The identification of Ftr1 and Zrt1 as novel micronutrient transceptors in S. cerevisiae

The identification of Ftr1 and Zrt1 as novel iron and zinc transceptors respectively in S. cerevisiae Joep Schothorst Supervisors: Prof. Dr. Johan Thevelein, Promotor Dr. Griet van Zeebroeck, Co-Promotor Members of the Examination committee Prof. Dr. Sandra Paiva Prof. Dr. Patrick van Dijck Prof. Dr. Filip Rolland Prof. Dr. Koen Geuten Dr. Marta Rubio-Texeira Dr. Erwin Swinnen June 2016 © 2016 KU Leuven, Science, Engineering & Technology Uitgegeven in eigen beheer, Joep Schothorst, Leuven, België Alle rechten voorbehouden. Niets uit deze uitgave mag worden vermenigvuldigd en/of openbaar gemaakt worden door middel van druk, fotokopie, microfilm, elektronisch of op welke andere wijze ook zonder voorafgaandelijke schriftelijke toestemming van de uitgever. All rights reserved. No part of the publication may be reproduced in any form by print, photoprint, microfilm, electronic or any other means without written permission from the publisher. To all who have contributed… Acknowledgements After years of hard work I have finally arrived at this moment where I can take the time to thank everyone who has been part of my PhD, either as a contributor to my research or as a friend to share some drinks and good times with. It feels really exciting and strange to write these final words and I hope you will enjoy reading it as much as I have enjoyed writing it. First of all, I would like to thank my promotor Johan Thevelein for the trust you put in me as a scientist and the opportunity you gave me to perform a PhD within your lab. I have learned a lot from my time here and can honestly say that I have never met someone with a stronger passion for science, nor with a better knowledge of his fields. At certain times you appear to be a walking encyclopaedia of scientific knowledge. What I admire most is your passion and interest for science and your positive attitude and open mind towards whatever seemingly negative results come on your path. Thank you, Johan, for giving me the opportunity to be part of your lab. Secondly, I would like to give a special acknowledgement to my supervisor Griet Van Zeebroeck. Throughout my PhD, you have always welcomed me and provided me with council when I had something to discuss, independent of the matter. I have learned a lot from our academic discussions both on scientific and social level. Without your input, this thesis and my PhD experience as a whole would not have been the same. Thank you for all your efforts and support. I wish you all the best and hope you can pass on your knowledge to the new generation of PhD scientists. I would also like to thank Marta Rubio-Texeira, both for being part of my Examination Committee as well as for your social input and guidance during my PhD. I have learned a lot from you on scientific level and admire your passion and drive with regard to science. Furthermore I would like to thank the other members of my Examination Committee for their feedback and advice with regard to my thesis. Prof. Patrick van Dijck, Prof Filip Rolland, Prof. Sandra Paiva and Dr. Erwin Swinnen. I much appreciate all the time and energy you have invested in support of my thesis. This brings me to the people of the lab who have directly supported my PhD. First of all Hilde and Leni, you have taken so much work out of my hands and have taken care of so much administration that I would not have known what to do without you. Jan… there is a reason why I have repeatedly stated that you are the most important person in the lab! Thank you for all the small things you do on a daily basis and for the pleasant talks during the coffee-breaks. I would also like to thank Willy, Martine and Renata for their assistance in the experimental work and the constant willingness to provide feedback. And of course all the other technicians who keep the lab running! Furthermore I would like to thank all members of the FGM group, for the interesting discussion we have had and the good times we have spent. Michaela, Fenella, Thomas, Zhiqiang, Frederik, Sanne, Harrish, Jurgen and Dries. My time in the lab would not have been the same without you. The same is true for my office