Exploring the roles of FK506-binding proteins in chloroplast function and plant development

Peter J. Gollan

BSc (Hons)

This thesis is presented for the degree of Doctor of Philosophy

December 2010

Environment and Biotechnology Centre

Faculty of Life and Social Sciences

Swinburne University of Technology

Melbourne, Australia

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Abstract

The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl cis/trans isomerase

(PPIase) enzymes found in all organisms, comprising large protein families that are

localised throughout the cell. FKBPs possess a well conserved FK506-binding domain

that forms the active site for PPIase activity, although many isoforms are PPIase-

deficient. The FKBP families of higher plants include over twenty members with

established roles as molecular chaperones in hormone trafficking and stress response.

The chloroplast thylakoid contains half of the plant FKBP population, which have been

tentatively linked to regulating photosynthetic membrane function. The aim of this

study was to characterise the FKBP families occurring in wheat and other cereals, with

particular focus on elucidating their functions in the thylakoid.

Identification of the FKBP multigenes in the rice genome uncovered the largest FKBP

family so far reported. Bioinformatics characterisation of their proteins revealed unique

isoforms in rice and showed that gene duplications have driven expansion of the plant

FKBP families. Ten isoforms predicted in the thylakoid of rice, wheat and other cereals

were highly conserved with their counterparts in other plants, suggesting specialised

and conserved roles for this subfamily. Of these, FKBP13, FKBP16-1 and FKBP16-3

were isolated from the wheat genome and found to be expressed in leaf tissue.

Transcription regulation analyses identified a novel promoter element that linked

FKBP16-1 with chloroplast biogenesis, while FKBP16-3 expression indicated a role in

carbohydrate synthesis. In vitro PPIase assays demonstrated an absence of enzyme

activity in both FKBP16-1 and FKBP16-3, suggesting alternative roles in the thylakoid

for these isoforms, while FKBP13 was shown to be the first active PPIase reported in a

cereal chloroplast.

FKBP13, FKBP16-1 and FKBP16-3 were found to interact with specific chloroplast

proteins in yeast two-hybrid analysis, indicating roles as specialised chaperones in the

thylakoid that may serve to regulate photosynthetic acclimation processes. Mechanisms

linking these FKBPs with photosynthetic state transitions and cyclic electron flow

around photosystem one are described here, and a hypothesis linking FKBP function to

protein phosphorylation is suggested.

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“One should never underestimate the unimportance of almost everything”

Daniel Beaty

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Acknowledgments

Although I am the sole author accredited for this work, many people have contributed to

its completion in many different ways, and I am pleased to acknowledge some of most

valuable contributions here.

I am especially grateful to my supervisor Professor Mrinal Bhave, for inspiring me to

take on this project and for teaching me the beautiful art of creative research. I sincerely

appreciate her generosity with time, her enthusiastic reception of my (often wild and

fantastical) postulations, and her detailed and engaging critique of my work. She

tirelessly motivated me to dig a little deeper, to think a little harder, to give a little more,

and that has enriched the quality of this work.

To my co-supervisor Dr. Patrick Romano, for inventing opportunities for me that have

been, and continue to be simply priceless.

This study was made possible through financial support from the Grains Research and

Development Corporation (GRDC), to whom I am grateful.

I am grateful to the Faculty of Life and Social Sciences at Swinburne University of

Technology, where I learned to be a scientist and where I carried out this work. I truly

appreciate the Faculty’s support through the funding and resources provided to me. I

owe particular thanks to Chris Key, Soula Mougos and Ngan Nguyen for technical

support during my research.

I am fortunate to have shared my PhD years with many exceptional fellow candidates

and I have benefited from their experience, expertise, perspective and support.

Outstanding in their contributions were Dr. Mark Ziemann, Sarah McLean, Dr. Huimei

Wu, Shee Ping Ng and Dr. Kerrie Forrest.

I was privileged to conduct part of my PhD research in the Horton labs at the

Department of Molecular Biology and Biochemistry at the University of Sheffield. I am

forever indebted to Professor Peter Horton for creating a place for me in his group and

for his generous support of my work. I am also grateful to Dr. Paul Davison, Pam

Scholes, Dr. Derren Heyes, Dr. Marisa Perez-Bueno and Dr. Daniel Kinsman for their

generosity with time and technical assistance in my work at Sheffield.

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Many thanks to Meisy and her team at Mario’s Hawthorn, for a happily endless supply

of that wonderful elixir of creativity and motivation.

Over the years, various experimental results, journal decisions, deadlines and quantities

of sleep have made my PhD experience at times edifying and exhilarating, and on other

occasions traumatic and tiring, and I have experienced all moods between these

extremes many times over. This has been sometimes difficult and taxing for my closest

friends and loved ones, from whom I have demanded patience, understanding and

perseverance. I am deeply humbled by the unwavering support received from Daniel

Beaty, Fiona Lynch, Julian Bettiol, Dr. Jaimey Tucker, Ciaran Bateman, Dr. Will

Ainslie, Fiona Sheil, Dr. Anett Kiss, Mum and Dad.

Finally to Jo Long, who has shared this journey with me and supported me ceaselessly

from beginning to end, and to whom I dedicate this thesis.

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Declaration

I, Peter Gollan, declare that the thesis entitled “Exploring the roles of FK506-binding

proteins in chloroplast function and plant development” is no more than 100,000 words

in length, exclusive of tables, figures, appendices, references and footnotes. This thesis

contains no material that has been submitted previously, in whole or in part, for the

award of any other academic degree or diploma, and has not been previously published

by another person. Except where otherwise indicated, this thesis is my own work.

Peter J. Gollan

December 2010

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Original publications arising from this work

Refereed journal articles

Gollan PJ, Bhave M. 2010 Genome-wide analysis of genes encoding FK506-binding

proteins in rice. Plant Mol. Biol. 72: 1-6

Gollan PJ, Bhave M. 2010 A thylakoid-localised FK506-binding protein in wheat may

be linked to chloroplast biogenesis. Plant Phys. Biochem. 48: 655-662

Book Chapters

Gollan PJ, Bhave M. 2009 The FK506-binding proteins (FKBPs) of the thylakoid:

Emerging roles in plant photosynthesis. In: Photosynthesis: theory and applications in

energy, biotechnology and nanotechnology TB Buchner, NH Ewingen (Eds) Nova

Publishing, New York 43-79

Conference proceedings

Thylakoid-localised FKBPs regulating photosynthetic membrane architecture. In: Proceedings of the 60th Australian Cereal Chemistry Conference 2010 Melbourne

Isolation of wheat FK506-binding proteins (FKBPs): Exploring roles for FKBPs in the wheat thylakoid. In: Proceedings of 2008 AACC International Meeting 2008 Honolulu

Introducing the cereal thylakoid FK506-binding proteins: little enzymes with a bright future. In: Proceedings of 58th Australian Cereal Chemistry Conference 2008 Gold Coast

Isolation and characterisation of the genes encoding a thylakoid FK506-binding protein in wheat. In: Proceedings of 58th Australian Cereal Chemistry Conference 2008 Gold Coast

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Abbreviations

Standard chemical symbols and SI units are used without definition. Full names of

genes and proteins are stated at first mention.

3AT 3-amino-1,2,4-triazole Ade adenine Ala (or A) alanine Arg (or R) arginine Asn (or N) asparagine Asp (or D) aspartic acid At Arabidopsis thaliana ATP adenosine triphosphate BLAST Basic Local Sequence Alignment Search Tool bp nucleotide base pair C-terminal peptide or protein carboxyl terminal cDNA complementary DNA CDS coding sequence Col-0 Columbia-0 Arabidopsis thaliana ecotype cv. cultivar CsA cyclosporin CYP cyclophilin Cys (or C) cysteine DNA deoxyribonucleic acid DNase deoxyribonuclease dNTP any deoxyribonucleotide dTTP (or dT) deoxythymidine triphosphate EDTA ethylenediaminetetraacetic acid ER endoplasmic reticulum EST expressed sequence tag FKBP FK506-binding protein FKBd FK506-binding domain g centrifugal force gDNA genomic DNA Gln (or Q) glutamine Glu (or E) glutamic acid Gly (or G) glycine His (or H) histidine HSP heat shock protein Hv Hordeum vulgare Ile (or I) Isoleucine IPTG isopropyl β-D-1-thiogalactopyranoside

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kb kilobase pair kDa kiloDaltons Leu (or L) leucine Lys (or K) lysine Met (or M) methionine mRNA messenger RNA MW molecular weight n ploidy N-terminal protein or peptide amino terminal NADH nicotinamide adenine dinucleotide NADPH nicotinamide adenine dinucleotide phosphate OD optical density ORF open reading frame Os Oryza sativa PAGE polyacrylamide gel electrophoresis PCR polymerase chain reaction Phe (or F) phenylalanine Pro (or P) proline PSI photosystem I PSII photosystem II PPIase peptidyl prolyl cis/trans isomerase RNA ribonucleic acid RNase ribonuclease RT PCR reverse transcriptase polymerase chain reaction Sc Saccharomyces cerevisiae SDS sodium dodecyl sulphate Ser (or S) serine SQ semi-quantitative SV splice variant Ta Triticum aestivum Tat twin-argnine motif Thr (or T) threonine TM transmembrane TPR tetratricopeptide Tris tris(hydroxymethyl)aminomethane Trp (or W) tryptophan Tyr (or Y) tyrosine UTR untranslated region Val (or V) valine Y2H yeast two-hybrid Zm Zea mays

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Table of contents

Chapter 1 General introduction and literature review 1

1.1 Discovering the immunophilins 2

1.1.2 Immunophilin-mediated immunosuppression 4

1.2 Isomerisation of prolyl bonds 5

1.2.1 Immunophilins are peptidyl prolyl cis/trans isomerase enzymes 5

1.2.2 Parvulins; the non-immunophilin PPIase 6

1.2.3 Mechanisms of prolyl bond rotation 6

1.2.4 Measuring PPIase activity 7

1.3 FKBP sequences, structures and domains 8

1.3.1 The FK506-binding domain: Conserved sequence and structure 8

1.3.2 The FKBP active site 10

1.3.3 Additional domains in FKBPs 11

1.4 Analysing the FKBP multigene families 12

1.4.1 The FKBP families of Homo sapiens and other vertebrates 12

1.4.2 FKBPs in Saccharomyces cerevisiae and lower eukaryotes 17

1.4.2.1 Immunophilin knockout yeast 17

1.5 Immnunophilins in Arabidopsis thaliana and higher plants 20

1.5.1 FKBPs in plant development 20

1.5.2 FKBPs in plant heat stress response 25

1.5.3 FKBPs in transcription regulation 26

1.5.4 FKBPs in the chloroplast 27

1.5.5 The cyclophilin family in Arabidopsis thaliana 28

1.5.6 Cyclophilins in the chloroplast 28

1.5.7 Non-chloroplastic plant cyclophilins 29

1.6 Photosynthesis and the chloroplast 30

1.6.1 Photosynthetic electron flow 31

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1.6.2 Photosynthetic acclimation mechanisms 32

