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REGULATION of MAIZE (Zea Mays L.) STARCH SYNTHASE Iia by PROTEIN PHOSPHORYLATION

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REGULATION of MAIZE (Zea Mays L.) STARCH SYNTHASE Iia by PROTEIN PHOSPHORYLATION Regulation of Maize (Zea mays L.) Starch Synthase IIa by Protein Phosphorylation by Usha P. Rayirath A Thesis presented to The University of Guelph In partial fulfillment of requirements for the degree of Doctor of Philosophy in Molecular and Cellular Biology Guelph, Ontario, Canada © Usha P. Rayirath, May, 2014 ABSTRACT REGULATION OF MAIZE (Zea mays L.) STARCH SYNTHASE IIa BY PROTEIN PHOSPHORYLATION Usha P. Rayirath Advisor: Dr. Michael J. Emes University of Guelph, 2014 Starch is a significant carbohydrate reserve in plants and has enormous use in both food and non-food industries. Biosynthesis of storage starch in maize (Zea mays L.) occurs in the amyloplasts of the developing endosperm, through the coordinated actions of several enzymes, including ADP-glucose pyrophosphorylase (AGPase), starch synthases (SS), starch branching enzymes (SBE) and debranching enzymes (DBE). Starch synthase IIa (SSIIa) catalyzes the synthesis of intermediate glucan chains (DP 12- 25) and plays a significant role in starch biosynthesis. Previous studies indicate that in cereal endosperm, protein phosphorylation plays a major role in regulating the formation of functional multi-enzyme complexes between SSs and SBEs during starch biosynthesis and that SSIIa forms the core of such a functional protein complex, with SSI and SBEIIb. The present study investigated the specific role of protein phosphorylation on the regulation of SSIIa, in the amyloplasts of developing maize endosperm, during starch biosynthesis. In vitro phosphorylation of stromal SSIIa in maize amyloplasts was detected by phosphate affinity Mn2+ Phos-tagTM gel electrophoresis, Pro-Q® Diamond phospho-protein gel staining, and by autoradiography following radio labelling with γ- [32-P] ATP. The results indicated that granule bound SSIIa exists in the phosphorylated state. In vitro phosphorylation of recombinant maize SSIIa and immunopurified SSIIa ii occurred only in the presence of amyloplast lysates and could be inhibited by protein kinase inhibitors, suggesting the presence of one or more protein kinase(s) in amyloplasts. ATP caused marked shifts in the electrophoretic mobility of SSIIa in non- denaturing polyacrylamide gels, and also in phosphate affinity (Phos-tag) gels, further suggesting the role of post-translational protein phosphorylation in regulating maize SSIIa. Protein phosphorylation significantly enhanced (12-fold), and dephosphorylation substantially reduced the catalytic activity (Vmax) of maize SSIIa, whereas its dissociation constant (Kd) and affinity for amylopectin was not affected. Depending on the phosphorylation status, stromal maize SSIIa existed in two distinct protein complexes, a LMW (260 kDa) protein complex was formed with SSI and SBEIIb under conditions favouring phosphorylation; whereas under conditions favouring dephosphorylation, this 260 kDa complex of SSIIa-SSI-SBEIIb possibly associated with other starch synthesizing enzymes and/or itself to form HMW complexes of 670 kDa or more. iii Acknowledgements I humbly bow my head before the God Almighty, who blessed me with the will power and courage to complete this endeavor. I submit this small venture before God, for His grace in providing me with the health and strength throughout the study. Firstly, I express my profound gratitude to my supervisors Dr. Michael J. Emes, and Dr. Ian J. Tetlow, for their excellent guidance, valuable suggestions, constructive criticism, constant encouragement and above all, for their patience, understanding and whole-hearted co-operation rendered throughout the course of my PhD program. I wish to extend my sincere gratitude to my advisory committee members, Dr. Annette Nassuth, and Dr. Bernard Grodzinski, for their time, help and constant support rendered during my study. I also extend my sincere thanks to the members of the examination committee, Dr. Ravi Chibbar (University of Saskatchewan) (External), Dr. Andrew Bendall (Chair), Dr. Annette Nassuth and Dr. Nina Jones. The funding support from NSERC Discovery Grant to Dr. M. Emes is greatly acknowledged. I also deeply acknowledge the Alexander Graham Bell Canada Graduate Scholarship (Doctoral) from NSERC, the Ontario Graduate Scholarship (declined) from Govt. of Ontario, and Deans’ Tri-council Scholarship from the University of Guelph, provided to me to conduct my PhD studies. My sincere thanks go to the past and present members of Emes/Tetlow lab, at the Department of Molecular and Cellular Biology, University of Guelph for their help, support and constant encouragement. I am especially grateful to Dr. Amina iv Makhmoudova, for her timely and constant help and advice, whenever I needed. Special