mates, who have made the long hours of a PhD a lot more bearable. I would also like to thank the students who have performed their master thesis under my supervision. Yannick, Nils and Wouter, I have been lucky to be able to guide you and have learned a lot about myself in the process. I have enjoyed working together with each of you and wish you all the best for your future. Which brings me to all the other members of our lab. I will always remember the good times we have shared both in- and outside of the lab, especially with the ´old-crew´: Jurgen, Dries, Harish, Georg, Dorotha, Jean-Paul, Frederique and the Toms! I will never forget the ´legendary´ lab meetings in Blankenberge and the trips we have embarked on. The atmosphere within the Laboratory of Molecular Cell Biology has always been amazing and is something I will dearly miss. Therefore I would like to thank all members of the lab for their enthusiasm and the good times inside the lab. I would also like to thank my Dutch friends, who have always kept me included within their friend-groups even when I was off in the ‘far south’. Especially a big thank you goes out to the chemistry-related group, Jeroen, Ingrid, Korneel, Emma, Bram, Lianne, Joris, Tine, Joris, Marcel, Heleen, Gert-Jan and Tobias, you guys are amazing! Furthermore I would like to thank Vincent for providing the necessary amount of ‘chill’ when called upon and Jasper for always being there A big thank you goes out to my family, who have supported me all the way during my PhD. Mum, Dad, Anita, my brother Bram, Oma, Yolanda, Rolf, Jan and Marina, thank you for always making time for me! A special thank you also goes out to Mariella, Mois and Boyko, who have openly welcomed me into their family and made Bulgaria feel like a second home. This brings me to the last person I have to thank, my lovely Vessy. You have always been there for me, supporting me and pushing me forward. As we have met at the start, you have always been a part of my PhD and I could not imagine it without you. Even in my most difficult times, you have managed to put a smile on my face. I clearly remember how you once came with me to the lab on a Sunday since I had to go ´feed my little yeasties’, while I really did not want to be there. You decided to come along to support me and have a look around in the lab. Your pure enthusiasm when walking throughout the lab followed by your excitement when I showed you some simple yeast cells under the microscope has really made me realize how fun my PhD actually has been. I know I have promised you at least a whole chapter as acknowledgement, but that would still not do right to the time and effort you have put in as well in support of me and this thesis. Therefore I would like to hereby dedicate this thesis in your name! Thank you all again for everything! Table of contents Acknowledgements Table of contents I List of Abbreviations VII Summary IX Samenvatting XI Chapter I Literature overview 1 1. General introduction S. cerevisiae 3 1.1. S. cerevisiae 3 1.2. S. cerevisiae´s nutrient regulated cell cycle control 4 2. Nutrient sensing pathways in S. cerevisiae 5 2.1. The cAMP-PKA pathway 5 2.1.1. PKA and its regulation 5 2.1.2. Glucose signal transduction in the cAMP-PKA pathway 7 2.1.3 Intracellular Ras-mediated signalling for activation of the cAMP-PKA pathway 8 2.1.4. GPCR-mediated signalling for activation of the cAMP-PKA pathway 9 2.1.5. Interplay between the different components of the cAMP-PKA pathway 10 2.2. Regulation of glucose uptake: the hexose transporters 11 2.2.1. The Snf3/Rgt2 glucose sensing pathway 11 2.3. Hxt5, a peculiar glucose transporter 13 2.4. FGM pathway 14 2.4.1 Nutrient transceptors 14 2.4.2 Potential signal transduction pathways in the FGM pathway 15 2.4.3. Nutrient transceptors – Gap1 17 2.4.3.1. Nitrogen homeostasis and Gap1 regulation 17 2.4.3.2. Gap1 as transceptor 18 2.4.4. Nutrient transceptors – Mep2 19 2.4.5. Nutrient transceptors – Pho84 20 2.4.5.1. Phosphate homeostasis and Pho84 regulation 21 2.4.5.2. Pho84 as transceptor 22 2.4.6. Nutrient transceptors - Sul1-Sul2 23 2.4.6.1 Sulfate homeostasis and Sul1-2 regulation 23 2.4.6.2 Sul1/2 transceptors 23 2.5. TOR Pathway 24 2.5.1 TORC1 – regulation