1.6.3 Thylakoid phosphorylation for acclimation 33

1.6.4 Thylakoid macrostructure 33

1.6.5 Chloroplast biogenesis 33

1.6.6 Molecular transport in and around the chloroplast 35

1.6.7 PPIase activity in the chloroplast 36

1.7 Introduction to wheat 37

1.7.1 Introduction to wheat cultivation 37

1.7.2 Photosynthesis in cereals 38

1.8 Project aims 38

Chapter 2 Materials and methods 41

2.1 Materials 42

2.1.1 Equipment 42

2.1.2 Commercial kits, reagents and materials 43

2.1.3 Prepared solutions and materials 44

2.1.3.1 Buffers and solutions 44

2.1.3.2 Media and solutions for growth of bacteria and yeast 46

2.1.4 Plant materials 47

2.1.5 Microbial strains 48

2.1.6 Cloning vectors 48

2.2 Methods 49

2.2.1 Plant propagation 49

2.2.2 General molecular methods 49

2.2.2.1 Genomic DNA extraction 49

2.2.2.2 RNA extraction from wheat plants 50

2.2.2.3 DNase treatment of RNA 51

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2.2.2.4 Single-stranded cDNA synthesis 51

2.2.2.5 Phenol/chloroform extraction and ethanol precipitation 51

2.2.2.6 Cloning 52

2.2.2.7 Plasmid purification 52

2.2.2.8 Restriction digestions 53

2.2.2.9 Purification of DNA from agarose gels 54

2.2.2.10 Protein gel electrophoresis 54

2.2.2.11 Protein concentration measurements 55

2.2.2.12 Western blotting 55

2.2.3 Polymerase chain reaction 56

2.2.3.1 Primer design 56

2.2.3.2 PCR conditions 56

2.2.3.3 Constructing wheat gDNA ligation libraries for inverse PCR 58

2.2.3.4 Inverse PCR conditions 59

2.2.3.5 Semi-quantitative reverse transcriptase PCR 60

2.2.4 DNA sequencing 61

2.2.5 Over-expression, purification and quantification of FKBPs 62

2.2.6 PPIase assays 63

2.2.7 Analyses of carbon partitioning in atfkbp16-3 Arabidopsis mutant 64

2.2.7.1 Measuring flux of 14C into carbohydrates 64

2.2.7.2. Measuring starch accumulation during the photoperiod 65

2.2.8 Analysis of thylakoid membrane complexes 66

2.2.8.1 Thylakoid isolation 66

2.2.8.2 Thylakoid membrane fractionation 66

2.2.9 Yeast two-hybrid methods 67

2.2.9.1 Transforming yeast with bait and prey vectors 67

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2.2.9.2 Yeast mating 68

2.2.9.3 Construction of a wheat leaf cDNA library 68

2.2.9.4 Screening the library with FKBP baits 71

2.2.9.5 Identifying strong interactions on 3AT plates 72

2.2.9.6 Amplification of library inserts using colony PCR 72

2.2.9.7 Segregation of library constructs for control reactions 72

2.3 Methods for computer-based analysis 73

2.3.1 Bioinformatics methods 74

2.3.1.1 Isolating sequences from genome databases 73

2.3.1.2 EST retrieval and coding sequence construction 74

2.3.1.3 Identification of protein domains 75

2.3.1.4 Signal peptide and phosphorylation prediction 75

2.3.1.5 Prediction molecular weight and isoelectric point of proteins 75

2.3.1.6 Protein 3D structure analysis 75

2.3.1.7 Microarrays 76

2.3.1.8 Identification of putative cis elements 76

2.3.2 Software 76

Chapter 3 Isolation and analysis of the FKBP families in cereals and other plants using bioinformatics techniques 78

3.1 Introduction 79

3.2 Results 80

3.2.1 Analysis of the FKBP multigene family in rice 88

3.2.1.1 Cytosolic FKBPs in rice 82

3.2.1.2 Nuclear FKBPs 88

3.2.1.3 FKBPs in the ER 89

3.2.1.4 FKBPs in the chloroplast 90

3.2.2 Analysis of putative active sites in the rice FKBPs 92

3.2.3 Thylakoid-localised FKBPs in cereals and other plants 95

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3.2.3.1 Paralogues among the lumenal FKBPs 97

3.2.3.1 Isolation of the thylakoid-localised FKBPs in wheat 99

3.2.4 Evolution of the thylakoid-localised FKBPs 101

3.2.4.1 FKBPs in cyanobacteria and their contribution to plant FKBP evolution 101

3.2.4. FKBPs in algae and their contribution to plant FKBP evolution 102

3.3 Discussion 104

3.3.1 Expansion of the rice FKBP family through gene duplication 104

3.3.2 Chaperone roles for FKBPs outside of PPIase activity 106

3.3.3 Explaining the evolution of the lumenal isoforms 107

3.3.3 Features and functions of the lumenal FKBPs 108

3.3.3.1 Conserved orthologues in a variable subfamily 108

3.3.3.2 Redox regulation through disulphide bonding 108

3.3.3.3 The lumenal FKBPs in T. aestivum; candidates for characterisation 109

Chapter 4 Characterising FKBP13, FKBP16-1 and FKBP16-3 genes in Triticum aestivum 109

4.1 Introduction 110

4.2 Results 111

4.2.1 Primer design for amplification of TaFKBP13, TaFKBP16-1 and TaFKBP16-3 genes from genomic DNA 111

4.2.2 FKBP13 genes isolated from T. aestivum 111

4.2.3 FKBP16-1 genes isolated from T. aestivum 117

4.2.4 FKBP16-3 genes isolated from T. aestivum 134

4.2.5 Analysis of the putative wheat FKBP protein sequences 142

4.2.5.1 Features of the translated TaFKBP13 CDS 142

4.2.5.2 Features of the translated TaFKBP16-1 splice variants 143

4.2.5.3 Features of the translated TaFKBP16-3 CDS 143

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4.3 Discussion 148

4.3.1 FKBP13, FKBP16-1 and FKBP16-3 proteins are conserved in wheat 148

4.3.2 Genes and expression 149

Chapter 5 Investigating transcriptional regulation of FKBP16-1 and FKBP16-3 expression in wheat and other cereals 151

5.1 Introduction 152

5.2 Results 153

5.2.1 RT PCR analysis of TaFKBP16-1 and TaFKBP16-3 expression 153

5.2.2 Light and stress regulation of TaFKBP16-1 and TaFKBP16-3 expression 156

5.2.3 Isolation of the TaFKBP16-1 and TaFKBP16-3 promoters 158

5.2.3.1 Constructing wheat ligation libraries 158

5.2.3.2 Isolation of genomic sequences flanking TaFKBP16-1 159

5.2.3.3 Isolation of genomic sequences flanking TaFKBP16-3 161

5.2.4 Analysis of the TaFKBP16-1 and TaFKBP16-3 promoters 163

5.2.4.1 Features of the FKBP16-1 promoters of wheat and other cereals 163

5.2.4.2 Features of the FKBP16-3 promoters of wheat and other cereals 167

5.3 Discussion 171

Chapter 6 Exploring the functions of FKBP13, FKBP16-1 and FKBP16-3 in wheat and Arabidopsis 175

6.1 Introduction 176

6.2 Results 177

6.2.1 Over-expression and purification of wheat FKBP13, FKBP16-1 and FKBP16-3 177

6.2.2 Assaying PPIase activity of the TaFKBPs 185

6.2.3 Over expression and purification of Arabidopsis FKBP16-1 and FKBP16-3 186

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6.2.4 Sub-chloroplastic localisation of FKBP orthologues in Arabidopsis 188

6.2.5 Western blotting for AtFKBP16-1 and AtFKBP16-3 localisation 191

6.2.6 Investigating the role of AtFKBP16-3 through gene knockout 193

6.2.6.1 Carbohydrate partitioning in the atfkbp16-3 mutant 194

6.3 Discussion

6.3.1 PPIase activity discovered in the wheat thylakoid 197

6.3.2 AtFKBP16-1 and AtFKBP16-3 are thylakoid proteins 198

6.3.3 AtFKBP16-3 in chloroplast signalling 199

Chapter 7 Yeast two-hybrid analysis of FKBP interactions in the wheat chloroplast 201

7.1 Introduction 202

7.2 Results 203

7.2.1 Construction of a wheat leaf cDNA library for yeast two-hybrid screens 203

7.2.2 FKBP bait constructs used for Y2H screens 206

7.2.3 Isolation and cloning of TaRieske CDS for Y2H screens 206

7.2.4 Screening the wheat library for interactions with wheat FKBPs 209

7.2.5 Detection and analysis of TaFKBP13 interactors 211

7.2.6 Exploring interactions between TaFKBP13 and TaRieske 218

7.2.7 Detection and analysis of TaFKBP16-1 interactors 220

7.2.8 Detection and analysis of TaFKBP16-3 interactors 226

7.2.9 Pro residues in TaFKBP13, TaFKBP16-1 and TaFKBP16-3 interaction sites 231

7.2.10 Predicted phosphorylation sites in peptide interactors 231

7.3 Discussion 233

7.3.1 The wheat FKBPs exhibit interaction specificity 233

7.3.2 TaFKBP13 operates in electron transfer in the thylakoid membrane 234

7.3.3 TaFKBP16-1 interacts with the PsaL subunit of PSI 236

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7.3.4 TaFKBP16-3 chaperone activity in thylakoid biogenesis 238

