Studies of Intracellular Transport and Anticancer Drug Action by Functional Genomics in Yeast

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

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]

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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 pleasure 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 organism 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 specifically nitrogen starvation, yeast cells switch to filamentous growth by forming 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 (Amon, 1996). The resulting diploid can then generate haploid offspring in the form of ascospores through a meiosis event called sporulation (Neiman, 2005). The yeast haploid genome is approximately 13 Mbp in size and has around 6000 open reading frames (ORFs) dispersed over 16 chromosomes (Goffeau et al., 1996). About 70 years ago, S. cerevisiae was adopted for genetic studies in several laboratories, something which has undeniably proven to be a success story. Intense and elaborate research using yeast has provided us with in- valuable knowledge about the eukaryotic cell, in terms of understanding its genetic, cytological and physiological features. The importance of yeast as a model system was further manifested when S. cerevisiae was selected to be the first eukaryotic organism to have its genome fully sequenced (Goffeau et al., 1996). Subsequently, science has used yeast for the development of an

9 array of genomic screening methodologies, and yeast now serves as a re- nowned in vivo test system for drug target identification.

Yeast: a model system in drug target identification Identifying precise molecular targets of drug compounds is fundamental for the success of clinical therapy and for the development of novel drugs. De- spite an already extensive and long-time use of many well-established drugs, their targets are controversial or remain unspecified. More knowledge is thus required to circumvent negative side-effects and improve drug performance. Moreover, a substantial amount of molecules with therapeutic prospective value never attain pharmaceutical status since the underlying mechanism of action is unknown. In an ideal situation, a drug would target one protein or be limited to a defined cellular pathway accountable for a particular disease. Unfortunately, this is seldom the case, but with the rapid advancement of genomics and proteomics technologies together with computational methods, it is now possible to assess the global effects of drug action. Only 2% of the proteins in human cells are today recognized as drug targets, which leaves a remaining 98% as candidate targets of possible therapeutic signifi- cance (Hughes, 2002). Evaluation of drugs intended for clinical use is a complex, costly and te- dious procedure, lasting approximately 10-15 years (Auerbach et al., 2005). By integrating high throughput screening and the use of simple model organisms in the research strategy, this process can be significantly shortened. The non-pathogenic yeast Saccharomyces cerevisiae currently offers a superior non-mammalian model system for molecular studies due to a number of characteristics. Firstly, the advantageous innate features of this organism includes its rapid cell cycle of 90 minutes and the ease by which it can be grown, something that clearly provides a time- and cost-effective system in comparison to other more complex model organisms of higher order. Sec- ondly, the power of S. cerevisiae as a model system is reinforced by its genome being easily manipulated, which has generated a multitude of molecular genetic tools. Thus, yeast exhibits relatively high levels of homologous recombination enabling complete gene disruptions or gene targeting, a method that was discovered in yeast (Orr-Weaver et al., 1981). With the entire yeast genome having been sequenced (Goffeau et al., 1996), an association of laboratories commenced an imposing mission of creating a complete yeast “disruptome” i.e. knocking out all non-essential yeast genes (5000). This produced a now commercially available set of haploid and diploid deletion strains. In addition, annotated information on gene and protein functions can be found through several databases including the Sac- charomyces Genome Database (SGD) (Ball et al., 2001). Thirdly, yeast as a model system benefits from the fact that approximately 30% of all yeast

10 proteins possess human orthologues, which reflects the fact that major parts of the fundamental cellular machinery, such as the cell cycle network and the metabolic pathways, are highly conserved in all eukaryotes (Menacho- Márquez and Murguía, 2007; Bjornsti, 2002). Furthermore, as a validation of its role in medical research, almost half of the genes known to be connected to human diseases have orthologues in yeast (Menacho-Márquez and Mur- guía, 2007). The ultimate objective of anti-cancer drug discovery is to find small molecules that exclusively target properties of the tumour cell. Nevertheless, most of the prevailing anti-cancer drugs affect both tumours and normal tissue. To selectively increase the susceptibility of malignant cell to drugs, it is crucial to decipher the molecular processes that determine the cell’s sensitivity or resistance to chemotherapy. Although small and not by far as complex as a human being, yeast has gained expanding focus as a model in cancer therapeutics. A key attribute of a cancer cell is the acquisition of mutations causing an uncontrolled proliferation. Yeast and humans share several genes that cause cancer when mutated in human cells, and in cases where yeast does not possess the gene of interest, it can be humanized by introduc- ing human DNA fragments. Initially, yeast was not considered a particularly useful model system in anti-cancer research since it exhibits insensitivity to many anti-cancer agents. This problem has to large extent been resolved by deleting genes that restrain uptake of the drug or mediate drug efflux (Mena- cho-Márquez and Murguía, 2007). But undoubtedly, the extensive availability in yeast of high throughput genomics methodology must be regarded as the strongest argument for employing yeast in anti-cancer drug research.

