IDENTIFICATION AND CHARACTERIZATION OF THE ARABIDOPSIS GAUT1:GAUT7 PECTIN HOMOGALACTURONAN:GALACTURONOSYLTRANSFERASE COMPLEX by MELANI AGUSTINA ATMODJO (Under the Direction of Debra Mohnen) ABSTRACT Pectin is a family of structurally complex polysaccharides defined by the presence of α- 1,4-linked galacturonic acid (GalA) residues. It is present in all plant cell walls and contributes substantially to normal plant growth and development. Prior research has elucidated the intricate structure of pectin, yet a detailed understanding of pectin biosynthesis remains elusive. The membrane-bound Arabidopsis GALACTURONOSYLTRANSFERASE 1 (GAUT1) catalyzes the biosynthesis of homogalacturonan (HG), a linear homopolymer of α-1,4-linked GalA that makes up ~65% of total pectin. GAUT1 is an α-1,4-galacturonosyltransferase (GalAT) that transfers GalA from UDP-GalA onto HG acceptors to synthesize HG oligomers and polymers. This study presents the identification of an Arabidopsis HG:GalAT complex composed of GAUT1 and its homolog GAUT7, both of which are Golgi-localized, type II transmembrane proteins. Co-immunoprecipitation using anti-GAUT1 and anti-GAUT7 polyclonal antibodies and bimolecular fluorescent complementation demonstrated protein-protein interaction between GAUT1 and GAUT7. Transcript analyses indicate GAUT1 and GAUT7 co-expression in most tissues and developmental stages of Arabidopsis. Upon non-reducing SDS-PAGE, both GAUT1 and GAUT7 resolve at ~185 kDa, a size larger than their monomeric forms (~60 kDa and ~75 kDa, respectively) observed by reducing SDS-PAGE, thus indicating disulfide bond involvement in GAUT1:GAUT7 complex formation. Proteomics analyses identified GAUT1 and GAUT7 as the only components of the non-reducing SDS-PAGE-stable GAUT1:GAUT7 complex. The observed size of monomeric Arabidopsis GAUT1 (~60 kDa) is smaller than its predicted mass (77.4 kDa). N-terminal sequencing of GAUT1, mass spectrometry protein sequence coverage, and primary structure-dependent GAUT1 reactivity against a series of anti-GAUT1 antibodies, all suggest post-translational cleavage of Arabidopsis GAUT1 between Met167 and Arg168, resulting in a mature GAUT1 with no transmembrane domain. Transient expression of several GFP fusion constructs of GAUT1 in N. benthamiana leaves, in the presence or absence of GAUT7, showed a GAUT7-dependent Golgi accumulation of GAUT1-GFP. It is concluded that Golgi retention of GAUT1 is due to the formation of a complex with GAUT7. Models of the GAUT1:GAUT7 HG:GalAT complex, with GAUT1 as the catalytic subunit and GAUT7 as the membrane anchor, are proposed. Twelve additional proteins were also identified by proteomic analyses and proposed as GAUT1:GAUT7 complex putative interacting proteins. INDEX WORDS: plant cell wall, pectin, homogalacturonan, biosynthesis, glycosyltransferase, galacturonosyltransferase, Golgi apparatus, Arabidopsis thaliana, GAUT, protein complex, membrane anchor IDENTIFICATION AND CHARACTERIZATION OF THE ARABIDOPSIS GAUT1:GAUT7 PECTIN HOMOGALACTURONAN:GALACTURONOSYLTRANSFERASE COMPLEX by MELANI AGUSTINA ATMODJO B.S., Bogor Agricultural University, Indonesia, 1997 M.S., University of Adelaide, Australia, 2001 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2010 © 2010 Melani Agustina Atmodjo All Rights Reserved IDENTIFICATION AND CHARACTERIZATION OF THE ARABIDOPSIS GAUT1:GAUT7 PECTIN HOMOGALACTURONAN:GALACTURONOSYLTRANSFERASE COMPLEX by MELANI AGUSTINA ATMODJO Major Professor: Debra Mohnen Committee: Maor Bar-Peled Michael Pierce Lance Wells Electronic Version Approved: Maureen Grasso Dean of the Graduate School The University of Georgia December 2010 DEDICATION This doctoral work is dedicated to my husband Frank V. Gagliano, my family back home in Bandung, Indonesia, and my new family here in Athens and Crawford, Georgia, U.S. Their continuous support, love, and prayers are what kept me going and made all this possible. iv ACKNOWLEDGEMENTS I would like to thank my major professor, Dr. Debra Mohnen, for giving me the opportunity to learn and work on this project. I appreciate her guidance, friendship, and encouragement throughout my time in her laboratory, as well as the knowledge, experience, and inspiration that she shared me. I would also like to thank my committee members Dr. Maor Bar- Peled, Dr. Michael Pierce, and Dr. Lance Wells, for their support, advice, and constructive criticism that helped me get to where I am now. It was a great honor for me to have the chance to collaborate with these excellent researchers: Dr. Yumiko Sakuragi (University of Copenhagen, Denmark), Dr. Henrik Scheller (Joint BioEnergy Institute), Xiang Zhu (CCRC), Dr James Atwood III (Bioinquire), and Dr. Ron Orlando (CCRC). I am indebted as well to many people who