7.3.6 Linking FKBP chaperone activity and membrane phosphorylation 239

Chapter 8 Final discussion and conclusions 240

8.1 Contributions of this work 241

8.2 Discussion and conclusions 243

8.2.1 A general role for the eukaryotic FKBPs in phosphorylation 243

8.2.2 On the emergence of FKBP multigene families in eukaryotes 246

8.2.3 Tracking the evolution of the FKBP multigenes in higher plants 247

8.2.4 The FKBP population in the thylakoid is important for state transitions and cyclic electron flow 249

8.3 Final comments and future directions 251

Bibliography 253

Appendices 286

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List of figures

Figure 1.1 Molecular structures of FK506 and Rapamycin 3

Figure 1.2 The mechanism of FK506- and CsA-mediated immunosuppression 4

Figure 1.3 Prolyl bond configurations 5

Figure 1.4 The protease-couple PPIase assay 7

Figure 1.5 Conservation in the FKBP12 orthologues among eukaryotes 9

Figure 1.6 The conserved tertiary structure of the FKBd 10

Figure 1.7 Cellular transport of GR to the nucleus by FKBP52 16

Figure 1.8 The FKBP family in Arabidopsis thaliana 22

Figure 1.9 Electron flow pathways in photosynthesis 31

Figure 1.10 Thylakoid structures 34

Figure 1.11 Tat mechanism of thylakoid transport 36

Figure 2.1 Ligation library construction 58

Figure 2.2 IPCR for amplifying genomic regions flanking from ligation libraries 60

Figure 2.3 Apparatus for 14C fixing in leaf discs 65

Figure 2.4 Synthesis of double-stranded cDNA for Y2H library construction 69

Figure 2.5 Recombination of pGADT7-Rec and ds cDNA in yeast 71

Figure 3.1 Genomic loci of twenty-nine FKBP genes in O. sativa 80

Figure 3.2 Domain structures of cytosolic FKBPs in rice 83

Figure 3.3 Conserved features of plant FKBPs 85

Figure 3.4 HSP90-binding residues in the rice FKBP TPR domains 86

Figure 3.5 A putative chaperone domain identified in OsFKBP75 86

Figure 3.6 Hydropathy of the PAS1, OsFKBP72 andOsFKB75 C-termini 87

Figure 3.7 Domain structures of FKBPs in the rice nucleus 88

Figure 3.8 FKBPs in the rice ER 89

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Figure 3.9 Domain structures of FKBPs localised to the rice chloroplast 90

Figure 3.10 Phylogenetic relationships of individual FK506-binding domains in rice FKBPs 94

Figure 3.11 Gene structures of the lumenal FKBP subfamilies 96

Figure 3.12 Phylogenetic relationships among the lumenal FKBPs subfamilies 97

Figure 3.13 Exon fusion and the novel WEPT motif in cereal FKBP13 98

Figure 3.14 The cyanobacterial FKBPs compared with FKBP12 and FKBP13 from plants 100

Figure 4.1 Alignment of FKBP13 gene sequences 112

Figure 4.2 Isolation of TaFKBP13 from wheat gDNA 115

Figure 4.3 Gene structures of TaFKBP13 genomic sequences 115

Figure 4.4 Isolation of TaFKBP13 from T. aestivum cDNA 116

Figure 4.5 Alignment of FKBP16-1 gene sequences 118

Figure 4.6 Isolation of TaFKBP16-1 from T. aestivum gDNA 130

Figure 4.7 Structures of TaFKBP16-1 genes 130

Figure 4.8 Isolation of FKBP16-1 from wheat progenitor gDNAs 131

Figure 4.9 Splice variants amplified from TaFKBP16-1 cDNA 132

Figure 4.10 Predicted gene structures of the TaFKBP16-1 splice variants 133

Figure 4.11 Alignment of FKBP16-1 gene sequences 135

Figure 4.12 Isolation of TaFKBP16-3 from T. aestivum gDNA 140

Figure 4.13 Structures of TaFKBP16-3 genes 140

Figure 4.14 Isolation of FKBP16-3 from wheat progenitor gDNAs 141

Figure 4.15 Transcripts amplified from TaFKBP16-3 cDNA 142

Figure 4.16 FKBP13 amino acid sequence alignment 144

Figure 4.17 FKBP16-1 amino acid sequence alignment 145

Figure 4.18 FKBP16-3 amino acid sequence alignment 146

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Figure 5.1 Development and tissue regulation of TaFKBP16-1 and TaFKBP16-3 expression 153

Figure 5.2 Examples of semi-quantification of RT PCR products 154

Figure 5.3 Tissues regulation of TaFKBP16-1 and TaFKBP16-3 expression 155

Figure 5.4 Light and stress regulation of TaFKBP16-1 and TaFKBP16-3 expression 157

Figure 5.5 T. aestivum gDNA digested for ligation library construction 158

Figure 5.6 IPCR amplification of genomic sequences flanking TaFKBP16-1 159

Figure 5.7 KpnI digestion of TaFKBP16-1 IPCR product 160

Figure 5.8 Genomic regions flanking TaFKBP16-1 isolated by IPCR 160

Figure 5.9 Genomic regions flanking TaFKBP16-3 isolated by IPCR 161

Figure 5.10 IPCR amplification of genomic sequences flanking TaFKBP16-3 162

Figure 5.11 DNA sequence of the putative TaFKBP16-1 gene promoter 163

Figure 5.12 The chloroplast biogenesis module 166

Figure 5.13 DNA sequence of the putative TaFKBP16-3 gene promoter 168

Figure 6.1 TaFKBP13 expression constructs 178

Figure 6.2 TaFKBP16-1 expression construct 179

Figure 6.3 TaFKBP16-3 expression construct 180

Figure 6.4 Preparation of TaFKBP13 expression constructs 181

Figure 6.5 Preparation of TaFKBP16-1 and TaFKBP16-3 expression constructs 181

Figure 6.6 SDS PAGE analyses of TaFKBP over-expression and purification 183

Figure 6.7 Bradford assay of purified protein concentrations 184

Figure 6.8 In vitro assays of PPIase activity in recombinant TaFKBPs 186

Figure 6.9 Amino acid sequences of AtFKBP expression constructs 187

Figure 6.10 SDS PAGE analyses of AtFKBP over-expression and purification 188

Figure 6.11 FPLC fractionation of purified thylakoids 189

Figure 6.12 Absorption spectra of thylakoid membrane fractions 190

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Figure 6.13 Western blots of AtFKBP16-1 and AtFKBP16-3 in thylakoid fractions 192

Figure 6.14 Preliminary characterisation of the atfkbp16-3 knockout mutant 193

Figure 6.15 Photosynthesis rates in atfkbp16-3 194

Figure 6.16 Starch synthesis in the atfkbp16-3 mutant 195

Figure 6.17 Starch accumulation in atfkbp16-3 196

Figure 7.1 PCR amplifications of cDNA used in library construction 204

Figure 7.2 PCR amplification of tagged ds cDNA using the MatchMaker primers 204

Figure 7.3 Dilution plates to determine yeast library titre 205

Figure 7.4 Isolation to the TaRieske coding sequence 207

Figure 7.5 CDS of TaRieske used in yeast two-hybrid prey construct 208

Figure 7.6 Yeast transformants grown on selective media 209

Figure 7.7 Estimation of mating efficiencies 210

Figure 7.8 Example library screen plate showing positive matTaFKBP13 interactors 211

Figure 7.9 Selection of strong matTaFKBP13 interactors on 3AT media 212

Figure 7.10 Library inserts amplified from matTaFKBP13 and preTaFKBP13 interactors 213

Figure 7.11 Sites of interaction between matTaFKBP13 and prey proteins 216

Figure 7.12 Yeast two-hybrid screens of matTaFKBP13 and preTaFKBP13 219

Figure 7.13 Selection of strong matTaFKBP16-1 interactors on 3AT media 220

Figure 7.14 Library inserts amplified from matTaFKBP16-1 interactors 221

Figure 7.15 Region of PsaL interacting with matTaFKBP16-1 224

Figure 7.16 Yeast two-hybrid screens of matTaFKBP16-1 and matTaFKBP16-3 225

Figure 7.17 Selection of strong matTaFKBP16-3 interactors on 3AT media 226

Figure 7.18 Library inserts amplified from matTaFKBP16-3 interactors 228

Figure 7.19 Region of matTaFKBP16-3 interaction with library preys 228

Figure 7.20 Possible roles for FKBP13 in regulating the Pro loop of plastid Rieske 235

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Figure 7.21 Schematic representation of the LHCII docking site at PSI 237

Figure 8.1 Predicted scheme for FKBP-mediated phosphorylation 245

Figure 8.2 Immunophilins involved in thylakoid membrane architecture 251

 

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List of tables

Table 1.1 Summarised characteristics of the FKBP families of H. sapiens and other vertebrates 13

Table 1.2 The FKBP family of S. cerevisiae 19

Table 1.3 The FKBP family in Arabidopsis 23

Table 2.1 Instruments and apparatus used 42

Table 2.2 Commercial kits and solutions used 43

Table 2.3 Restriction enzymes used 53

Table 2.4 Details of primers used in standard PCR, RT PCR, directional cloning and sequencing 57

Table 2.5 Details of primers used in inverse PCR 59

Table 2.6 Oligos used in wheat leaf cDNA library construction 69

Table 2.7 Sequenced genome databases BLASTed to identify FKBPs and other genes 74

Table 3.1 Characteristics of FKBPs in rice 81

Table 3.2 Features of FKBP expression in rice from microarray data 84

Table 3.3 Key residues in the rice FKBPs 91

Table 3.4 Conserved features of thylakoid-localised FKBPs in rice, sorghum, maize, Arabidopsis and Physcomitrella 95

Table 3.5 Details of the putative lumenal FKBPs isolated from the wheat EST database 99

Table 3.6 FKBPs O. lucimarinus and C. reinhardtii and photosynthetic bacteria 101

Table 3.7 Suspected FKBP duplicates in the rice genome 103

Table 5.1 Cis elements in the putative TaFKBP16-1 promoter sequence 165

Table 5.2 Cis elements in the putative TaFKBP16-3 promoter sequence 169

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Table 6.1 Details of purified recombinant TaFKBPs 184

Table 6.2 Details of recombinant AtFKBPs 188

Table 6.3 Details of fractions collected from FPLC of thylakoids 189

Table 7.1 Primers and templates used for PCR verification during cDNA synthesis 204

Table 7.2 Titre determination of the yeast library shown in Figure 7.3 205

Table 7.3 Details of the constructs used in Y2H 209

Table 7.4 Details of the library inserts amplified from strong positive matTaFKBP13 and preTaFKBP13 interactors 214

Table 7.5 Details of cultures spotted onto selective plates in Figure 7.12 219

Table 7.6 Details of the library inserts amplified from strong positive matTaFKBP16-1 interactors 222

Table 7.7 Details of cultures spotted onto selective plates in Figure 7.16 225

Table 7.8 Details of the library inserts amplified from strong positive matTaFKBP16-3 interactors 229

Table 7.9 Phosphorylation prediction in FKBP interactors in the chloroplast 232

Table 8.1 Phosphoprotein chaperone partners of eukaryotic FKBPs 243

Table 8.2 Summary of findings from investigations into plant FKBPs 248

1

Chapter 1

General introduction and literature review

2

1 Introduction

1.1 Discovering the immunophilins

In 1976 Borel, Stähelin and co-workers reported their discovery of immune-inhibiting

properties of cyclosporin A (CsA), a cyclic peptide metabolite originally isolated from

the fungus Tolypocladium inflatum (Borel et al. 1976). CsA that was approved for use

as an immunosuppressant in human patients undergoing organ transplants and led to a

search throughout the 1980s for other safe and effective immunosuppressant

compounds (Sigal and Dumont 1992). In 1987 Goto and co-workers reported the

immunosuppressant properties of a macrolide lactone they had previously isolated from

fermentation broth of soil containing the bacterium Streptomyces tsukubaensis that was

collected from Mount Tsukuba, Japan (Goto et al. 1987; Tanaka et al. 1987). This novel

immunosuppressant was called FK506 (tacrolimus), which proved to be a potent

inhibitor of T-cell proliferation, and a more potent immunosuppressive agent than the

unrelated CsA (Sawada et al. 1987; Arai et al. 1989; Ochiai et al. 1989). Tanaka et al.