5-Fluorouracil The antimetabolite 5-fluorouracil (5-FU) is historically one of the most pre- scribed anticancer drugs in the treatment of solid malignant tumours such as breast cancer, head and neck cancers and cancer in the gastrointestinal tract (Longley et al., 2003; Malet-Martino et al., 2002). In 2002, more than 2 mil- lion patients around the world was administered 5-FU (Seiple et al., 2006). It was introduced already in 1957 after the discovery that the utilization of radiolabelled uracil was more pronounced in rat hepatomas compared to normal cells (Rutman et al., 1954, Heidelberger et al., 1957). Despite the fact that it was one of the first anticancer drugs to be developed, the mechanism of action of 5-FU is not yet completely understood, which to some extent limits its usability in cancer therapy. Single-agent 5-FU chemotherapy is associated with an overall response-rate of only 10-15% in colorectal cancers and the poor responsiveness is greatly influenced by tumour resistance mechanisms (Longley et al., 2003). In addition, catabolic pathways remove

11 more than 80% of the administered 5-FU before it can act on target cells (Malet-Martino et al., 2002). 5-FU is synthesized by substituting hydrogen for a fluorine atom at the carbon-5 position of uracil (Figure 1) (Heidelberger et al., 1957). Since it is a pyrimidine derivate, 5-FU is subject to the same uptake mechanisms, and anabolic and catabolic pathways as uracil. However, due to its structure, 5- FU (or rather some of its metabolites) inhibits several enzymatic reactions. 5-FU in itself is thus not a cytotoxic substance, rather it depends on uptake and metabolic conversion in order to inhibit cellular proliferation (reviewed by Grem, 2000). In mammalian cells, 5-FU is anabolised to the nucleotide level resulting in three main active metabolites, fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP) (Figure 2). FdUMP and FdUTP act on DNA metabolism while FUTP interferes with RNA metabolism.

O O

H F HN HN

O N H O N H H H URACIL 5-FLUOROURACIL

Figure 1. Structures of uracil and 5-fluorouracil.

The conversion of 5-FU to FdUMP is a two step reaction starting with the formation of fluorodeoxyuridine (FdUrd) from deoxyribose-1-phosphate (dRib-1-P) and 5-FU base by thymidine phosphorylase (TP) (Figure 2) (reviewed by Longley et al., 2003). FdUrd is further phosphorylated by thymidine kinase (TK), thus producing FdUMP. FdUMP binds to thymidylate synthase (TS) which is the key enzyme in the de novo synthesis of thymidylate (dTMP). Under normal conditions, TS catalyzes the transfer of a methyl group to its natural substrate, deoxyuridine monophosphate (dUMP), in the presence of a methyl donor, 5,10-methylene tetrahydrofolate (CH2THF). The formation of dTMP is needed for DNA synthesis and repair. However, when cells are exposed to 5-FU, FdUMP instead forms a ternary

12 complex with TS and CH2THF, thereby inhibiting any further methyl transfer and dTMP synthesis (Santi et al., 1974; Sommer and Santi, 1974). Deple- tion of dTMP ultimately leads to a state referred to as thymineless death (Ahmad et al., 1998). Many organisms are also able to synthesize dTMP directly from exogenous thymidine via TK (the salvage pathway) which would be a possible way to overcome that part of the toxic effect of 5-FU which is caused by TS inactivation (Grem, 2000). In addition, inhibition of TS also leads to an accumulation of dUMP and conversion of dUMP to deoxyuridine triphosphate (dUTP), which in turn may lead to misincorporation of dUTP into DNA, thus causing DNA damage (Ingraham et al., 1982).