thanks to Drs. Nadya Romanova, Wendy Allan and Fushan Liu, for their help and support. I am very much grateful to my seniors, Dr. Renuka Subasinghe and Dr. Zaheer Ahmed for their enormous help and support given to me, whenever needed. I am equally thankful to my other lab mates, Lily Navanosky (especially for her immense help with recombinant protein work), Jenelle Patterson (especially for her help with radioactive experiments, and during thesis preparation), Qianru Zhao (Ruby), Sarah Massey and You Wang. My special thanks to all the staff members of the Department of Molecular and Cellular Biology, New Science Complex, for their help and encouragement during the entire course of study. I express my sincere thanks to all the fellow graduate students in the department for sharing experiences, and developing valuable friendships. I am in dearth of words to express my gratitude and indebtedness to my loving husband for his constant support, understanding and love in all my endeavors. Words can’t express my boundless love to my son, for his understanding, sacrifice and support given to me during this endeavor. Last, but not least, I owe my profound gratitude to my father, mother, brother and in-laws, relatives and friends for their boundless affection, constant prayers, moral support, and unfailing inspiration, throughout my career. v Affectionately Dedicated to my Loving Family To my loving husband, my son and my loving parents and in laws and all who were understanding and supportive helping me to accomplish this… vi Table of Contents Abstract ii Acknowledgements iv Dedication v Table of Contents vii List of Figures xiv List of Tables xviii List of Abbreviations xix CHAPTER 1: GENERAL INTRODUCTION 2 1. Introduction 2 1.1 Starch 2 1.2 Molecular structure and composition of Starch 3 1.2.1 Starch granule 3 1.2.2 Starch composition- Amylose and Amylopectin 5 1.3 Biosynthesis of starch 8 1.3.1 Starch biosynthetic enzymes 9 1.3.1.1 ADP-glucose pyrophosphorylase (AGPase EC 2.7.7.27) 9 1.3.1.2 Starch synthases (SSs, EC 2.4.1.21) 12 1.3.1.2.1 Granule bound starch synthases (GBSS) 16 1.3.1.2.2 Starch synthase I (SSI) 17 1.3.1.2.3 Starch synthase II (SSII) 19 vii 1.3.1.2.4 Starch synthase III (SSIII) 21 1.3.1.2.5 Starch synthase IV (SSIV) 23 1.3.1.3 Starch Branching Enzymes (SBEs, EC 2.4.2.18) 24 1.3.1.3.1.Starch Branching Enzyme I (SBEI) 25 1.3.1.3.2 Starch Branching Enzyme II (SBEII) 26 1.3.1.4 Starch Debranching Enzymes (DBEs, EC 3.2.1.41 and EC 3.2.1.68) 27 1.3.1.5 Starch Phosphorylase (SP,EC 2.4.1.1) 29 1.3.1.6 Disproportionating enzyme (D-enzyme, EC 2.4.1.25) 31 1.4 Regulation of starch biosynthetic enzymes 32 1.4.1 Regulation at the transcriptional level 32 1.4.2 Allosteric regulation and redox modulation 34 1.4.3 Protein phosphorylation 35 1.4.3.1 Protein phosphorylation – a universal post-translational regulatory 35 mechanism 1.4.3.2 Protein phosphorylation and plant metabolism 39 1.4.3.3 Carbohydrate metabolism is regulated by protein phosphorylation – 41 from bacterial glycogen to plant 1.4.4 Phosphorylation dependent protein-protein interactions in starch 47 biosynthesis 1.4.5 Regulation of starch turnover by protein phosphorylation 52 1.5 Importance of starch synthase IIa (SSIIa) in starch biosynthesis 55 1.6 Hypothesis and Objectives of the study 58 CHAPTER 2: INVESTIGATION OF PROTEIN PHOSPHORYLATION 60 OF STARCH SYNTHASE IIa IN MAIZE ENDOPSERM 2.1 Introduction 60 viii 2.2 Materials and Methods 67 2.2.1 Materials 67 2.2.1.1 Plant materials 67 2.2.1.2 Chemicals 67 2.2.2 Methods 67 2.2.2.1 Isolation of amyloplasts from maize endosperms 67 2.2.2.2 Preparation of maize whole cell extracts 68 2.2.2.3 Isolation of starch granule bound proteins 69 2.2.3 Proteomic analysis 70 2.2.3.1 Quantification of proteins 70 2.2.3.2 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- 70 PAGE) 2.2.3.3. Coomassie blue staining 71 2.2.4 Immunological techniques 72 2.2.4.1 Preparation of peptides and antisera 72 2.2.4.2 Purification of polyclonal maize antibodies 72 2.2.4.3 Immunoblot analysis 73 2.2.4.4 Immunopurification of SSIIa from maize amyloplast lysates 74 2.2.5 Detection of phosphorylation of SSIIa in maize endosperm 75 2.2.5.1 Phosphorylation and dephosphorylation of SSIIa in maize amyloplast 75 stroma 2.2.5.2 Phos-tag TM phosphate affinity acrylamide gel electrophoresis 76 2.2.5.3 Pro-Q diamond phospho-protein gel staining 79 ix 2.2.5.4 In vitro phosphorylation of maize amyloplasts using γ- [32-P] ATP 80 2.2.5.5 Autoradiography 80 2.2.5.6 Expression and purification of recombinant maize SSIIa in Escherichia 81 coli 2.2.5.7 Immobilization of recombinant maize SSIIa on S-protein agarose beads 83 and pull down assay 2.2.5.8 In vitro phosphorylation of S-tag immobilized recombinant SSIIa using 83 γ- [32-P] ATP and autoradiography 2.3 Results 85 2.3.1 Detection of starch synthase IIa in the amyloplast stroma and in starch 85
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