(1987) identified homology between the FK506 chemical structure (Fig. 1.1a) and

another macrolide compound ‘Rapamycin’ (Fig. 1.1b) which had been previously

isolated from the fermentation of soil collected from Rapa Nui (Easter Island).

Rapamycin (Rap) had been investigated as an antifungal antibiotic (Vezina et al. 1975;

Findlay, 1980), but proved to be another powerful agent for immunosuppression

following allografts (Calne et al. 1989). FK506 and Rap remain the drugs of choice for

immunosuppression in transplantation surgery (Gillard 2008; Gralla et al. 2009).

The cellular targets of the immunosuppressants were sought to explain the mechanisms

of immunosuppression. A novel 18 kDa protein isolated from CsA-charged columns

was named ‘cyclophilin’ (CYP; Handschumacher et al. 1984) and was identified in

various eukaryotes (Handschumacher et al. 1984; Koletsky et al. 1986). Harding and

co-workers (1989) described a 14 kDa protein that was isolated from bovine thymus and

human spleen using FK506-derivatised matrices, while at the same time Siekierka et al.

(1989a) eluted a protein of 10-11 kDa from Jurkat cells using a similar method. Both

groups demonstrated separately that the cellular receptor for FK506 was unrelated to

cyclophilin due to difference in size, heat stability and CsA binding ability (Siekierka et

al. 1989a), and through antigenic assays (Harding et al. 1989). The novel protein was

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called the FK506-binding protein (FKBP; Harding et al. 1989; Siekierka et al. 1989a)

and proved to be identical to the cellular target of Rap (Bierer et al. 1990; Fretz et al.

1991). The term ‘immunophilin’ was coined to describe both FKBP and cyclophilin

(Bierer et al. 1990).

Figure 1.1 Molecular structures of FK506 and Rapamycin (Galat 2003)

The protein sequence of human FKBP deduced cDNA comprised 108 amino acids

(Harding et al. 1989; Standeart et al. 1990), although the initial Met residue was

consistently absent and designated residue ‘0’. The molecular weight of bovine FKBP

was determined at 11.8 kDa through protein sequencing and electrospray ionisation

(Lane et al. 1991), and differed in sequence from human FKBP at only three residues

(Standaert et al. 1990; Lane et al. 1991). Additional protein species with different

molecular weights eluted from FK506 and Rap columns (see below) indicated the

existence of multiple FKBP isoforms in a single organism (Fretz et al. 1991), requiring

development of a nomenclature identifying the size and organism of origin of the

FKBP. Accordingly, the original 12 kDa FKBP isolated from human T-cells was called

‘hFKBP12’.

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1.1.2 Immunophilin-mediated immunosuppression

In a normal immune response, foreign antigens stimulate T-cell receptors and initiate a

calcium-mediated signal transmission that activates the calmodulin (CaM)-dependent

serine/threonine phosphatase calcineurin (CaN). Activate CaN dephosphorylates

cytosolic ‘nuclear transcription factor of activated T-cells’ (NF-AT), translocating it to

the nucleus where it upregulates expression of T-cell activation genes like the T-cell

growth factor interleukin-2 (IL-2) and γ-interferon (reviewed in Macian 2005). FK506

binds to cellular FKBP12 at the ‘FK506-binding domain’ (FKBd) and the drug-protein

complex interacts with CaN, blocking its activity and thereby inhibiting nuclear

translocation of cytosolic NF-AT (Bierer et al. 1990b; Flanagan et al. 1991). Through

this mechanism, FK506 prevents the expression of genes involved in early activation of

T-cells and effectively inhibits an immune response (Fig. 1.2) (reviewed in Gothel and

Marahiel 1999). An identical mechanism of immunosuppression is effected by the CsA-

cyclophilin complex.

Figure 1.2 The mechanism of FK506- and CsA-mediated immunosuppression Immunosuppressant-immunophilin complexes block calcineurin activity, inhibiting dephosphrylation of NF-AT and preventing transcription of IL-2 (adapted from Lazarus and Kerdel 2002)

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Rap-mediated immunosuppression occurs via a separate mechanism from that of FK506

and CsA. The Rap-FKBP complex binds to a serine/threonine kinase known as ‘target

of rapamycin’ (TOR; Heitman et al. 1991), also called ‘rapamycin and FKBP12 target

1’ (RAFT1; Sabatini et al. 1994) and ‘FKBP12-rapamycin-associated protein 1’

(FRAP1; Brown et al. 1994) Mammalian TOR (mTOR) regulates numerous cell growth

and proliferation processes and inactivation of mTOR caused by binding of the FKBP-

rap complex interrupts a cell signalling pathways and leads to in inhibition of gene

expression and protein translation, and ultimately in cell cycle arrest at the G1 phase

(reviewed in Sehgal 2003).

1.2 Isomerisation of prolyl bonds

1.2.1 Immunophilins are peptidyl prolyl cis/trans isomerase enzymes

Peptide bonds exist in cis and trans configurations, and bond rotation usually occurs

spontaneously during protein folding due to the relative instability of the cis conformer

(Zimmerman and Scheraga, 1976). In the case of proline however, the ring structure

confers partial double-bond character to the Xaa-Pro linkage and as a result, rotation of

this bond (Fig. 1.3) is a rate-limiting step in protein folding (Brandts et al. 1975). In

1984, Gunter Fischer and co-workers reported their discovery of an enzyme isolated

from pig kidney that increased the rate of cis-to-trans conversion of a proline-

containing peptide and refolded denatured ribonuclease A (Fischer et al. 1984; 1985). It

was dubbed the ‘peptidyl prolyl cis/trans isomerase’ (PPIase; EC 5.2.1.8).

Figure 1.3 Prolyl bond configurations A peptidyl Pro residue shown in cis (A) and trans (B) configurations (Gothel and Mahariel 1999)

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The amino acid sequence of porcine PPIase was identical to bovine CYP, furthermore

PPIase activity was inhibited by CsA (Fischer et al. 1989; Takahashi et al. 1989),

proving that the isolated PPIase was in fact CYP. Harding et al. (1989) showed that

bovine and human FKBP were also PPIase enzymes were inhibited by FK506 (Harding

et al. 1989; Siekierka et al. 1989).

1.2.2 Parvulin; the non-immunophilin PPIase

Rahfeld et al. (1994) reported the discovery of a PPIase in E. coli with a mass of 10

kDa, which was smaller than any of the previously reported immunophilins. The novel

enzyme was dissimilar in sequence to CYP and FKBP, and was resistant to inhibition

by FK506 and CsA (Rahfeld et al. 1994). This third type of PPIase dubbed ‘parvulin’

(from the Latin ‘parvulus’ = small; Rahfeld et al. 1994b). Homologues of E. coli

parvulin (Par10) were discovered in yeast (ESS1; Hani et al. 1995), Drosophila (Dodo;

Maleszka et al. 1997), Arabidopsis thaliana (PIN1At; Landrieu et al. 2000) and humans

(Pin1; Lu et al. 1996). As non-receptors for any immunosuppressant compound,

parvulins are excluded from the category of immunophilin, although inhibition of the

PPIase activity of parvulin by the natural dye compound juglone has been demonstrated

(Hennig et al. 1998; Wang and Etzkorn 2006). Unlike FKBPs and CYPs, parvulin

PPIase activity is specific to substrates with phosphorylated serine or threonine residues

preceding the target proline (Ranganathan et al. 1997; Yaffe et al. 1997). Also unlike

the other PPIases, parvulin was essential for survival in yeast (Hani et al. 1995;

Dolinski et al. 1997), operating as a vital regulator of cell cycle progression from G2 to

M phase during mitosis (Lu et al. 1996).

1.2.3 Mechanisms of prolyl bond rotation

Prolyl isomerisation mechanisms are not fully understood. Rotation by FKBP and CYP

occurs by distorting the substrate towards the transition state in a mechanism called

‘catalysis by distortion’ (Harrison and Stein 1990; Fanghanel and Fischer 2004), but

unlike CYP, FKBP demonstrates selectivity for substrates possessing a bulky

hydrophobic amino acid such as Leu or Phe preceding Pro (P1 position) over charged or

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small residues at P1 (Albers et al. 1990; Park et al. 1992; Golbik et al. 2005). Early

analyses indicated that FKBP stabilised a ‘twisted amide’ configuration of Pro substrate

intermediates through desolvation of the target peptide bond in the hydrophobic active

site of the enzyme (Albers et al. 1990; Park et al. 1992; Fischer et al. 1993; Orozco et

al. 1993), and the affinity of the FKBP for FK506 and Rap was attributed to

conformational similarity between the drug ligands and a ‘twisted amide’ substrate

(Albers et al. 1990; Rosen et al. 1990; Wilson et al. 1995).