FUrd

5-FU FUMP FUDP FUTP RNA TP OPRT

dRib-1-P FdUrd FdUDP

FdUMP FdUTP CH2THF TS

dUMP dTMP DNA

Figure 2. Intracellular metabolism of 5-FU. 5-FU is converted to active metabolites that interfere with DNA and RNA metabolism. Abbreviations: 5-FU, 5-fluorouracil; dRib-1-P, deoxyribose-1-phosphate; FdUrd, fluorodeoxyuridine; FdUMP, fluorodeoxyuridine monophosphate; CH2THF, 5,10-methylene tetrahydrofolate; dUMP, deoxyuridine monophosphate; dTMP, thymidylate; FUMP, fluorouridine monophosphate; FUrd, fluorouridine; FUDP, fluorouridine diphosphate; FdUDP, fluorodeoxyuridine diphosphate; FdUTP, fluorodeoxyuridine triphosphate; FUTP, fluorouridine triphosphate; TP, thymidine phosphorylase; TK, thymidine kinase; TS, thymidylate synthase; OPRT, orotate phosphoribosyl transferase. Enzymes are en- circled (Modified from Longley et al., 2003).

The conversion of 5-FU to FdUTP starts with the synthesis of fluorouridine monophosphate (FUMP) from 5-FU (Figure 2). FUMP is either produced directly by the orotate phosphoribosyl transferase (OPRT) enzyme or via an intermediate product, fluorouridine (FUrd) (reviewed in Malet-Martino et

13 al., 2002). FUMP is in turn converted to fluorouridine diphosphate (FUDP). FUDP is further metabolized into fluorodeoxyuridine diphosphate (FdUDP) and finally FdUTP. FdUTP can, similarly to dUTP, be misincorporated into DNA, causing DNA fragmentation and inhibition of DNA synthesis (Ingra- ham et al., 1982). Inhibition of TS by FdUMP has long been assumed to be the primary mechanism of 5-FU action. However, this classical explanation of the anti- proliferative capacity of 5-FU has recently been challenged. For example, Pritchard et al. (1997) found that 5-FU cytotoxicity in the mouse could not be relieved by the addition of thymidine, but the toxicity was significantly reduced in presence of uridine, clearly indicating that the RNA-mediated pathway for 5-FU action is important for its cytotoxicity. Clinical trials suggest that while both mechanisms contribute to cytotoxicity, TS inhibition correlates with the response to 5-FU therapy, indicating that tumour cells are particularly sensitive to TS inhibition (Noordhuis et al., 2004). Administra- tion of uracil has therefore been used to reduce 5-FU toxicity towards other tissues, enabling the use of higher concentrations of 5-FU (Peters and van Groeningen, 1991). The conversion of FUDP to FUTP (Figure 2) creates a substrate for the RNA polymerase and FUTP is readily incorporated into all species of RNA (Parker and Cheng, 1990). Early on it was demonstrated that the formation of both high and low molecular-weight RNA is inhibited in yeast cells grown in the presence of 5-FU (Hendricks et al., 1969). Over the years, nu- merous studies have confirmed that a correlation exists between 5-FU incorporation into RNA and cytotoxicity. Inhibition of mRNA splicing and rRNA processing are some of the effects of 5-FU exposure (Longley et al., 2003). Recent results from two genome-wide yeast screens using haploinsuffiency profiling have provided evidence that lack of rRNA processing components cause hypersensitivity to 5-FU (Giaever et al., 2004; Lum et al., 2004). One such component is Rrp6p, a 3´-to-5´ exoribonuclease which is part of the nuclear exosome, a 10 component complex found both in the cytoplasm and in the nucleus (Briggs et al, 1998; Van Hoof and Parker, 1999). The exosome performs multiple tasks including processing and degradation of RNAs (van Hoof and Parker, 1999). Degradation of non-coding RNAs is facilitated by a quality control system where malfunctioned RNAs are polyadenylated and subsequently degraded by the nuclear exosome. Yeast cells subjected to 5-FU treatment accumulate pre-rRNA intermediates and in strains mutated for RRP6, the accumulation of pre-rRNA in polyadenylated form is enhanced, which suggests that 5-FU may target RNA maturation and surveillance by disturbing the function of the nuclear exosome (Lum et al., 2004; Fang et al., 2004). Interestingly, HeLa cells depleted of the human counterpart to Rrp6p, hRrp6 (PM/Scl100), are also more sensitive to 5-FU than control cells (Kammler et al., 2008).

