An ancestral role in de novo peroxisome assembly is retained by the divisional peroxin Pex11 in

303 Pages·2014·62.68 MB·English

by  Mary Klute

An ancestral role in de novo peroxisome assembly is retained by the divisional peroxin Pex11 in
the yeast Yarrowia lipolytica

by

Mary Jessica Klute

A thesis submitted in partial fulfillment of the requirements for the degree of

Master of Science

Department of Cell Biology
University of Alberta

© Mary Jessica Klute, 2014
Abstract
This thesis is on peroxisome evolution and reports my findings with regard to two major aspects of
this process. First, I investigated the evolution of a peroxisomal protein family for which large
expansions had been noted in diverse eukaryotes from a comparative genomic perspective and
combined these findings with functional data delving into the evolutionary history of this protein
family. Second, I studied the peroxisomal protein complement of three newly available eukaryotic
genomes.
The peroxin (protein required for peroxisome biogenesis) Pex11p has a well recognized
role in peroxisome division in diverse eukaryotes. Pex11p remodels and elongates the membranes
of peroxisomes prior to the recruitment of dynamin-related GTPases that act in membrane scission
to physically divide peroxisomes. I performed a comprehensive comparative genomics survey to
understand the significance of the evolution of the Pex11 protein family in yeast and other
eukaryotes. Pex11p itself is highly conserved and ancestral, and has undergone numerous lineage-
specific duplications, while other Pex11 protein family members are fungal-specific innovations.
Functional characterization of the in silico predicted Pex11 protein family members of the yeast
Yarrowia lipolytica, i.e. Pex11p, Pex11Cp and Pex11/25p, demonstrated that Pex11Cp and
Pex11/25p function in the regulation of peroxisome size and number characteristic of Pex11
protein family members. Unexpectedly, deletion of PEX11 in Y. lipolytica produces cells that lack
morphologically identifiable peroxisomes, mislocalize peroxisomal matrix proteins, and show
preferential degradation of peroxisomal membrane proteins, i.e. they exhibit the classical pex
mutant phenotype, which has not been reported for any other eukaryotic cell deleted for the
PEX11 gene. My results demonstrate an unprecedented role for Pex11p in de novo peroxisome
assembly.
I also studied the peroxisomal protein complement of three medically and/or evolutionarily
relevant eukaryotic genomes: the intestinal parasite Blastocystis hominis, Bodo saltans, a close relative
of the parasitic trypanosomatids which contains a divergent peroxisome known as the glycosome,
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and Naegleria fowleri, better known as the ‘brain-eating amoeba’. I showed that Blastocystis lacks
peroxisomal proteins, consistent with parasites generally lacking the peroxisome organelle. I
demonstrated that B. saltans and N. fowleri encode a relatively complete PEX gene complement,
with certain losses. I wrote a program, PTS Finder, to predict proteins targeted to the peroxisome,
and used it during these analyses.

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Preface
Chapters 1 and 3 of this thesis have been submitted for publication as:
Klute, M.J., Chang, J., Tower, R.J., Mast, F.D., Dacks, J.B., and R.A. Rachubinski. 2014. An ancestral role
in de novo peroxisome assembly is retained by the divisional peroxin Pex11 in the yeast Yarrowia
lipolytica. J. Cell Sci.