1.2.4 Measuring PPIase activity

The original method for determining the activity of the PPIase enzymes, developed by

Fischer et al. (1984), exploits the specificity of a protease for the trans configuration of

Pro-containing tetrapeptides, which have the general composition of succinyl-Ala-Xaa-

Pro-Phe-nitroanilide. Selective hydrolysis of the anilide group from trans substrates

generates free nitroanilide that can be detected spectrophotometrically at 390 nm and is

monitored over the time course of the reaction (Fig. 1.4). The inclusion of an active

PPIase in the reaction accelerates the cis to trans conversion, and thus increases the

signal.

Figure 1.4 The protease-couple PPIase assay The tetrapeptide substrate shown is Ala-Ala-Pro-Phe-nitroanilide, PPIase catalyses rotation of prolyl bond as shown, converting the substrate from cis to trans configuration. Proteolysis of the trans form of the substrate is indicated by the scissors. Peptide model adapted 1RMH

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Kofron et al. (1991) improved the sensitivity of the PPIase assay using the ‘solvent

jump’, where introduction of the substrate to aqueous solution disrupted the cis/trans

equilibrium and increased the concentration of cis conformer from 10% to around 50%

of the total substrate, significantly improving the signal/noise ratio. The role of helper

protease is most often performed by serine proteases such as chymotrypsin, trypsin and

subtilisin, which exhibit relatively broad substrate specificity (reviewed in Hedstrom,

2002). The adverse effect of a protease has been a limitation of the traditional system

(Fischer et al. 1984; Garcia-Echeverria et al. 1992; Kern et al. 1995; Hani et al. 1995;

Fischer and Aumuller 2003), limiting this assay to PPIases that are not themselves

susceptible proteolysis. Janowski et al. (1997) devised a protease-free alternative, taking

advantage of a minor difference in the absorption coefficients of the cis and trans

conformers of the tetrapeptide substrate and allowing prolyl isomerisation to be follwed

directly at a wavelength specific to the substrate. Another protease-free assay measures

the refolding of denatured RNase T1 protein, which contains two proline residues in the

cis conformation (Kiefhaber et al. 1990).

1.3 FKBP sequences, structures and domains

1.3.1 The FK506-binding domain: Conserved sequence and structure

Human FKBP12 and its orthologues, which have been identified in all eukaryotes and

some prokaryotes (Galat 2003), represent the simplest known FKBP isoform

comprising a single FKBd which is also the active site for PPIase activity. The FKBP12

sequence is extremely well conserved among diverse organisms (Fig.1.5). Mammalian

FKBP12 protein orthologues are 95-100% identical (Kay 1996), while over FKBP12

from 19 diverse animal taxa shared over 30% identity (Somarelli and Herrera 2007). At

the other end of the size spectrum, the FKBds in the high molecular weight FKBP

isoforms, many of which contain additional domains (see below) also demonstrate a

high degree of sequence conservation with the archetypal FKBd (Galat 2003; Patterson

et al. 2002; Somarelli et al. 2008).

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Figure 1.5 Conservation in the FKBP12 orthologues among eukaryotes Aligned FKBP12 isoforms from Homo sapiens (Hs), Oryza sativa (Os), Arabidopsis thaliana (At), Drosophila melanogaster (Dm), Caenorhabditis elegans (Ce) and Saccharomyces cerevisiae (Sc). Positioning corresponds to human FKBP12 (Standaert et al. 1990). Asterisks denote residues important for PPIase activity in mammalian FKBP12 (DeCenzo et al. 1996; Tradler et al. 1997). Residues highlighted blue are involved in FK506 and/or rapamycin binding in mammalian FKBP12 (Van Duyne et al. 1993). Beta sheet (β) and alpha helix (α) elements and loops are numbered according to the conserved FKBP tertiary structure shown in Figure 1.6

The tertiary structures of numerous FKBPs have been solved, including those

originating from mammals (Michnick et al. 1991; Van Duyne et al. 1991a; Wilson et al.

1995; Liang et al. 1996; Li et al. 2003; Sinars et al. 2003; Maestre-Martinez et al.

2006), plants (Gopalan et al. 2004; Granzin et al. 2006; Unger et al. 2010), bacteria and

archea (Suzuki et al. 2003; Saul et al. 2004), and various others (Somarelli et al. 2008).

These structures have shown that the overall conformation of all FKBds is also

extremely well conserved, primarily comprising six anti-parallel beta sheets connected

by a number of solvent exposed loops, one of which contains a short alpha helix (Fig.

1.6; Van Duyne et al. 1993; Wilson et al. 1995). The beta sheets assume a concave

surface opposite the helix with hydrophobic sidechains projecting towards the helix,

creating the hydrophobic enzyme core that has been likened to an empty ice-cream cone

(Van Duyne et al. 1993), or a bowl with hydrophobic sides and bottom, and a

hydrophilic edge (Ikura et al. 2008). The loop regions in the FKBd tertiary structure,

which are designated the 40s, 50s and 80s loops according to their location in hFKBP12

(Fig. 1.5), provide the sites for FKBP interaction with various other proteins, as detailed

below.

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Figure 1.6 The conserved tertiary structure of the FKBd A. Side view of the hFKBP12 tertiary structure (2PPN.pdb) showing beta strands (blue), helices (red) and loops (white), which correspond with Figure 1.5. B. Top view showing the hydrophobic active site pocket and the sidechains of five hydrophobic core residues. Adapted from protein model 2PPN (Szep et al. 2009)

1.3.2 The FKBP active site

The possibility that specific residues in the conserved FKBd core were important or

vital for PPIase activity has been investigated through several site-direct mutagenesis

studies, amounting to almost 30% of the entire FKBP12 active site (Aldape et al. 1992;

Park et al. 1992; Yang et al. 1993; Futer et al. 1995; Timerman et al. 1995; DeCenzo et

al. 1996; Tradler et al. 1997). These studies demonstrated that a considerable proportion

of wildtype PPIase activity was retained in almost all mutants tested. The greatest

reductions in activity resulted from separate single mutations to Y26, D37, F48, W59

and F99 (shown in Fig. 1.6) (DeCenzo et al. 1996). Furthermore, these studies showed

that the nature of the substituted residues influenced the activity of the mutant. For

example, the W59L mutant retained a significantly higher proportion of wildtype

PPIase activity than W59A (DeCenzo et al. 1996) or W59H (Timerman et al. 1995).

More recently, an FKBP12 mutant with six active site residues replaced with glycine

exhibited only a small reduction in PPIase activity on a tetrapeptide substrate (Ikura and

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Ito 2007), supporting the proposal that the side-chains of conserved residues were

dispensable for catalysing proline rotation (Fanghanel and Fischer 2004). Mounting

evidence suggests that the size and overall hydrophobicity of the FKBP active site may

be the major determinant of PPIase capability (Li et al. 2003; Ikura et al. 2008; Szep et

al. 2009; Unger et al. 2010).

1.3.3 Additional domains in FKBPs

Large, multi-domain FKBP isoforms contain at least one, and up to three FKBds, and an

array of additional functional domains have been described. Commonly occurring

additional domains are described below;

Tetratricopeptide repeat (TPR) regions are degenerative motifs of 34 amino acids that

form anti-parallel alpha-helical structures and provide sites for protein-protein

interactions (reviewed in Blatch and Lassle 1999). TPR domains have been implicated

in HSP90-binding in the FKPBs

Calmodulin-binding domain (CaMBd) is the site of interaction with the calcium sensor

protein calmodulin (CaM). Bound calcium causes a conformation change in CaM,

which then binds to helical CaMBd (reviewed in Bhattacharya et al. 2004). CaMBds

consist of around 20 amino acids, conserving bulky hydrophobic residues that facilitate

CaM interaction (Yap et al. 2000)

The EF hand is a helix-loop-helix motif that binds calcium and undergoes a

conformational change. EF hands occur in calcium-regulated proteins including

calmodulin (CaM) (reviewed in Bhattacharya et al. 2004)

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1.4 Analysing the FKBP multigene families

Strong homologies existing among FKBPs from diverse organisms can allow certain

well characterised isoforms, such as those of mammals, to operate as analogues for less

understood isoforms including those in higher plants. The characteristics of certain

isoforms that hold particular relevance to plant homologues have been reviewed below,

and summaries of entire FKBP families of selected organisms are presented in the

accompanying tables.

1.4.1 The FKBP families of Homo sapiens and other vertebrates

The paralogues FKBP12 and FKBP12.6 interact with ryanodine receptors (RyRs)

(Collins 1991; Jayraman et al. 1992), which are channels that regulate the release of

calcium ions from the sarcoplasmic reticulum in muscle tissue (reviewed in Fleischer

2008). FKBP12-binding stabilises the RyR in both the closed and open configurations,

thought to prevent untimely ‘leakage’ of calcium ions known as ‘sparks’ (Zalk et al.

2007; Fleischer 2008). Analogous interaction was found between FKBP12 and the

inositol 1,4,5-trisphosphate receptor (IP3R), which forms calcium release channels in

the endoplasmic reticulum (Cameron et al. 1995). FKBP12 also interacts with a

transforming growth factor-β (TGF-β) type I receptor. The TGF-β hormone signals

many developmental processes (reviewed in Massague 2000) by docking at the type II

TGF-β receptor, a kinase which then activates the nearby type I TGF-β receptor by

phosphorylation. In the absence of hormone activation, FKBP12-bindng prevents type I

TGF-β receptor phosphorylation, while activation removes bound FKBP12 and allows

to phosphorylate other signal transducers, eventually promoting cellular transcription

response (Wang et al. 1994). The role of FKBP12 is thought to prevent ‘leaky’ signa1

transduction in the absence of ligand (Wang and Donahoe 2004). The sites of FKBP12-

binding in IP3R and TGF-β receptor were Leu-Pro moieties (Schiene-Fischer and Yu

2001).