14 Besides its effect on rRNA processing, 5-FU also acts by inactivating post-transcriptional modification enzymes that modify non-coding RNAs. Among the various species of RNA, most work regarding the effect of 5-FU on post-transcriptional modifications has focused on tRNA, specifically in prokaryotic systems. Uridines in tRNAs are easily replaced by its fluorinated analogue (Kaiser, 1971). Furthermore, the formation of tRNA modifications, in particularly pseudouridine, and 5-methyluridine and 5,6-dihydrouridine, are substantially reduced when cells are grown in presence of 5-FU (Kaiser, 1971; Tseng et al., 1978; Kaiser and Kladianos, 1981; Frendewey et al., 1982). Interestingly, the decrease in uridine modification was shown to be larger than the physical substitution of fluorouridine into tRNA (Tseng et al., 1978). These data suggested that 5-FU-containing tRNA could directly inhibit the enzymatic reactions, thus preventing further modifications. Indeed, similar to the action of FdUMP on thymidylate synthase, pseudouridine synthases and methyltransferases acting on uridine form highly stable complexes with RNAs containing fluorinated uridines (Santi and Hardy, 1987; Samuelsson, 1991). Furthermore, Hoskins and Butler (2008) recently suggested that the enhanced 5-FU toxicity in an rrp6 mutant is mediated by the tight binding of the Cbf5p pseudouridine synthase to 5-FU-containing rRNAs. tRNA modifications The smallest of the non-coding RNA species is tRNA, which is employed as an adaptor molecule in translation of mRNA into protein. The tRNAs repre- sent a highly abundant class of RNAs, due to a high rate of production, but also stability. tRNAs are highly stable structures with a half-life of hours or even days. A linear tRNA molecule folds to form a secondary cloverleaf structure made up of four prominent domains, the acceptor stem, the D-arm, the anticodon arm and TC-arm (Figure 3) (Nakanishi and Nureki, 2005). In addition, tRNAs may have an extra variable loop located between the anticodon arm and the TC-arm. The cloverleaf further folds to adopt a tertiary L-shaped structure. tRNA synthesis in eukaryotic organisms starts with transcription of tRNA genes by RNA polymerase III (Sprague, 1995). The Saccharomyces cerevisiae nuclear genome contains 274 tRNA encoding genes, of which 59 codes for intron-containing tRNAs, and they are divided into 42 different families based on codon specificity (Hani and Feldmann, 1998; Abelson et al., 1998). Precursor tRNAs mature trough several ordered processing events (Wolin and Matera, 1999). Processing of pre-tRNAs starts with exo- and endonucleolytic trimming of the 5´leader and 3´trailer of the tRNA and is followed by the addition of a CCA trinucleotide to the 3´ end of the tRNA acceptor stem, a reaction performed by the ATP(CTP):tRNA nucleotidyl-

15 transferase Cca1p (Aebi et al., 1990). Nascent tRNAs with intervening sequences must also undergo splicing.

3´ A C ACCEPTOR STEM C 5´

TC ARM D ARM

VARIABLE LOOP

ANTICODON ARM

Figure 3. A schematic picture of the cloverleaf structure of tRNA.

As part of the tRNA processing pathway, nucleosides in tRNAs are subject to a large number of posttranscriptional chemical modifications (Figure 4). Although nucleoside modifications are present in all non-coding RNAs, tRNAs are by far the most decorated species and to date, 86 tRNA modifications have been identified (Johansson and Byström, 2005). tRNA modifications are consistently found in all organisms and yeast cytoplasmic tRNAs possess 25 of the known nucleoside modifications (Sprinzl et al., 1998). Certain modifications exist at more than one position in the tRNA while others are limited to one specific location. The latter type of position-specific modifications are often evolutionary conserved (Agris, 2008). Enigmatically, many tRNA modifications appear to be redundant for tRNA function, at

16 least under standard conditions, since mutants of the corresponding modification enzymes lack any apparent phenotypes (Hopper and Phizicky, 2003). For that reason, the biological role of many tRNA modifications remains to be determined. It is, however, well established that certain modifications of nucleosides in the anticodon loop or its surroundings are critical for reading frame maintenance and correct aminoacylation (Urbonavicius et al., 2001; Nakanishi and Nureki, 2005). Proposed roles of modifications in the remaining parts of the tRNA, specifically in the D-arm and TC-arm, are im- provement of tRNA stability as well as a function in tRNA export to the cytoplasm (Alexandrov et al., 2006; Helm, 2006; Lipowsky et al., 1999). The discussion below will primarily focus on those modification enzymes which are discussed in paper II of this thesis.

