX19 was shown to interact with three
peroxisomal ABC half-transporters (Adrenoleukodystrophy protein ALDP,
Adrenoleukodystrophy-related protein ALDRP, and the 70 kDa peroxisomal membrane
protein PMP70), confirming its role as a broad specific peroxisomal membrane-protein
binding-protein. As a prerequisite for these protein-protein interaction assays, the human
ALDR gene was characterized. Proteins encoded by two PEX19 splice variants showed a
considerable functional diversity as to peroxisomal membrane protein binding and with
respect to induction of peroxisomal formation in PEX19-deficient human fibroblasts.
Farnesylation of PEX19 was shown not to be essential for these functions. These data provide
the first experimental evidence for specific biological functions of the different predicted
domains of the PEX19 protein. In order to investigate the molecular details of peroxisomal
assembly and to evaluate experimental treatment strategies for the peroxisomal biogenesis
disorders, data useful for the future generation of a mouse model with targeted disruption of
the PEX3 gene were presented. The human PEX3 gene was characterized with the goal to
identify a yet unknown human PEX3-deficient phenotype. Two inactivating PEX3 mutations
were detected in two PBDs-patients exhibiting a severe and lethal Zellweger syndrome
phenotype. They showed a total lack of morphologically recognizable peroxisomal membrane
remnants in their fibroblasts. Expression of the wild type PEX3 cDNA in the mutant cell lines
restored peroxisomal biogenesis, establishing PEX3 as a key factor in early human
peroxisome synthesis. Taken together, the combined data presented here provide evidence for
the essential role of PEX3 and PEX19 in the initial steps of an alternative human peroxisome-
formation pathway. In contrast to the theory that peroxisomes can arise exclusively by growth
and division of preexisting peroxisomes, this alternative pathway does not require
morphologically recognizable peroxisomal membranes.ZUSAMMENFASSUNG v
Zusammenfassung