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Table 1.1 Summarised characteristics of the FKBP families of H. sapiens and other vertebrates

Mammalian FKBP 

Gene/ synonyms 

PPIase activity

FK506‐binding Domain structure  Subcellular locus*

Operations

FKBP12  FKBP1A  Yes 1 Yes 1 FKBd C (muscle tissue) 

Binds to RyR1 and IP3R, prevents Ca2+ leakage 2, 3Binds to TGF‐β, regulates phosphorylation 4

FKBP12.6  FKBP1B FKBP12A 

Yes 5 Yes 5 FKBd C (cardiac tissue)

Binds to RyR2 in cardiac muscle, prevents Ca2+ leakage 5

FKBP12c  FKBP12c  Unknown Unknown FKBd Unknown Gene identified on chromosome six, expression undetected 6

FKBP13  FKBP2  Yes 7 Yes 7, 8 FKBd; ER sig. (N); ER reten. (C)  

ER Protein folding 9

Interacts with membrane cytoskeleton 10 and C1q in complement protein 11

FKBP19  FKBP11  Unknown Yes (weak) 8 FKBd; ER sig. (N)   ER Highly expressed in secretory tissues 8

FKBP22  FKBP14  Unknown Unknown  FKBd; ER sig. (N); EF hand; ER reten. (C)

ER Unknown

FKBP23  FKBP7  Yes 12 Yes (weak) 12 FKBd; ER sig. (N);  EF hand; ER reten. (C)

ER Binds to HSP70, regulates BiP‐mediated Ca2+ storage in ER 12 

FKBP25  FKBP3  Yes 13 Yes 13 FKBd; DNA‐binding (N); NLS 

C/No 14 Interacts casein kinase, nucleolin 15 and HMGII 16 Probable role in transcription regulation

FKBP36  FKBP6  No 17 No 17 FKBd (N); TPR; CaMBd (C) 

C/N FKBd interacts with clathrin heavy chain, TPR binds HSP72, HSP90, GAPDH 17 Vital for spermatogenesis 18

FKBP37  AIP XAP2, APA9 

No 19 No 20 FKBd (N); TPR; CaMBd (C)

C/M/N TPR binds AhR transcription regulator 19 and HSP90 20

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FKBP38  FKBP8  Yes 21 Yes (Ca2+/CaM‐dependent) 21 

FKBd (N); TPR; CaMBd (C) 

C (M&ER membrane) (neural tissue) 

Ca2+‐dependant binding of anti‐apoptotic proteins Bcl‐2 and Bcl‐XL at FKBd 22 Regulates signalling and apoptosis in neuronal cells, linked to Alzeimer’s Disease  

FKBP51  FKBP5  DI: Yes 23

DII: No 23DI: Yes 24

DII: unknown2 FKBPds (N); TPRs; CaMBd (C) 

C Antagonises FKBP52‐mediated transport of GR to nucleus 25

FKBP52  FKBP4 see text 

DI: Yes 26

DII: minor 26

DI: Yes 27

DII: unknown 2 FKBPds (N); TPR; CaMBd (C) 

C/N 

TPR bind HSP90‐GR complex, FKBdI attaches to dynein to transport receptor complex to nucleus 25

FKBP60  FKBP9  Yes 27 Yes (weak) 27 4 FKBds; ER sig. (N); EF hand; ER reten. (C)

ER Ca2+‐dependant chaperone 27

FKBP65  FKBP10  Yes 28 Yes 29 4 FKBds; ER sig. (N); EF hand; ER reten. (C)

ER Regulates folding of tropoelastin prior to secretion 30 

FKBP133  WAFL, KIAA0674 

Unknown Unknown FKBPdWH1

C Binds with WIP, involved in actin filamentation in nerve cells 31

*C- cytosol; N- nucleus; ER- endoplasmic reticulum; M- mitochondria 1Standeart et al. 1990; 2Timerman et al. 1993; 3Cameron et al. 1995; 4Wang et al. 1994; 5Lam et al. 1995; 6Galat 2003; 7Rosborough et al. 1991; 8Rulten et al. 2006; 9Bush et al. 1994; 10Walensky et al. 1998; 11Neye and Verspohl 2004; 12Wang et al. 2007; 13Jin et al. 1992; 14Galat et al. 1992; 15Jin and Burakoff 1993; 16Leclercq 2000; 17Jarczowski et al. 2008; 18Crackower et al. 2003; 19Carver et al. 1998; 20Laenger et al. 2009; 21Edlich et al. 2005; 22Shirane and Nakayama 2003; 23Sinars et al. 2003; 24Davies et al. 2002; 25Davies and Sanchez 2005; 26Chambraud et al. 1993; 27Shadidy et al. 1999; 28Coss et al. 1995; 29Ishikawa et al. 2009; 30Patterson et al. 2000; 31Viklund et al. 2008

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FKBP52 was first identified as a component of steroid receptor complexes in

conjunction with heat shock proteins (Tai et al. 1986; Renoir et al. 1990; Sanchez et al.

1990), and has been intensively investigated in numerous vertebrates, including rabbits

(Renoir et al. 1990), cows (Peattie et al. 1992; Yem et al. 1992), rats (Ruff et al. 1992),

mice (Alnemri et al. 1993), chickens (Smith et al. 1993), squirrel monkeys (Denny et al.

2005) and humans (Sanchez et al. 1990; Peattie et al. 1992). FKBP52 functions as an

adapter protein in the nuclear transport of cytosolic steroid receptors, most notably the

glucocorticoid receptor (GR) (reviewed in Davies and Sanchez 2005). The first of two

consecutive FKBds in FKBP52 is PPIase active and binds to dynamitin, a subunit of the

dynein molecular motor complex, while three TPR units bind the HSP90 chaperone

complex. The function of a CaMBd that occurs at the FKBP52 C-terminus (Callebaut et

al. 1992) is unclear, however this region was also vital for HSP90-binding (Cheung-

Flynn et al. 2003) and has been recently shown to interact with polymerised tubulin in

FKBP52 (Chambraud et al. 2007). The dynein complex facilitates retrograde movement

of the FKBP52-bound receptor complex along the actin cytoskeleton to the nucleus

(Harrell et al. 2004), where nuclear translocation of the complex is thought to occur via

one of two nuclear localisation signals (NLSs) in the GR (Picard and Yamamoto 1987;

Echeverria et al. 2009). Once in the nucleus, GR regulates the expression of genes

involved in anti-inflammatory stress response (reviewed in Rhen and Cidlowski 2005).

FKBP52 also facilitates retrograde transport of the tumour-suppressor transcription

factor p23 via an identical mechanism (Galigniana et al. 2004), and has been implicated

in nuclear localisation of the androgen and progesterone receptors (Cheung-Flynn et al.

2005; Yang et al. 2006; Banerjee 2008).

A close homologue of FKBP52, FKBP51 shares 50-60% amino acid identity with

FKBP52 and has identical domain structure, including multiple FKBds and a three-unit

TPR region (Baughman et al. 1995; Yeh et al. 1995; Sinars et al. 2003). Similar to

FKBP52, PPIase activity is restricted to the N-terminal FKBdI of FKBP51 (Sinars et al.

2003) although, unlike its homologue, this domain lacks the capacity to interact with

dynein (Wochnik et al. 2005).

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Figure 1.7 Cellular transport of GR to the nucleus by FKBP52 FKBP52 binds the HSP90-GR-steroid complex, attaches to the dynein complex and then enters the nucleus through retrograde motion along mictotubules (Davies and Sanchez 2005)

Overlapping or cooperative functionality between of FKBP51 and FKBP52 was initially

suggested in the observation that FKBP52-bound receptors had a higher affinity for

steroid-binding than FKBP51-bound receptors (Denny et al. 2005; Davies and Sanchez.

2005). Riggs et al. (2003) showed that FKBP52 potentiated GR-responsive

transcription, while co-expression of FKBP51 inhibited FKBP52-mediated GR

response. The function as either agonist (FKBP52) or antagonist (FKBP51) on GR

activation was localised to single Pro residue in the 80s loop of FKBP52, which is

substituted for Leu in FKBP51, that interacts with the receptor ligand-binding domain

(Riggs et al. 2003; Wochnik et al. 2005; Riggs et al. 2007). Steroid-binding to the

receptor is thought to displace FKBP51 in favour of FKBP52, which attaches to dynein

and transports the receptor complex to the nucleus (Davies et al. 2002; Wolf et al.

2009), although further details of this mechanism are anticipated.

The mammalian endoplasmic reticulum (ER) contains a large subset of FKBPs, with at

least six isoforms targeted to this organelle (Patterson et al. 2002; Rulten et al. 2006).

The ER-localised FKBPs have been predicted as important for folding and trafficking of

secreted proteins. FKBP13 is an active PPIase thought to operate as a folding chaperone

for ER proteins, as FKBP13 expression was upregulated under treatments causing

protein misfolding in the ER (Bush et al. 1994). Specific interaction between FKBP13

and the 4.1G protein, part of the membrane cytoskeleton (Walensky et al. 1998), and

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also with C1q, a subunit protein of the C1 component of the complement system (Neye

and Verspohl 2004), indicate that FKBP13 may be also operational outside of the ER.

The FKBP13-C1q association was not disrupted by FK506, suggesting that this

interaction occurs outside of the PPIase active site (Neye and Verspohl 2004).

A PPIase domain at the C-terminus of the nuclear FKBP25 contains a unique lysine

motif ‘KK(X)7KK(X)26KKK’ that acts as a nucleus localisation signal (NLS) (Galat et

al. 1992; Riviere et al. 1993; Jin and Burakoff 1993; Mas et al. 2005). The N-terminal

domain contains a high concentration of charged and basic residues (Galat et al. 1992)

and this region was predicted to form a helix-loop-helix secondary structure, possibly

conferring DNA-binding activity to nuclear FKBP25 (Riviere et al. 1993). Interactions

have been detected between mammalian FKBP25 and several nuclear proteins,

including casein kinase II, the nuclear phosphoprotein nucleolin (Jin and Burakoff

1993) and the DNA-binding high mobility group (HMG) II (Leclercq et al. 2000).

Although undetermined, a specific role for FKBP25 was suspected to involve

transcriptional regulation (Leclercq et al. 2000).

1.4.2 FKBPs in Saccharomyces cerevisiae and lower eukaryotes

FKBP exist as conserved protein families in yeast and other fungi (Pemberton 2006;

Pinto et al. 2008), although they lack large multi-domain isoforms found in higher

eukaryotes. This has been suggested to indicate that roles for the fungal FKBPs relate

mainly to protein folding (Pemberton 2006). In support of this, each FKBP in the

budding yeast S. cerevisiae, a model lower eukaryote, is an active PPIase (see Table

1.2).

