PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/113351 Please be advised that this information was generated on 2021-10-10 and may be subject to change. GALACTOSYLTRANSFERASES OF CHLOROPLAST ENVELOPES Galactolipid metabolism in Spinacia olerácea L. JOHAN HEEMSKERK GALACTOSYLTRANSFERASES OF CHLOROPLAST ENVELOPES Galactolipid metabolism in Spinacia olerácea L. PROEFSCHRIFT ter verkrijging van de graad van doctor in de wiskunde en natuurwetenschappen aan de Katholieke Universiteit te Nijmegen, op gezag van de Rector Magnificus prof. dr. J.H.G.I. Giesbers volgens besluit van het College van Dekanen in het openbaar te verdedigen op donderdag 5 juni 1986 des namiddags te 4.00 uur door JOHAN WILLEM MARIE HEEMSKERK geboren te Arnhem Promotores, co-referent J.F.G.M. Wintermans H.F. Linskens R. Douce GALACTOSYLTRANSFERASES OF CHLOROPLAST ENVELOPES Galactolipid metabolism in Spinacia olerácea L. CIP-DATA KONINKLIJKE BIBLIOTHEEK, DEN HAAG Heemskerk, Johan Willem Mane Galactosyltransfcrascs of chloroplast envelopes galactohpid metabolism in Spinacia olerácea L / Johan Willem Mane Heemskerk - [S 1 s η ] 111 Thesis Nijmegen - With ref - With summary in Dutch ISBN 90-9001193-5 SISO 583 UDC 581 1 Subject headings glycohpids / galactosvltransferases / chloroplast envelope membranes Aan Miep, Wim Marly Cover: Scanning micrograph of isolated spinach chloroplasts. The intact chloroplasts were fixed for 2 h (4°C) in 2.5% glutaraldehyde, containing 0.33 M sorbitol and 25 mM Hepes/NaOH (pH 7.2). After several washings in the same medium without glutaraldehyde, samples were post-fixed for 2 h (40C) in a slightly hypertonic buffer containing 0.4 M sorbitol, 25 mM Hepes/NaOH (pH 7.2) and 1% Os04. After dehydration in ethanol, the samples were shadowed and observed with a Jeol JSM-T300 Scanning Mi­ croscope (10000 x ). vi DANKWOORD Een proefschrift schrijf je niet alleen. Het overgrote deel van hetgeen hier beschreven en hetgeen hier niet beschreven is, was dan ook het werk van anderen. Mijn hartelijke dank aan iedereen voor zijn of haar bijdrage aan de totstandkoming van dit boekje. In het bijzonder wil ik bedanken al de medewerkers van Botanie I en III en van de proeftuin, voor ondersteuning en hulp in velerlei vorm en voor de aangename sfeer. Talloze experimenten en veel denkwerk werd uitgevoerd door Monique Aarnink, Peter van de Akker, Felix van Bavel, Gerard Bögemann, Gerda Caerteling, Peter Fijneman, Frans Jacobs, Ton Peeters, Martin Scheijen, Vincent Tromp en Willy Wolfs. Technische ondersteuning van Theo Laeyendecker en Jean-Marie Vijverberg (ultracentrifuges) was onontbeerlijk. Alard Glaap en medewerkers en de firma Gerrits & Zn. (Nijmegen) zorgden voor een constante aanvoer van spinazie. Gerard Bögemann, Frans Jacobs en José Broekmans hebben talloze zaken geregeld en in goede banen geleid. Daarnaast verrichtten José Broekmans en Marga Jansen veel typewerk. Illustraties werden vakkundig verzorgd door de afdelingen Illustratie en Fotografie. Dolf Dicke maakte de omslagfoto, Theo Laeyendecker de overige foto's en Bert Boone tekende de cartoons. Merci, Messieurs Roland Douce et Jacques Joyard pour votre acceuil cordial pendant des périodes que j'ai pu travailler dans le laboratoire de Physiologie Cellulaire Végétale du Centre d'Études Nucléaires de Grenoble (France). Merci, Jacques, pour avoir achevé le Chapitre 4, et, merci Marise Block et Albert-Jean Dorne, pour la collaboration amicale. A part of the work described in this thesis (Chapter 4) was supported by the European Molecular Biology Organisation, Heidelberg, FRG (grant ASTF4572). Een gedeelte van dit proefschrift is verwerkt door de computers van de afdeling Informatie-Verzameling/Verwerking, en afgedrukt met een Linotron 202 fotozetmachine. Tenslotte bedankt ook vrienden en kennissen die op een niet- botanische manier hebben meegewerkt aan dit proefschrift. Nijmegen, 5 juni 1986 Johan Heemskerk vu Abbreviations Abbreviations throughout the text were according to suggestions by Biochimica et Biophysica Acta (1982) 715, 1-23. Non-standard abbrevia­ tions are defined in each chapter separately. Vili CONTENTS OUTLINE OF THIS THESIS 1 CHAPTER 1 General introduction. 