M.J. Klute, J. Chang, R.J. Tower, and F.D. Mast performed the experiments. M.J. Klute, J. Chang, R.J.
Tower, F.D. Mast, J.B. Dacks, and R.A. Rachubinski provided a conceptual framework for
the study, interpreted data and wrote the manuscript.
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Dedication
For Mom and Dad.
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Acknowledgements
Firstly, I would like to thank my co-supervisors, Dr. Richard Rachubinski and Dr. Joel Dacks. I
appreciate all of the opportunities that you have given me. Thank-you for allowing me to share in
your passion for basic science research.
Rick, it has been a privilege to be a part of your lab for the past three years. I feel like I am
leaving my graduate program as a much more confident and opinionated scientist and person.
Thank-you for allowing me the freedom to explore various aspects of this project.
Joel, I am truly grateful for your guidance not only throughout my graduate program, but
also throughout my three summer research projects, three undergraduate research courses, and
undergraduate degree. Your constant encouragement and words of faith in my abilities have been
very much appreciated. I would not be where I am today without you.
I would also like to thank Dr. Kinga Kowalewska-Grochowska for serving on my supervisory
committee, and Dr. Andrew Simmonds and Dr. Paul LaPointe for serving on my thesis defense
committee.
Words cannot express how grateful I am for the friendship of Emily Herman. I consider
myself very lucky to have worked alongside you throughout our undergraduate and graduate
studies. Thank-you very much for all of your advice, whether it be science-related or not.
Fred Mast, I truly appreciate your involvement in and support throughout my graduate
program, and everything you have taught me. Thank-you for always being willing to take the time to
answer my questions, assist with my experiments, and provide helpful discussion and insights.
Thank-you for being such an incredible mentor in both my academic and personal life.
I would like to thank all of the members of the Rachubinski lab, including Hiren Banerjee,
Jenny Chang, Barbara Knoblach, Hanna Kroliczak, David Lancaster, Sandrine Lepine, Fred Mast, Rick
Poirier, Elena Savidov and Rob Tower. Thank-you for your assistance with experimental
troubleshooting and for keeping the lab running smoothly. In particular, I would like to thank Rob
Tower for initial training and proposing many of the intial experiments for my graduate project, as
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well as my benchmate, Jenny Chang, for answering many questions and being a great resource for
everything Yarrowia-related.
I would also like to thank the members the Dacks lab, including Maria Aguilar González,
Lael Barlow, Emily Herman, Chris Klinger, Alex Schlacht and Jeremy Wideman. Your enthusiam for
science is both contagious and inspiring. It has been a pleasure working with all of you.
I would like to acknowledge the Department of Cell Biology, as well as the financial support
provided by the Faculty of Medicine and Dentistry, the Faculty of Graduate Studies and Research,
Alberta Health Services and the Government of Alberta.
My final thanks goes to my family: Mom, Dad and Stephen. I truly appreciate your support
of all of my academic endeavours and your involvement in everything I do.

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Technical Acknowledgements
The genome of Bodo saltans was analyzed with Dr. Fred Mast, Seattle Biomedical Research Institute.
The PTS Finder program was written in collaboration with Emily Herman, Department of Cell
Biology, University of Alberta. Affinity purification of antibodies against Pex2p was performed by
Elena Savidov, Department of Cell Biology, University of Alberta.