in response to nutrient availability 25 2.5.2 TORC1 downstream effectors: Sch9 and Tap42 26 3. Regulation of micronutrient homeostasis in S. cerevisiae 27 3.1. Iron homeostasis, regulation and the putative iron transceptor Ftr1 28 3.1.1. Cellular responses upon iron limitation 28 3.1.2. Ftr1 – high affinity iron transporter 29 3.1.3. Ftr1 transcriptional and post-translational regulation 30 3.2. Zinc homeostasis, regulation and the putative zinc transceptor Zrt1 32 3.2.1. Cellular responses upon zinc limitation 32 3.2.2. The high affinity zinc uptake system – Zrt1 33 3.3. Copper homeostasis, regulation and the putative copper transceptor Ctr1 34 3.3.1. Cellular responses upon copper limitation 35 3.3.2. The high-affinity copper uptake system 35 4. Aims of this thesis and outlook 36 Chapter II Materials & Methods 39 2.1. Strains and plasmids 41 2.1.1. Yeast strains 41 2.1.2 Bacterial strains 41 2.1.3. Yeast plasmids 42 2.2. Materials 43 2.2.1. Different media used 43 2.2.1.1. Bacterial medium 43 2.2.1.2. Yeast medium Zrt1-Ftr1-Ctr1 research – Medium 43 2.2.1.3. Yeast Medium Zrt1-Ftr1-Ctr1 research – Chelators 44 2.2.1.4. Yeast medium Hxt research 44 2.2.1.5. Yeast medium uracil – adenine research 44 2.3. Methods 45 2.3.1. E. coli culture conditions 45 2.3.2. Yeast culture conditions 45 2.3.3. Molecular Biology Methods 45 2.3.3.1. Cloning and transformations 45 2.3.3.2. DNA Purification techniques 46 2.3.3.3. Site directed mutagenesis 46 2.3.3.4. Growth measurements 46 2.3.3.5. Trehalose and glycogen content determination 46 2.3.3.6. RNA extraction and qPCR 47 2.3.3.7. Trehalase assay 48 2.3.3.8. Trehalase phosphorylation assay 49 2.3.3.9. Fe55 & Zn65 uptake 49 2.3.3.10. cAMP assay 50 2.3.3.11. Expression of yeast HA-constructs 50 2.3.3.12. Expression of E. coli GST constructs 50 2.3.3.13. GST-pulldown 50 2.3.3.14. SDS-PAGE and Western Blotting 51 2.3.3.15. Budding assays - microscopy 52 2.3.3.16. Localization studies - Fluorescence microscopy 52 Chapter III Optimization of iron, zinc and copper depletion conditions in S. cerevisiae 53 3.1. Introduction 55 3.2. Results 55 3.2.1. Mere omitment of iron, zinc or copper from the media is not sufficient for deprivation 55 3.2.2. Inhibition of cell growth by the different deprivation conditions 56 3.2.3. Inhibition of budding by the different metal ion deprivation conditions 57 3.2.4. Trehalose accumulation under the different metal ion deprivation conditions 59 3.2.5. Glycogen accumulation under the different metal ion deprivation conditions 59 3.2.6. The deprivation phenotypes are specific for the deprived nutrient 59 3.2.7. Expression level of the putative transceptors under metal ion deprivation conditions as determined by qPCR 61 3.2.8. Expression level of the putative transceptors under metal ion deprivation conditions as determined by fluorescence microscopy 63 3.3 Discussion 64 3.3.1. iron-, copper- and zinc-deprivation trigger a low-PKA phenotype 64 3.3.2. The metal ion depletion conditions trigger a specific metal ion deprivation 65 3.3.3. Expression of the putative transceptors is properly induced in response to the metal ion deprivation 65 3.4. Conclusion 66 Chapter IV Characterization of Ftr1 as an iron transceptor triggering PKA activation in response to iron replenishment 67 4.1. Introduction 69 4.2. Results 69 4.2.1. Iron replenishment triggers a rapid activation of PKA 69 4.2.2. Iron mediated PKA activation is dependent on Ftr1 70 4.2.3. Ftr1 does not signal the presence of iron through the TOR or Ras-cAMP pathway 71 4.2.4. No correlation between iron uptake, intracellular iron levels and iron signalling 72 4.3. Discussion 74 4.3.1. Iron mediated activation of PKA is faster than the activation through the established macronutrient transceptors 74 4.3.2. Iron-mediated activation of PKA is independent of the TOR- and Ras-cAMP pathway 75 4.3.3. Characteristics of the iron mediated activation of PKA 75 4.3.4. What is the actual iron substrate that triggers Ftr1 mediated signalling? 