O O O H3 C H C C + 3 N N N HN HN N

N N N H N H3C N H N 2 N N 2 N

H3C 1 2 7 m G m 2G m G O O

HN NH HN

O O N

CH 3 HNCOCH HNCH2CH C 3 CH3 N N N

N N N O

6 4 i A ac C

Figure 4. A subset of modified nucleosides found in yeast tRNA. Abbreviations: 1 2 2 2 7 m G, 1-methylguanosine; m 2G, N N -dimethylguanosine; m G, 7- methylguanosine; , pseudouridine; D, dihydrouridine; i6A, N6- isopentenyladenosine; ac4C, N4-acetylcytidine (Modified from Johansson and By- ström, 2005).

17 tRNA pseudouridine synthases Pseudouridine () was discovered in yeast in 1957 as the “fifth nucleotide” and it was the first nucleoside modification ever to be identified (Davis and Allen, 1957). Pseudouridine is the most prevalent modification found in tRNA, rRNA, snRNA and snoRNA (Charette and Gray, 2000). Modification of uridine residues into pseudouridine does not involve any addition of new side chains or atoms, since pseudouridine just is a C-C glycosyl isomer of the native uridine (Foster et al., 2000). Isomerization starts with the cleavage of the glycosidic bond linking the base via the N1 position to the ribose. The uracil base is rotated 180 and the C5 carbon is then connected to the C1´ carbon of the sugar leaving an extra N1-H group on the base accessible for hydrogen bonding. The family of enzymes responsible for catalyzing the formation of pseudouridine is referred to as pseudouridine synthases. The first identification and characterization of a tRNA pseudouridine synthase was done in Salmo- nella typhimurium (Chang et al., 1971; Singer et al., 1972; Cortese et al., 1974) and soon after in Escherichia coli (Bruni et al., 1977). Sequence differences have been used to divide the pseudouridine synthases into five distinct families, named after the E. coli representative for each class, TruA, TruB, TruD, RsuA, RluA (Hur et al., 2006). Pseudouridine is ubiquitous in mature tRNAs and at position 55 is a universally conserved modification throughout the kingdoms of life and also the reason for naming the TC stem loop (Charette and Gray, 2000). The biological function of in tRNA (or other non-coding RNAs) is not completely understood, but it has been proposed to be involved in tRNA structural maintenance, accurate ribosomal binding, and anti-codon specificity. Seven genes predicted to encode tRNA pseudouridine synthases which act on cytoplasmic tRNAs have been identified in Saccharomyces cerevisiae: PUS1, DEG1 (PUS3), PUS4, PUS6, PUS7, RIB2 (PUS8) and PUS9 (Johansson and Byström, 2005). Some of the tRNA:-synthases are substrate- and site-specific, while others are remarkably promiscuous. Consider- ing the widespread occurrence of pseudouridine in tRNAs, it is noteworthy that many pseudouridine synthases are not required for viability. Synthetic lethality data does, however, suggest that some of the enzymes redundantly provide essential functions in yeast (see below). Pus1p is a multi-site specific pseudouridine synthase which introduces at positions 1, 26, 27, 28, 34, 35, 36, 65 and 67 of tRNAs and also at position 44 of U2 snRNA (Simos et al., 1996; Motorin et al., 1998; Massenet et al. 1999; Behm-Ansmant et al., 2006). The Pus1 protein is located in the nucleus, which correlates with the observation that formation of 34, 35 and 36 in some tRNA species is intron-dependent (Simos et al., 1996; Motorin et al., 1998). Pus1p is also linked to the nuclear export machinery. Thus, Simos et al. (1996) identified PUS1 as a suppressor of a temperature sensi-