generously provided me with assistance and materials to do my research: Drs. Carl Bergmann and Gerardo Gutierrez-Sanchez (CCRC–for exo- and endopolygalacturonase enzymes), Stefan Eberhard (CCRC–for Arabidopsis suspension culture, RG-I and RG-II polysaccharides), Sarah Inwood (CCRC–for technical assistance and enzyme), Drs. Jae-Min Lim and Lance Wells (CCRC–for LC-MS/MS identification of GAUT1 and GAUT7), Dr. Mary Tierney (The University of Vermont-for pBI101 vector containing AtPRP3 promoter), Dr. Malcolm O’Neill (CCRC–for RG-II monomer and dimer), Dr. Henk Schols (Wageningen University–for RG-I hydrolase enzyme), Dr. Simon Turner (University of Manchester, UK-for KOR1 antibody), and Sangita Mohanty (CCRC–for technical assistance). v My gratitude goes to all past and present members of the Mohnen lab, whom I have been privileged to know, work with, and learn from over the years, and to all the undergraduate students who contributed to my research, especially Amy Burrell and Ioana Petrascu, for their excellent work. I would also like to acknowledge people at CCRC for their administrative (especially Sheilah Dixon-Huckabee, Karen Howard, and Carol Connelly) and technical assistance, scientific advice, and friendships. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS.............................................................................................................v INTRODUCTION ...........................................................................................................................1 CHAPTER 1 LITERATURE REVIEW: BIOSYNTHESIS OF PECTIN HOMOGALACTURONAN .................................................................................................4 Introduction ...............................................................................................................4 Overview of Pectin Structure and Functions.............................................................5 Biosynthesis of Homogalacturonan.........................................................................11 Synthesis of Homogalacturonan Backbone.............................................................13 Arabidopsis Galacturonosyltransferase 1 (GAUT1) and the GAUT Gene Family.18 Modifications of Homogalacturonan During Synthesis .........................................29 Conclusions and Relevance ....................................................................................41 2 LITERATURE REVIEW: EUKARYOTIC POLYSACCHARIDE BIOSYNTHESIS – MECHANISM AND THE ROLE OF PROTEIN COMPLEXES...................................43 Introduction .............................................................................................................43 Cellulose Biosynthesis in Higher Plants .................................................................45 Starch Biosynthesis in Higher Plants ......................................................................64 Hyaluronan Biosynthesis in Vertebrates .................................................................81 Heparan Sulfate Biosynthesis in Vertebrates ..........................................................93 vii Protein N-Glycosylation: Role of the Oligosaccharyltransferase Complex..........109 Conclusions and Relevance...................................................................................120 3 GAUT1 AND GAUT7 ARE THE CORE OF A PLANT CELL WALL PECTIN BIOSYNTHETIC HOMOGALACTURONAN:GALACTURONOSYLTRANFERASE COMPLEX........................................................................................................................124 Abstract .................................................................................................................125 Introduction ...........................................................................................................126 Results and Discussion..........................................................................................128 Methods.................................................................................................................146 Supporting Information .........................................................................................149 DISCUSSIONS AND CONCLUSIONS .....................................................................................179 REFERENCES ............................................................................................................................199 APPENDICES .............................................................................................................................238 A Characterization of Anti-GAUT1, Anti-GAUT7, and Anti-GAUT4 Polyclonal Antibodies..................................................................................................................238