76 4.3.5. Iron signalling does not correlate with iron uptake or intracellular iron levels 77 4.4. Conclusion 77 Chapter V Characterization of Zrt1 as a zinc transceptor triggering PKA activation in response to zinc replenishment 79 5.1. Introduction 81 5.2. Results 81 5.2.1. Zinc replenishment triggers a rapid increase in PKA activation 81 5.2.2. Zinc mediated PKA activation is largely dependent on Zrt1 82 5.2.3. Zrt1 does not signal the presence of zinc through the TOR or Ras-cAMP pathway 83 5.2.4. No correlation present between zinc uptake and zinc signalling 84 5.2.5. Point-mutation analysis of Zrt1 to gain insight into its signalling mechanism 85 5.2.6 Identification of cadmium, cobalt and manganese as potential Zrt1 signalling agonists 87 5.3. Discussion 88 5.3.1. zinc replenishment to zinc-deprived cells triggers a rapid Zrt1 dependent increase in PKA activity 88 5.3.2. Zrt1 acts as a transceptor in the transduction of the zinc signal to PKA 89 5.3.3. Zrt1 point mutation analysis to gain insight into its signalling mechanism 90 5.4. Conclusion 91 Chapter VI The existence of putative copper and nucleobase transceptors 93 6.1. Introduction 95 6.1.1. Copper – Ctr1 95 6.1.2. Uracil – Fur4 95 6.1.3. Adenine - cytosine/adenine permeases 96 6.2. Results 97 6.2.1. Copper replenishment triggers a weak, Ctr1 independent PKA activation 97 6.2.2. Uracil replenishment triggers Fur4-independent activation of PKA 98 6.2.3. Adenine replenishment triggers PKA activation 100 6.3. Discussion 101 6.3.1. We provide evidence to support the existence of a copper transceptor 101 6.3.2 Nucleobase re-addition to nucleobase-starved cells stimulates PKA activation 102 6.4. Conclusion 103 Chapter VII The ongoing search for the glucose transceptor in S. cerevisiae 105 7.1. Introduction 107 7.1.1. The existence of a glucose transceptor 107 7.1.2. Hxt5 as a potential glucose transceptor 107 7.2. Results 108 7.2.1. HXT5 is expressed under all the different nutrient depletion conditions known to allow transceptor mediated activation of PKA 108 7.2.2. Characterization of the influence of a hxt5 on nutrient starvation phenotypes 109 7.2.3. Hxt5 plays a role in growth recovery after nutrient starvation 110 7.2.4. Hxt5 does not influence glucose mediated signalling to PKA 111 7.2.5. Hxt5 does not influence transceptor mediated signalling to PKA 111 7.3. Discussion 112 7.3.1. Hxt5 fulfils a role in the start-up of growth upon restoration of a favourable growth medium 112 7.3.2. Hxt5 influences neither glucose nor Gap1 mediated amino acid signalling 113 7.4. Conclusion 113 Chapter VIII Interaction between the eIF2 eukaryotic translation initiation system and our identified (putative) transceptors 115 8.1. Introduction 117 8.1.1. Translation initiation 118 8.1.2. eIF2 & eIF2B 119 8.2. Results 120 8.2.1. Overcoming protein stability issues due to cation starvation 121 8.2.2. Only Ftr1 shows strong interaction with eIF2/eIF2B in vitro 122 8.2.3. The glucose transporters Hxt1 and Hxt2, but not Hxt5, interact in vitro with eIF2/eIF2B. 124 8.3. Discussion 126 8.3.1. Ftr1 strongly interacts in vitro with subunits of eIF2/eIF2B 126 8.3.2. A weak interaction established in vitro between Ctr1 and eIF2/eIF2B 127 8.3.3. No strong interaction established in vitro between Zrt1 and eIF2/eIF2B 128 8.3.4. Glucose transporters but not Hxt5 interact with eIF2/eIF2B in vitro 128 8.3.5. In vivo interaction assay necessary to confirm interaction: BiFC 129 8.3.6. Transceptor - eIF2/eIF2B system seems to be related to protein synthesis initiation rather than signal transduction towards PKA 129 8.4. Conclusion 129 Chapter IX General Discussion 131 9.1. General discussion of our results 133 9.1.1. The aim and background of our research 133 9.1.2. The transceptor concept extends towards micronutrients (and nucleobases) 133 9.1.3. Ftr1 – a novel iron transceptor 134 9.1.4. Zrt1 – a novel zinc transceptor 135 9.1.5. Copper signalling and the involvement of Ctr1 137 9.1.6. Nucleobase signalling 137 9.1.7. The elusive glucose transceptor 137 9.1.8. Signal transduction from the transceptor towards PKA – the eIF2 system 138 9.1.9. The transceptors might directly regulate protein synthesis through interaction with eIF2/eIF2B 139 9.1.10 Physiological relevance of nutrient transceptors for S. cerevisiae 140 9.1.11. The importance of our identification of novel micronutrient transceptors 141 9.2. Conclusion and outlook 142 References 143nrpages: 182status: publishe