5 CHAPTER 2 Turnover of galactolipids incorporated into chloroplast en- 41 velopes: an assay for galactolipid : galactolipid galactosyl- transferase. Biochim. Biophys. Acta (1983) 754, 181-189 CHAPTER 3 Spinach chloroplasts: localization of enzymes involved in 53 galactolipid metabolism. Biochim. Biophys. Acta (1985) 835, 212-220 CHAPTER 4 Localization of galactolipid : galactolipid galactosyl- 65 transferase and acyltransferase in outer envelope mem­ brane of spinach chloroplasts. Biochim. Biophys. Acta (1986) (in press) CHAPTER 5 Regulation of the galactolipid synthesis in spinach chloro- 83 plasts. Advances in Photosynthesis Research (1984) (Sybesma, C, ed.), pp. Ш.2.147-150, NijhofflJunk Pubi., The Hague (The Netherlands) CHAPTER 6 Galactolipid : galactolipid galactosyltransferase can regulate 91 the galactolipid synthesis in isolated spinach chloroplasts. (submitted) CHAPTER 7 Separation of chloroplast polar lipids and measurement of 103 galactolipid metabolism by high-performance liquid chromatography Anal Biochem 153 (1986) (in press) CHAPTER 8 Galactolipid synthesis in chloroplast envelopes a specific 113 assay for UDPGal diacylglycerol galactosyltransferase (submitted) CHAPTER 9 Characterization of galactosyltransferases in spinach chloro- 129 plast envelopes (submitted) CHAPTER 10 UDPGal-independent formation of monogalactosyldiacyl- 153 glycerol, an enzymatic activity of the spinach chloroplast envelope (in preparation) CHAPTER 11 Conclusions 165 SAMENVATTING 175 FURTHER PUBLICATIONS 179 CURRICULUM VITAE 181 x OUTLINE OF THIS THESIS Some aspects of the galactolipid metabolism in spinach leaves are described, with particular emphasis on the galactosyltransferases responsible for the synthesis and the turnover of galactosyldiacylglycerols. Two of such galactosyltransferases have been demonstrated unequivocally in the chloro­ plast envelope membranes, viz. UDPGal : diacylglycerol galactosyl- transferase (UDGT) and galactolipid : galactolipid galactosyltransferase (GGGT). The presence of a third enzyme, UDPGal : monogalactosyldia- cylglycerol galactosyltransferase seems unprobable yet. The following chapters are arranged partly in a historical order, partly according to their thematic context. Chapter 1 was written quite recently (January, 1986) and it includes some of the results described in the subsequent chapters. It considers the plant galactolipid metabolism within the broader concept of the acyllipid synthesis (glycerolipid synthesis) in the leaf. Chapter 2 describes a specific assay for GGGT in isolated chloroplast envelopes, and shows the range of experimental conditions at which this en­ zyme works. Chapter 3 presents a separation method for the two envelope membranes of the chloroplast, and describes an attempt to localize UDGT and GGGT in the outer or in the inner envelope membrane. Whereas lo­ calization of UDGT was quite straight-forward, it appeared more difficult to localize GGGT unequivocally. Chapter 4 indicates that those difficulties were a consequence of the specific properties of GGGT. Both UDGT and GGGT are undoubtedly active in isolated intact spinach chloroplasts; this is described in Chapters 5 and 6. Chapter 7 returns to the envelope membranes. It presents a separation method for galactolipids and other acyllipids in the envelope, using high- performance liquid chromatography. This method was used in a quantita­ tive way for the measurement of both galactosyltransferases. An assay for UDGT, developed quite recently is described in Chapter 8. Chapter 9 presents a characterization study of both galactosyltransferases, using the specific assays for UDGT and GGGT. Next, Chapter 10 brings together some observations on monogalactolipid formation in the absence of the common precursor UDPGal. Some general conclusions are presented in Chapter 11, which is followed by a short resumé. 1 CHAPTER 1 GENERAL INTRODUCTION GENERAL INTRODUCTION Role of the chloroplast in acyllipid synthesis The current knowlegde of the acyllipid metabolism in plant leaf cells is summarized here, with special attention to the contribution of the chloro­ plast. Recent reviews are given in Refs. (1-10). It is now widely accepted that the lipid metabolic machinery is distri­ buted over the different compartments of the green leaf cell (1-3,11-13). With recent progress in the separation of plant cell organelles (14), more detailed information is becoming available. In general, phospholipid syn­ thesis appears to be concentrated in the microsome-rich cell fractions, en­ riched in endoplasmic reticulum (11,15), and lipid degradation in the perox­ isomes annex glyoxisomes (16,17). The role of mitochondria is probably more restricted in lipid metabolism: generation of acetate for fatty acid syn­ thesis (12,18) and involvement in the synthesis of the mitochondrial phos- phatidylglycerol (PG*) and diphosphatidylglycerol (DPG) (12,13). Finally, the chloroplast is considered the major site of fatty acid (1,18), glycolipid (2,3,5-9) and isoprenoid (8-10) biosynthesis. With such a compartmentaliza- tion, the cytosol must act as a connecting substance, converting and distri­ buting the necessary metabolic intermediates. Lipophilic compounds like fatty acids, their Co A derivatives