18 tive mutant of nsp1, a gene encoding a nucleoporin participating in nucleocytoplasmic transport. They were also able to show that cells with a disruption of both pus1 and los1 have a reduced growth rate at 30 ºC and viability is completely lost at 37 ºC. The role of the export receptor Los1p is to transport tRNAs over the nuclear membrane, it has therefore been proposed that formation by Pus1p helps to distinguish between functional and non- functional tRNAs prior to Los1p-mediated export (Sarkar and Hopper, 1998; Wolin and Matera, 1999). The association of Pus1p to tRNA transport it further strengthened by evidence that the minor tRNAIle(UAU) accumulates in the nucleus in a pus1 mutant (Grosshans et al., 2001). Considering the fact that few tRNA:-synthases display any mutant phenotype, it is intriguing that a pus1 pus4 double mutant is inviable (Grosshans et al., 2001). The non- essential Pus4p enzyme modifies the U55 position of cytoplasmic and mitochondrial tRNAArg (Becker et al., 1997). The synthetic lethality of the pus1 pus4 double mutant suggests that at least some tRNA pseudouridinylation is essential in yeast. However, to decipher the biological role Pus1-mediated formation in tRNAs, more work needs to be done. tRNA methyltransferases Besides pseudouridine, methylation is the most frequent and widely distributed modifications of tRNA nucleosides. This modification is found at multiple positions on all four bases of tRNA, and methylation can also occur at the 2-O position of riboses (Johansson and Byström, 2005). The enzymes responsible for this modification are methyl transferases (MTases) and MTa- ses that modify tRNA are designated as Trm (for tRNA methyltransferase) in yeast. tRNA MTases are typically more site-specific than -synthases meaning that they exclusively modify only one or a few positions in the tRNA. RNA MTases make use of the methylated sulphur compound S-adenosyl- L-methionine (AdoMet or SAM) as a donor of methyl groups (Graham and Kramer, 2007). Transmethylation is catalyzed by the MTases after binding to the cofactor AdoMet and the RNA substrate, and most AdoMet-dependent MTases share a common AdoMet-binding motif. To date, twelve enzymes responsible for simple base methylations of cytoplasmic tRNAs have been identified in Saccharomyces cerevisiae. Seven of these are involved in modification of guanosine at various positions in 2 1 7 tRNA; Trm1p (m 2G26), Trm5p (m G37), Trm8p/Trm82p (m G46), Trm10p (m1G9) and Trm11p/Trm112p (m2G10) (Ellis et al., 1986; Björk et al., 2001; Alexandrov, et al., 2002; Jackman et al., 2003; Purushothaman et al., 2005). Furthermore, Trm6p/Trm61p (m1A58) is responsible for adenosine methylation and Trm4 (m5C34, 40, 48, 49) catalyzes modifications of cytidine (Andersson et al., 1998; Motorin and Grosjean, 1999). Finally, methylation of uridine is catalyzed by Trm2p (m5U54) and Trm9p (mcm5U34, mcm5s2U34) (Nordlund et al., 2000; Kalhor and Clarke, 2003). Notably,

19 Trm8p/Trm82p, Trm11p/Trm112p and Trm6p/Trm61p are heterodimeric methyltransferases. Of all of these MTases, only the Trm6p/Trm61p complex, which catalyzes the formation of m1A58 in the TC loop, is essential for viability (Andersson et al., 1998). Mutant alleles of either trm6 or trm61 with reduced functions cause a significant reduction in levels of mature Met tRNAi but do not affect any of the other tRNA species examined. As a Met high copy number plasmid carrying the initiator tRNA (tRNAi ) can sup- press the temperature-sensitive phenotype of a trm6 mutant it was suggested that the essential function of m1A58 is to maintain adequate levels of mature Met tRNAi in the cell. The TRM1 gene, which is responsible for N2,N2-dimethylguanosine 2 (m 2G) formation at position 26 of tRNAs, was isolated by genetic comple- mentation in 1986 by Ellis and partners. Later analysis of the TRM1 gene and its mRNA showed that this gene encodes two isozymes (Ellis et al., 1987). Translation initiated from either of two in-frame AUGs, separated by 15 codons, can thus give rise to two distinct TRM1 products. Thus, Trm1p 2 translated from the second AUG primarily catalyses the formation of m 2G in cytoplasmic tRNAs while modifications of mitochondrial tRNAs is catalyzed by both isozymes. The yeast Trm8p/Trm82p MTase contains two protein subunits. This enzyme catalyses the formation of 7-methylguanosine at the 46th position (m7G46), within the extra loop of tRNAs, both in vivo and in vitro (Alexan- drov et al., 2002). The m7G modification is highly conserved in both prokaryotes and eukaryotes and supposedly present in 11 yeast tRNAs. Interest- ingly, the m7G46 forming enzyme in bacteria is composed of only one subunit, called YggH in Escherichia coli, which is an orthologue of Trm8p (De Bie et al., 2003). Furthermore, Alexandrov et al. (2005) showed that YggH alone can restore m7G-methyltransferase activity in a yeast trm8 trm82 double mutant. The exact roles of Trm8p and Trm82p within the complex are still somewhat unclear. The amino acid sequence of Trm8p contains a SAM- binding domain and Trm8p, not Trm82p, is able to crosslink to tRNA, suggesting that Trm8p provides the catalytic activity (Alexandrov et al., 2005). This is also consistent with the fact that it is the Trm8p homolog alone that provides the catalytic activity in E. coli. Following the observation that the levels of endogenous Trm8p were diminished in a trm82 deletion strain, it was proposed that Trm82p primarily controls Trm8p protein levels in the cell. The TRM10 gene codes for the MTase responsible for m1G formation at position 9 (Jackman et al., 2003). The prevalence of m1G9 in many eukaryotic species indicates that this modification is highly conserved. None- theless, a yeast strain deleted for TRM10 does not show any apparent growth defect. Recently, Lee et al. (2007) performed protein expression profiling using a GFP library in order to survey protein levels in response to methyl methanesulphonate (MMS), a DNA-damaging agent, and Trm10p was iden-