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Table of Contents
Chapter 1: Introduction...................................................................................................................................1
1.1 Introduction to peroxisomes........................................................................................................................................2
1.2 PEX genes are necessary for peroxisome biogenesis.....................................................................................2
1.3 Peroxisome biogenesis disorders...............................................................................................................................4
1.4 Peroxisome matrix protein import...........................................................................................................................6
1.5 Peroxisome membrane protein import..................................................................................................................8
1.6 De novo synthesis from the ER..................................................................................................................................10
1.7 Regulation of the peroxisome population..........................................................................................................11
1.8 Peroxisome division........................................................................................................................................................12
1.8.1 Role of the Pex11 protein family in peroxisome division...................................................................12
1.8.2 Role of other peroxins in the regulation of peroxisome size and number...............................19
1.8.3 Constriction and scission of peroxisomes....................................................................................................19
1.8.4 Transcriptional regulation of peroxisome proliferation in yeasts....................................................20
1.8.5 Transcriptional regulation of peroxisome proliferation in other eukaryotes.............................24
1.9 Introduction to eukaryotic diversity.......................................................................................................................26
1.9.1 Introduction to Blastocystis hominis..............................................................................................................31
1.9.2 Introduction to Bodo saltans.......................................................................................................................32
1.9.3 Introduction to Naegleria fowleri....................................................................................................................32
1.10 Diverse peroxisomes for diverse eukaryotes...................................................................................................34
1.11 Peroxisome evolution....................................................................................................................................................34
1.12 Evolution of PEX genes.................................................................................................................................................36
1.13 Focus of this thesis...........................................................................................................................................................37
1.13.1 Specific Aim 1. Evolution of the Pex11 protein family, and functional characterization of
the Pex11 protein family in Yarrowia lipolytica...............................................................................................................37
1.13.2 Specific Aim 2. Comparative genomic survey of peroxisomal proteins in eukaryotic
genomes: Blastocystis hominis, Bodo saltans and Naegleria fowleri................................................................38
Chapter 2: Materials and methods...............................................................................................................39
2.1 Materials for molecular and cellular biology......................................................................................................40
2.2 Microorganisms and culture conditions...............................................................................................................48
2.2.1 Bacterial strains and culture conditions........................................................................................................48
2.2.2 Yeast strains and culture conditions...............................................................................................................48
2.3 DNA manipulation and analysis...............................................................................................................................51
2.3.1 Amplification of DNA by the polymerase chain reaction (PCR)....................................................51
2.3.2 Digestion of DNA by restriction endonucleases......................................................................................52
2.3.3 Dephosphorylation of 5’-ends............................................................................................................................52
2.3.4 Blunting 5’- and 3’-ends with DNA Polymerase I, Large (Kleenow) Fragment.....................52
2.3.5 Separation of DNA fragments by agarose gel electrophoresis.......................................................53
2.3.6 Purification of DNA fragments from agarose gel....................................................................................53
2.3.7 Purification of DNA fragments from solution............................................................................................53
2.3.8 Ligation of DNA fragments..................................................................................................................................53
2.3.9 DNA sequencing........................................................................................................................................................54
2.4 Introduction of DNA into microorganisms.......................................................................................................54
2.4.1 Chemical transformation of E. coli..................................................................................................................54
2.4.2 Chemical transformation of Y. lipolytica.....................................................................................................55
2.4.3 Electroporation of Y. lipolytica..........................................................................................................................55
2.5 Isolation of DNA from microorganisms..............................................................................................................56
2.5.1 Isolation of plasmid DNA from bacteria......................................................................................................56
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2.5.2 Isolation of chromosomal DNA from yeast................................................................................................56
2.6 Protein manipulation and analysis............................................................................................................................57
2.6.1 Preparation of yeast whole cell lysates.........................................................................................................57
2.6.2 Precipitation of proteins.........................................................................................................................................58
2.6.3 Determination of protein concentration.......................................................................................................58
2.6.4 Separation of proteins by electrophoresis...................................................................................................59
2.6.5 Detection of proteins by gel staining..............................................................................................................59
2.6.6 Detection of proteins by immunoblotting....................................................................................................59
2.7 Affinity purification of polyclonal antibodies......................................................................................................60
2.8 Subcellular fractionation................................................................................................................................................62
2.8.1 Peroxisome isolation from Y. lipolytica........................................................................................................62
2.8.2 Extraction and subfractionation of peroxisomes.....................................................................................64
2.9 Electron microscopy........................................................................................................................................................64
2.10 Microscopy...........................................................................................................................................................................66
2.10.1 3D confocal microscopy of living yeast.........................................................................................................66
2.10.2 Deconvolution and image processing............................................................................................................67
2.10.3 Quantification of peroxisome number...........................................................................................................67
2.11 Comparative genomic survey....................................................................................................................................67
2.11.1 Comparative genomic survey of the Pex11 protein family...............................................................67
2.11.2 Comparative genomic survey of Blastocystis hominis........................................................................75
2.11.3 Comparative genomic survey of Bodo saltans........................................................................................76
2.10.2 Comparative genomic survey of Naegleria fowleri..............................................................................78
2.12 Alignment..............................................................................................................................................................................78
2.12.1 Use of ZORRO in alignment masking..........................................................................................................78
2.13 Phylogenetic analysis.......................................................................................................................................................79
Chapter 3: An ancestral role in de novo peroxisome assembly is retained by the divisional
peroxin Pex11 in the yeast Yarrowia lipolytica............................................................................................80
3.1 A comparative genomic survey of the Pex11 protein family..................................................................81
3.2 Use of ZORRO for masking multiple sequence alignments....................................................................85
3.3 Evolution of the Pex11 protein family..................................................................................................................88
3.4 Conserved features of the Y. lipolytica Pex11 protein family................................................................100
3.4.1 The Y. lipolytica PEX11 family contains putative Far binding sites........................................103
3.4.2 Putative phosphorylation sites in the Y. lipolytica Pex11 protein family............................105
3.5 Deletion of the Y. lipolytica PEX11 family genes..........................................................................................106
3.6 Y. lipolytica Pex11 protein family members are peroxisomal integral membrane proteins
....................................................................................................................................................................................................................109
3.7 Peroxisomes are absent in pex11Δ cells, and are larger and fewer in number in pex11C Δ
delta and pex11/25Δ cells...........................................................................................................................................................112
3.8 Overexpression of PEX11C and PEX11/25 results in cells with increased number of smaller
peroxisomes........................................................................................................................................................................................118
3.9 pex11Δ cells exhibit abnormal localization of peroxisomal matrix and membrane proteins
....................................................................................................................................................................................................................118
3.10 Expression of PEX11, but not PEX11C or PEX1/25, complements the peroxisome
assembly defects of pex11Δ cells...........................................................................................................................................124
3.11 Pex11p, Pex11Cp and Pex11/2p are mislocalized in pex3Δ, pex16Δ and pex19Δ cells....124
3.12 Peroxisomal membrane proteins are mislocalized in pex11Δ cells..................................................132
3.13 Discussion...........................................................................................................................................................................132
Chapter 4: Comparative genomic survey of peroxisomal peroxisomal proteins in eukaryotic
genomes: Blastocystis hominis, Bodo saltans and Naegleria fowleri..........................................................144
4.1 PTS Finder: a new program to predict PTS-containing proteins........................................................145
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