Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 402 Studies of intracellular transport and anticancer drug action by functional genomics in yeast MARIE GUSTAVSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 UPPSALA ISBN 978-91-554-7360-0 2008 urn:nbn:se:uu:diva-9408 Dissertation presented at Uppsala University to be publicly examined in C10:301, BMC, Husargatan 3, Uppsala, Tuesday, December 16, 2008 at 13:00 for the degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted in Swedish. Abstract Gustavsson, M. 2008. Studies of intracellular transport and anticancer drug action by functional genomics in yeast. Acta Universitatis Upsaliensis. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 402. 56 pp. Uppsala. ISBN 978-91-554-7360-0. This thesis describes the use of functional genomics screens in yeast to study anticancer drug action and intracellular transport. The yeast Saccharomyces cerevisiae provides a particularly useful model system for global drug screens, due to the availability of knockout mutants for all yeast genes. A complete collection of yeast deletion mutants was screened for sensitivity to monensin, a drug that affects intracellular transport. A total of 63 deletion mutants were recovered, and most of them were in genes involved in transport beyond the Golgi. Surprisingly, none of the V-ATPase subunits were identified. Further analysis showed that a V-ATPase mutant interacts synthetically with many of the monensin-sensitive mutants. This suggests that monensin may act by interfering with the maintenance of an acidic pH in the late secretory pathway. The second part of the thesis concerns identification of the underlying causes for susceptibility and resistance to the anticancer drug 5-fluorouracil (5-FU). In a functional genomics screen for 5-FU sensitivity, 138 mutants were identified. Mutants affecting tRNA modifications were particularly sensitive to 5-FU. The cytotoxic effect of 5-FU is strongly enhanced in these mutants at higher temperature, which suggests that tRNAs are destabilized in the presence of 5-FU. Consistent with this, higher temperatures also potentiate the effect of 5-FU on wild type yeast cells. In a plasmid screen, five genes were found to confer resistance to 5-FU when overexpressed. Two of these genes, CPA1 and CPA2 encode the two subunits of the arginine-specific carbamoyl-phosphate synthase. The three other genes, HMS1, YAE1 and YJL055W are partially dependent on CPA1 and CPA2 for their effects on 5-FU resistance. The specific incorporation of [14C]5-FU into tRNA is diminished in all overexpressor strains, which suggest that they may affect the pyrimidine biosynthetic pathway. Keywords: 5-fluorouracil, tRNA modifications, Saccharomyces cerevisiae, carbamoyl phosphate synthase, V-ATPase Marie Gustavsson, Department of Medical Biochemistry and Microbiology, Box 582, Uppsala University, SE-75123 Uppsala, Sweden © Marie Gustavsson 2008 ISSN 1651-6206 ISBN 978-91-554-7360-0 urn:nbn:se:uu:diva-9408 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-9408) Distributor: Uppsala University Library, Box 510, SE-751 20 Uppsala www.uu.se, [email protected] ! " # $ % & '())*+ , - . &/.0 (*)'1+- (11.(2* & '())*+ % 30 4 5. 62- 777.782 9 $ % & 5. ' + Contents Introduction.....................................................................................................9 Saccharomyces cerevisiae..........................................................................9 Yeast: a model system in drug target identification .................................10 5-Fluorouracil...........................................................................................11 tRNA modifications .................................................................................15 tRNA pseudouridine synthases............................................................18 tRNA methyltransferases.....................................................................19 Other tRNA modifying enzymes .........................................................21 tRNA transport .........................................................................................21 tRNA stability and degradation................................................................22 Pyrimidine biosynthesis ...........................................................................23 Arginine biosynthesis...............................................................................26 Intracellular transport ...............................................................................28 The yeast vacuole ................................................................................29 Organelle acidification