1.4.2.1 Immunophilin knockout yeast

Several studies have use gene knockout mutant strains in attempts to define the in vivo

function of FKBPs in Saccharomyces cerevisiae (Heitman et al. 1991; Wiederrecht et

al. 1991; Nielsen et al. 1992; Benton et al. 1994; Dolinski et al. 1997) and other fungi

(Tremmel et al. 2007; Pinto et al. 2008). In each case, silencing of individual FKBP

18

expression did not produce a growth phenotype under normal growth conditions,

leading to questions regarding the importance of the FKBPs to eukaryotic viability and

growth. To clarify this, a landmark study conducted by Dolinski et al. (1997) produced

mutant strains lacking a single immunophilin, strains with the selective knockout of

immunophilins from cellular compartments including the cytosol and nucleus, and

strains in which all four FKBPs, or all eight CYPs were silenced. Each of the mutant

strains was viable and demonstrated no notable growth phenotype. Finally, a mutant

completely lacking in all twelve immunophilins was generated and this strain was also

viable, albeit with a phenotype of slower growth, but displayed no defects in mating,

sporulation or germination (Dolinski et al. 1997), although the dodecuplet mutant

exhibited limited sensitivity to extreme heat shock (48° C). The yeast mutant lacking

parvulin, on the other hand, did not survive. The authors concluded that the

immunophilins are nonessential to the viability of yeast and suggested an alternative

function as stress-responsive chaperones rather than critical protein folders, which was

more likely played by the essential parvulin (Dolinski et al. 1997).

19

Table 1.2 The FKBP family of S. cerevisiae

Isoform in  S. cerevisiae 

Fungal homologues 

PPIase activity 

FK506‐binding 

Domain structure 

Subcellular locus* 

Operations 

Fpr1  NcFKBP11  ScFKBP12 

Yes 1  Yes 1  FKBd  C  Binds aspartokinase 2, Hmo1 3 NcFKBP11 linked to sexual development 4 

Fpr2  NcFKBP22 5 Sc: none 6 

Yes 1  Yes 1  FKBd; ER sig. (N) 

ER  Upregulated by unfolded protein 7 NcFKBP22 interacts with BiP, involved in sporulation 8 

Fpr3  Nc: none  ScFKBP39 

Yes 9, 10  Yes 9, 10  FKBd; charged domain and NLS (N) 

N  FKBd interacts with cell cycle regulator 11

Interacts with S24 ribosomal subunit 12  

Fpr4  NcFKBP50 ScFKBP40 

Yes 13  Yes 13  FKBd; charged domain and NLS (N) 

Nol  Fpr3 paralogue, interacts with S24 ribosomal subunit 12 Charged N‐terminus binds histone, facilitates nucleosome assembly 13 

* C- cytosol; N nucleus; ER- endoplasmic reticulum; Nol- nucleolus 1Nielsen et al. 1992; 2Alarcon and Heitman 1997; 3Dolinski and Heitman 1999; 4Pinto et al. 2008; 5Solscheid and Tropschug 2000; 6Galat 2004; 7Partaledis and Berlin 1993; 8Tremmel and Tropschug 2007; 9Benton et al. 1994; 10Shan et al. 1994; 11Hochwagen et al. 2005; 12Dolinski et al. 1997; 13Kuzuhara and Horikoshi 2004

20

1.5 Immunophilins in Arabidopsis thaliana and higher plants

The first plant immunophilin to be characterised was an 18 kDa cyclophilin, transcripts

of which were detected in tomato, maize and Brassica napus cDNA, and were well

conserved in comparison to the orthologous mammalian CYPA (Gasser et al. 1990).

PPIase activity was later detected in the chloroplast and mitochondria of peas, attributed

to CYPs and FKBPs that were identified in those organelles (Breiman et al. 1992).

Immunophilins were also investigated in plants for their potential as inhibitors of plant

CaN, a regulator of ion efflux that controls stomatal aperture (Luan et al. 1993). More

recently, availability of the genome sequence of the model plant Arabidopsis thaliana

allowed identification of the entire immunophilin multigene family in a higher plant and

resulted in discovery of the largest immunophilin family of any organism (reviewed in

Romano et al. 2004; He et al. 2004; Romano et al. 2005), comprising twenty-three

FKBPs and twenty-eight CYPs.

1.5.1 FKBPs in plant development

AtFKBP42 was also called ULTRACURVATA2 (UCU2; Perez-Perez et al. 2001), and

is most commonly referred to as TWISTED DWARF1 (TWD1; Kamphausen et al.

2002) due to the phenotype of the twd1 mutant that displayed stunted growth and helical

rotation of roots and aerial organs. This was due to interrupted signalling of the growth

factors auxin and brassinosteroids (BR), which control cell elongation and division

(Perez-Perez et al. 2004). TWD1 possesses a single FKBd at the N-terminus that is

PPIase inactive and unable to bind FK506 (Kamphausen et al. 2002), and exhibits

significant structural heterogeneity with hFKBP12 in the 40s and 80s loop regions

(Weiergräber et al. 2006). The FKBd of TWD1 interacts with the P-glycoprotein pair

PGP1/PGP19 that are multidrug resistance (MDR)-like ATP-binding cassette (ABC)

transporters (Geisler et al. 2003). This interaction occurs at the plasma membrane

(Kamphausen et al. 2002; Geisler et al. 2003) for regulation of cellular auxin efflux

(Bouchard et al. 2006; Bailly et al. 2008). Three tandem TPRs occurring in TWD1

provide the site of interaction with another pair of ABC transporters, MRP12 and

MRP2, this time at the vacuolar membrane (Geisler et al. 2004), further implicating

TWD1 in auxin transport. Kamphausen et al. (2002) showed TWD1 binds to HSP90

21

and CaM, through the TPR region and CaMBd, respectively, although the effects of

these interactions are unknown. TWD1 terminates in a hydrophobic membrane anchor

(Kamphausen et al. 2002; Geisler et al. 2003) which aligns parallel to, rather than

spanning, membranes (Scheidt et al. 2007).

AtFKBP72 is encoded in Arabidopsis by the PASTICCINO1 (PAS1) gene, one of a

group of genes whose silencing contributed to cytokinin-induced developmental

mutations including thick hypocotyls, bushy rosettes and undeveloped cotyledons, in

Arabidopsis pas mutants (Faure et al. 1998; Vittorioso et al. 1998). PAS1 (also called

AtFKBP70; Carol et al. 2001) contains triplicate FKBds, a TPR regions, a CaMBd

(Carol et al. 2001) and a putative C-terminal trans-membrane anchor (He et al. 2004;

Geisler and Bailly 2007). PAS1 demonstrated only marginal PPIase activity that was

inhibited by FK506 (Carol et al. 2001), and interacts at a site C-terminal to the TPR

region with the NAC-like transcription factor FAN, which regulates cell proliferation

(Smyczynski et al. 2006). Both PAS1 and FAN are localised to the cytosol in

differentiated cells and the nuclear fraction of dividing cells (Carol et al. 2001;

Smyczynski et al. 2006), and it is suggested that PAS1 chaperones FAN to the nucleus

to inhibit cell division in response to cytokinin and auxin (Harrar et al. 2003;

Smyczynski et al. 2006).

FKBP12 is the smallest FKBP isoform in plants, originally purified from Vicia faba

leaves (Luan et al. 1994). FKBP12 from A. thaliana (AtFKBP12) and V. faba

(VfFKBP12) interacted with FK506 (Faure et al. 1998b; Xu et al. 1998) although,

unlike the homologous mammalian complex, the FKBP12-FK506 in plants

demonstrated only low affinity for the phosphatase CaN (Luan et al. 1993; Xu et al.

1998). Arabidopsis was resistant to the growth-inhibiting effects of Rap due to lack of

FKBP12-Rap-TOR complex formation (Menand et al. 2002), but Zea mays (maize) was

susceptible to Rap and this has been attributed to differences predicted in ZmFKBP12

structure compared with AtFKBP12 and VfFKBP12 (Agredano-Moreno et al. 2007).

Faure et al. (1998b) showed that AtFKBP12 interacts with a 37 kDa protein in

Arabidopsis called the FKBP-interacting protein (AtFIP37), a nuclear protein that is

essential for embryo development and was linked to regulation of gene splicing (Vespa

et al. 2004).

22

Figure 1.8 The FKBP family in Arabidopsis thaliana Scale representation of the domain structures of the Arabidopsis FKBP family (adapted from He et al. 2004). Domains shown are FKBd (blue), chloroplast target signal (light green), thylakoid target signal (dark green), ER target signal (orange), nuclear target signal (brown), Arg/Lys-rich region (pink), tetratricopeptide repeats (red), calmodulin-binding domain (yellow), transmembrane domain (black)

23

Table 1.3 The FKBP family in Arabidopsis

Arabidopsis isoform 

PPIase activity FK506‐binding Domains Subcellular locus* 

Results of functional characterisation

AtFKBP12  Yes 1  Yes 1 FKBd C   Interacts with FIP37 in Arabidopsis, involved in gene splicing 2 

AtFKBP13  Yes 3, 4  Yes (VfFKBP13) 5 FKBd;C/T sig. (N) 

TL 3, 5  Precursor interacts with Rieske subunit 5

PPIase activity is redox‐regulated 7 Non‐essential for normal plant function 8 

AtFKBP15‐1  Yes 9  Yes (VfFKBP15)5 FKBd; ER sig. (N); ER reten. (C) 

ER 9  Upregulated by heat shock, refolds denatured proteins in ER 9 

AtFKBP15‐2  Yes 9  Yes (VfFKBP15)5 FKBd; ER sig. (N);   ER reten. (C) 

ER 9  Upregulated by heat shock, refolds denatured proteins in ER 9 

AtFKBP15‐3  ND  ND FKBd; NLS (N) 

N  ND

AtFKBP16‐1  No 4  ND FKBd; C/T sig. (N) 

TL 10  ND

AtFKBP16‐2  No 4  ND FKBd; C/T sig. (N) 

TL/TM 11, 12 Part of NDH complex 13

AtFKBP16‐3  No 4  ND FKBd; C/T sig. (N) 

TL 6, 10  ND

AtFKBP16‐4  No 4  ND FKBd; C/T sig. (N) 

TL/TM 11, 12 ND

AtFKBP17‐1  No 4  ND FKBd; C/T sig. (N) 

TL  ND

AtFKBP17‐2  No 4  ND FKBd; C/T sig. (N) 

TL  ND

AtFKBP17‐3  No 4  ND FKBd; C/T sig. (N) 

TL  ND

AtFKBP18  No 4  ND FKBd; C/T sig. (N) 

TL 6  ND

AtFKBP19  No 4   ND FKBd; C/T sig. (N) 

TL 6, 11  ND

24

AtFKBP20‐1  ND  ND FKBd; NLS (C) 

N 14  ND; In rice, upregulated by heat treatment, chaperones Sce to nucleus 14 

AtFKBP20‐2  No 4  ND FKBd; C/T sig. (N) 