20 tified as one of the proteins that show an increased expression in presence of MMS. As discussed below, we found that a trm10 mutant was the most 5-FU sensitive mutant in the entire yeast knockout strain collection.

Other tRNA modifying enzymes A small set of yeast tRNAs contain a N6-isopentenyladenosine (i6A) at the 37th position adjacent to the 3´ end of the anticodon. Laten, Gorman and Bock (1978) isolated a S. cerevisiae mutant where levels of i6A were diminished to 1.5% of the wild type level and this mutant was deficient in i6A modification of both nuclear and cytoplasmic tRNAs (Martin and Hopper, 1982). The gene MOD5 was later cloned and shown to encode an enzyme that transfers isopentenyl to adenosine (Dihanich et al., 1987). MOD5 shares the intriguing feature of TRM1 in that is has multiple translation initiation sites, a mechanism for targeting products transcribed from one single gene to different subcellular compartments (Najarian et al., 1987). The rather unusual N4-acetylcytidine (ac4C) modification in yeast requires the product of the recently characterized TAN1 gene (Johansson and By- ström, 2004). Two yeast tRNA species, specific for serine and leucine, possess an ac4C modification at position 12. The involvement of TAN1 in ac4C formation in at least one of the two tRNAs is evident from the reduction of mature tRNASer(CGA) levels in a tan1 mutant strain. Despite the mounting evidence for a role of TAN1 in acetylation of cytosine, there is yet no evidence that Tan1p is an actual acetyltransferase. Given the fact that Tan1p can bind tRNA, Johansson and Byström (2005) did, however, propose that Tan1p is part of a tRNA acyltransferase complex. Finally, the known dihydrouridine (D) synthases in yeast comprise four enzymes, Dus1p (D16, D17), Dus2p (D20), Dus3p (D47) and Dus4p (D20), none of which can compensate for loss of any of the other three (Xing et al., 2002; Xing et al., 2004). Dihydrouridine is one of the most common modifications in tRNA and it is predominantly found in the D-arm, hence the name of its arm. Given how common Ds are in tRNAs, remarkably few studies have so far been done on the dihydrouridine synthases, particularly in yeast. tRNA transport In eukaryotic cells, the nucleus is spatially separated from the cytoplasm by a double-membrane nuclear envelope. Cytoplasmic RNAs that are transcribed in the nucleus must therefore move across the nuclear membrane in order to fulfil their functions in the cytoplasm. Movement of molecules across the membrane is facilitated by nucleocytoplasmic transport through a membrane-perforating nuclear pore complex (NPC) that is scattered throughout the nuclear envelope (Lim et al., 2008). The NPC is constituted