and the V-ATPase complex ...........................30 The CPY pathway................................................................................31 The ALP pathway................................................................................33 Endocytosis..........................................................................................34 The Cvt and autophagy pathways........................................................34 Retrograde transport ............................................................................34 Present Investigations ...................................................................................36 Aim...........................................................................................................36 Results ......................................................................................................36 Paper I..................................................................................................36 Paper II ................................................................................................38 Paper III ...............................................................................................39 Conclusions and Future perspectives............................................................41 Acknowledgements.......................................................................................43 References.....................................................................................................45 Abbreviations Pseudouridine 5-FU 5-Fluorouracil ALP Alkaline phosphatase AP Adaptor protein ATCase Aspartate transcarbamylase CH2THF 5,10-methylene tetrahydrofolate COP Coat protein CORVET Class C core vacuole/endosome tethering CP Carbamoyl phosphate CPase Carbamoyl phosphate synthase CPY Carboxypeptidase Y Cvt Cytoplasm-to-vacuole D Dihydrouridine DHOase Dihydroorotase DHODase Dihydroorotate dehydrogenase dRib-1-P Deoxyribose-1-phosphate dTMP Thymidylate dUMP Deoxyuridine monophosphate dUTP Deoxyuridine triphosphate EE Early endosome ER Endoplasmic reticulum ESCRT Endosomal Sorting Complexes Required for Transport FdUDP Fluorodeoxyuridine diphosphate FdUMP Fluorodeoxyuridine monophosphate FdUrd Fluorodeoxyuridine FdUTP Fluorodeoxyuridine triphosphate FUDP Fluorouridine diphosphate FUMP Fluorouridine monophosphate FUrd Fluorouridine FUTP Fluorouridine triphosphate GARP/VFT Golgi Associated Retrograde Protein/Vps Fifty-Three GDP Guanosine diphosphate GEF GDP/GTP exchange factor GGA Golgi-localized, gamma-ear-containing, ARF-binding GTP Guanosine triphosphate - HCO3 Bicarbonate HOPS Homotypic fusion and vacuole protein sorting LE Late endosome MTase Methyl transferase MVB Multivesicular body NPC Nuclear pore complex ODCase Orotidine-5´-phosphate decarboxylase OMP Orotidine-5´-phosphate OPRT Orotate phosphoribosyl transferase PVC Prevacuolar compartment RTD Rapid tRNA decay SNARE Soluble N-ethylmaleimide-sensitive factor attachment protein receptor TK Thymidine kinase TP Thymidine phosphorylase TS Thymidylate synthase UMP Uridine monophosphate uORF Upstream open reading frame UTP Uridine triphosphate VAM Vacuolar morphology VPS Vacuolar protein sorting Introduction Saccharomyces cerevisiae Oh, Yeast, What Art Thou? Well, for being such a small and at first glance rather insignificant little creature, yeast is an organism with a major impact on humans, giving pleas- ure as well as pain. Throughout the era of human civilization, yeast has been appreciated by mankind and the domestication of yeast has enabled us to take pleasure in what some would call the “joys of life”: wine, beer and bread. The most commercially exploited yeast and the species which people generally refer to when discussing yeast is Saccharomyces cerevisiae. S. cerevisiae is also called baker’s or brewer’s yeast, and is characterized by its capability to produce carbon dioxide or ethanol from sugar compounds. The preference of yeast for sugar as an energy source is reflected in its choice of natural habitat. Yeast frequently dwells in nature on sugary locations, and it thrives on fruits, for example grape skins. This eukaryotic, unicellular organ- ism belongs to the phylum Ascomycota of the kingdom Fungi (Guarro et al., 1999). S. cerevisiae is a budding yeast, meaning that it multiplies by asym- metrical division of the mother cell. Under certain conditions, more specifi- cally nitrogen starvation, yeast cells switch to filamentous growth by form- ing pseudohyphae (Gancedo, 2001). Although we may not consider yeast to be sexual beings, it does reproduce sexually. This is initiated by mating (or fusion) of opposing haploid cell types, a and