TL 6  Required for assembly of PSII supercomplexes 15 

AtFKBP42  No 16  No 16 FKBd (N); TPR; CaMBd;  Membrane anchor(C) 

C/PM/VM16, 17 

Interacts with ABC transporters at plasma membrane, required for auxin efflux 17, 18 Binds HSP90 and CaM 16 

AtFKBP43  ND  ND FKBd (C);Charged domain (N) 

N  

ND

AtFKBP53  ND  ND FKBd (C);Charged domain (N) 

N  Charged N‐terminus interacts with histones, regulates rRNA expression 19 

AtFKBP62  Yes (wFKBP73) 20 

Yes  (wFKBP73) 20 3 FKBd (N);  TPR; CaMBd (C) 

C/N  Expressed constitutively, TPR binds to HSP90 in presence of heat shock factor 21 

AtFKBP65  ND  ND 3 FKBd (N); TPR; CaMBd (C) 

C  Heat‐responsive expression, antagonises FKBP62 operation for acquired thermotolerance 22 

AtFKBP72  Yes (weak) 23 Yes 23 3 FKBds (N); TPR; CaMBd;  Mebrane anchor (C) 

C/NM 23 C‐terminus interacts with FAN transcription factor 24 

AtTIG  ND  ND FKBd (central)TIG domains (N&C) 

CS  ND

* C- cytosol, N- nucleus, ER- endoplasmic reticulum, TL- thylakoid lumen, TM- thylakoid membrane, PM- plasma membrane, VM- vaculor membrane, NM- nuclear membrane 1Faure et al. 1998b; 2Vespa et al. 2004; 3Gopalan et al. 2004; 4Shapiguzov et al. 2006; 5Gupta et al. 2002; 6Schubert et al. 2002; 7Gopalan et al. 2006; 8Ingelsson et al. 2009; 9Luan et al. 1996; 10Goulas et al. 2006; 11Peltier et al. 2002; 12Friso et al. 2004; 13Peng et al. 2009; 14Nigam et al. 2008; 15Lima et al. 2006; 16Kamphausen et al. 2002; 17Geisler et al. 2003; 18Bailly et al. 2008; 19Li and Luan 2010; 20Blecher et al. 1996; 21Meiri and Breiman 2009; 22Meiri et al. 2010; 23Carol et al. 2001; 24Smyczynski et al. 2006

25

1.5.2 FKBPs in plant heat stress response

AtFKBP62 and AtFKBP65 are more commonly known as ROF1 and ROF2,

respectively, which stands for ‘rotamase FKBP’ (Vucich and Gasser 1996). ROF1 and

ROF2 are duplicates sharing around 85% identity in Arabidopsis, and are homologues

of FKBP52 and FKBP51 in vertebrates (Aviezer-Hagai et al. 2007). ROF1 and ROF2

isoforms (Fig. 1.8) each contain three FKBds, plus a TPR region and CaMBd (Vucich

and Gasser 1996; Harrar et al. 2001; Kurek et al. 1999; 2000; Magiri et al. 2006).

PPIase activity is exclusive to the N-terminal FKBd in ROF1, ROF2 and their

orthologues in other plants (see below) (Blecher et al. 1996; Hueros et al. 1998; Meiri et

al. 2010). In Arabidopsis ROF1 was expressed constitutively in root and leaf tissues and

was upregulated by heat, while ROF2 is expressed solely in response to high

temperature (Aviezer-Hagai et al. 2007). In Arabidopsis, ROF1 formed a complex with

HSP90 and the heat shock transcription factor HsfA2, chaperoning the complex to the

nucleus and inducing expression of genes for plant thermotolerance (Aviezer-Hagai et

al. 2007; Meiri and Breiman 2009). Knockout of rof1 abrogated thermotolerance in the

mutant similar to knockout of HsfA2 (Meiri and Breiman 2009). ROF2, which was

unable to bind HSP90 in Arabidopsis, interacted with ROF1, while rof2 knockout plants

showed increased thermotolerance similar to ROF1 overexpression (Meiri and Breiman

2009; Meiri et al. 2010). ROF2 is thought to antagonise HsfA2 regulation of ROF1 in

the nucleus in a mechanism to regulate thermotolerance (Meiri et al. 2010). Orthologues

of the ROF1/ROF2 pair occur in wheat (Blecher et al. 1996; Kurek et al. 1999; Unger et

al. 2010) and rice (Magiri et al. 2006), and a ROF1 orthologue in maize, mzFKBP-66,

has also been identified (Hueros et al. 1998). The wheat and rice genes demonstrated

similar expression profiles to those of Arabidopsis, with rFKBP64 and wFKBP73

expressed under normal conditions, and rFKBP65 and wFKBP77 heat-induced. Both

wFKBP73 and wFKBP77 bind HSP90 at the TPR domain (Reddy et al. 1998). Over-

expression and subsequent accumulation of wFKBP77 led to increased cellular HSP90

levels and prompted developmental mutations such as dwarfism and sterility, while

increased wFKBP73 caused morphological abnormalities (Kurek et al. 2002). The

authors suggested that destabilisation of the equilibrium between HSP90 and one of its

binding partners can have a severe effect on signal transduction and normal

development, particularly in the seeds and reproductive organs of higher plants. CaM-

26

binding was demonstrated for mzFKBP-66 (Hueros et al. 1998) and wFKBP73 (Kurek

et al. 2002b) and deletion of the C-terminal CaM-binding domain from wFKBP73

expressed in rice had a negative impact on male fertility (Kurek et al. 2002b).

An FKBP localised to the ER of V. faba was isolated by Luan et al. (1996) and its

coding sequence used to identify two orthologues in Arabidopsis (Luan et al. 1996).

AtFKBP15-1 and AtFKBP15-2, as well as their bean orthologue, were homologous to

the ER-localised mammalian FKBP13, including conserved ER-localisation signals at

the N-termini and ER-retention motifs at the C-termini and a central FKBd that was

shown to be PPIase active in VfFKBP15. Expression of VfFKBP15 was upregulated by

heat-shock, and a role in the ER in refolding heat-denatured proteins has been proposed

(Luan et al. 1996).

The expression of OsFKBP20 in rice was upregulated by heat treatment (Nigam et al.

2008). OsFKBP20 possesses a single FKBd and an N-terminal nuclear localisation

signal (NLS), and was shown to interact with the SUMO-conjugating enzyme (Sce),

which attaches small ubiquitin-like modifier (SUMO) proteins to other target proteins.

The authors suggested that this interaction is important for heat response in rice. Two

isoforms with homologous domain structure exist in Arabidopsis (He et al. 2004).

AtFKBP15-3 and AtFKBP20-1 comprise a single FKBd and a lysine-rich nuclear target

at the N- and C- termini, respectively (Fig. 1.8).

1.5.3 FKBPs in transcription regulation

AtFKBP53 is localised to the nucleus, where it interacts with the H3 histone protein at

the 18S rRNA gene (Li and Luan 2010), thought to regulate deposition of histones to

DNA. AtFKBP53 possesses a C-terminal FKBd and has high concentrations of acidic

and basic residues in the N region. This domain structure is homologous with

AtFKBP43, another putatively nuclear isoform in Arabidopsis (He et al. 2004).

Separate knockout of AtFKBP53 and AtFKBP43 had no effect normal growth of the

mutants (Li and Luan 2010).

27

1.5.4 FKBPs in the chloroplast

Luan et al. (1994) found the most abundantly expressed FKBP in V. faba to be

FKBP13, which was localised to the chloroplast and upregulated by light. The proteome

of the Arabidopsis chloroplast showed that AtFKBP13 was localised to the thylakoid

(Schubert et al. 2002), and that seven other FKBPs were also found in this compartment

(Schubert et al. 2002; Peltier et al. 2002; Friso et al. 2004; Goulas et al. 2006). In total,

eleven FKBPs have been predicted in to be thylakoid-localised in the Arabidopsis (He

et al. 2004; Romano et al. 2005), accounting for almost half of the family of that plant.

The lumenal FKBPs in Arabidopsis are encoded on the nuclear genome and translocated

to the chloroplast and thylakoid through cleaved N-terminal, bipartite signal peptides in

the precursor proteins (He et al. 2004). The predicted thylakoid targets of the lumenal

FKBPs each contain a twin arginine (Arg-Arg) motif that suggested the use of the ‘twin-

arginine translocation’ (Tat) pathway of thylakoid entry, with the exception of

AtFKBP16-2, which possesses arginine-lysine at this site (He et al. 2004), although this

motif was also demonstrated as a functional Tat substrate in other thylakoid proteins

(Molik et al. 2001). He et al. (2004) detected the expression of each of the lumenal

FKBPs in Arabidopsis, with highest levels in green tissues.

AtFKBP13 is the most extensively characterised of the lumenal FKBPs. Gupta et al.

(2002) detected interaction between the N-terminal translocation signal of precursor

AtFKBP13 and the Rieske protein, the iron-sulphur subunit of the cytochrome b6/f

complex. In the same study, RNAi silencing of AtFKPB13 expression led to increased

accumulation of Rieske at the thylakoid membrane, and it was suggested that

AtFKBP13 is a stromal chaperone for Rieske operating to coordinate timely assembly

of the cytochrome complex (Gupta et al. 2002). No change in Rieske accumulation was

observed in recent characterisation of an atfkbp13 knockout (Ingelsson et al. 2009).

AtFKBP13 is the only FKBP in the chloroplast exhibiting PPIase activity (Gopalan et

al. 2004; Shapiguzov et al. 2006). The enzymatic activity of AtFKBP13 was shown to

be governed by the redox state of the protein, as activity was restricted to the oxidised

state (Gopalan et al. 2004; 2006). This was attributed two disulphide linkages in the N-

and C-terminal regions that to stabilise the ‘active’ configuration of the enzyme in

oxidising environments such as the thylakoid lumen. Site-directed mutations of

individual Cys residues indicate that the C-terminal disulphide, which supports the 80s

28

loop region at the top of the active site, is particularly important for PPIase activity in

AtFKBP13 (Gopalan et al. 2006). AtFKBP16-2 was recently detected as a subunit of

the NADPH dehydrogenase (NDH) enzyme complex in Arabidopsis on the lumenal

side of the membrane, which was required to stabilise the NDH-PSI supercomplex

(Peng et al. 2009). AtFKBP20-2 has been implicated in the stability of PSII in the

thylakoid membrane, as a higher concentration of