21 of nucleoporin (Nup) proteins, and active transport through the NPC is an energy-dependent process requiring a small Ran GTPase and shuttling cargo receptors. These receptors proteins predominantly belong to the importin family which in yeast is a family of 14 proteins (Hopper and Shaheen, 2008). As already mentioned above, Los1p, one yeast member of the importin family, functions as a nuclear exportin that translocates tRNAs to the cytoplasm. Originally, mutants of los1 were shown to accumulate pre-tRNAs with re- tained introns, which suggested that Los1p could play a role in tRNA splicing (Hopper et al., 1980; Sharma et al., 1996). However, Sarkar and Hopper (1998) found that efficient export of tRNAIle(AAU), a tRNA transcribed from a intronless gene, also relies on Los1p. Moreover, Los1p-dependent transport is restricted to a subset of tRNAs, as shown by the fact that tRNAGlu and tRNAGly are exported from the nucleus even in the absence of the LOS1 gene (Grosshans et al., 2000). Evidently, yeast cells must have optional routes for managing tRNA export, something which is further supported by the fact that LOS1 is a dispensable gene. Cca1p, the CCA adding enzyme which provides tRNAs with mature 3´ends, is also a nuclear/cytoplasm shuttling protein and can override the tRNA transport defects of a los1 mutant when overexpressed (Feng and Hopper, 2002). Considering the lack of synthetic interactions between los1 and cca1 mutants, additional pathways for tRNA export are likely to exist. Structural integrity of tRNAs seems to be a prerequisite for exportin recognition and interaction. In Xenopus laevis, mutations of nucleotides in the TC arms obstruct binding of Xpo-t (the orthologue to Los1p) to tRNA and subsequently prevent export of the tRNAs (Lipowsky et al., 1999). A similar mechanism may also exist in yeast. Thus, Cleary and Mangroo (2000) showed that point mutations in the D-loop of RNATyr lead to nuclear tRNA retention and that this effect is alleviated by overexpressing LOS1. tRNA stability and degradation The exosome operates as the RNA-surveillance apparatus of the cell and perform multiple tasks, including degradation of aberrant pre-RNAs (House- ley et al., 2006). Ten essential 3´5´exoribonucleases assemble to form the core exosome. However, one additional subunit binds to the complex, and this subunit is either Rrp6p or Ski7p, depending on whether the exosome is located in the nucleus or in the cytoplasm (Allmang et al., 1999; Araki et al., 2001). The co-factor complex TRAMP (Trf4 or Trf5/Air1 or Air2/Mtr4), adds poly(A) tails to non-coding RNAs destined for disposal and this tail acts as a signal for exosome activation (Schmid and Jensen, 2008). Two major pathways for degradation of hypomodified tRNAs are currently known. In 2004, Kadaba and coworkers made the discovery that hy-

22 pomodified tRNAs are subject to exosome-mediated degradation as part of a quality surveillance system in yeast. Moreover, in yeast cells lacking m1A58, Met a modification catalysed by the Trm6p/Trm61p MTase complex, tRNAi becomes unstable and is subject to degradation (Anderson et al., 1998). A subsequent genetic screen for spontaneous suppressors of the temperature sensitive phenotype of a trm6 mutant identified two members of the TRAMP/exosome machinery, the poly(A) polymerase TRF4, and the exoribonuclease RRP44 (Kadaba et al., 2004). Trf4p was shown to be required for Met polyadenylation of hypomodified pre-tRNAi and hypomodified pre- Met tRNAi which still contains its 5´leader and 3´extension is substrate for polyadenylation (Kadaba et al., 2004; Kadaba et al., 2006). Finally, degrada- Met tion of hypomodified tRNAi takes place in the nucleus, which is consistent with the fact that Rrp6p, the nucleus-specific subunit of the exosome, is re- Met quired for elimination of hypomodified pre-tRNAi (Kadaba et al., 2004). The recently discovered rapid tRNA decay (RTD) pathway is independent of Trf4p/Rrp6p and seems to act on hypomodified but mature tRNAs. As already mentioned, deletion of genes coding for tRNA modifying enzymes with targets outside the anticodon loop rarely result in any detectable growth phenotypes. In sharp contrast, Alexandrov et al. (2006) reported that the combined loss of either TRM8 or TRM82 and genes coding for other modification enzymes causes a severe growth defect 37 ºC. Northern Blot analysis revealed a surprisingly rapid reduction in steady-state levels of certain tRNAs, in particular tRNAVal(AAC), in the trm4 trm8 mutant, which was proposed to be due to structural instability of tRNAs lacking m5C49 and m7G46 modifications. In this case, neither Trf4p nor Rrp6p had an effect on tRNA degradation. It was further shown that tRNAVal(AAC) is subject to deacylation prior to degradation which strengthens the notion that the RTD pathway acts on mature tRNAs (Alexandrov et al., 2006; Chernyakov et al., 2008). Some of the proteins in the RTD pathway have recently been identified, and it appears that more than one tRNA species passes through this route (Chernya- kov et al., 2008).

Pyrimidine biosynthesis Pyrimidines nucleotides are fundamental building blocks in DNA and RNA synthesis, and two routes serve to provide the cell with the primary substrate for pyrimidine production, uridine monophosphate (UMP): the de novo pyrimidine biosynthetic pathway and the pyrimidine salvage pathway. The de novo synthesis of pyrimidines is a highly conserved pathway of six consecutive steps (Figure 5) (reviewed by Huang and Graves, 2002). Production of pyrimidines begins in the cytoplasm with ATP, glutamine and - bicarbonate (HCO3 ) which are used to form carbamoyl phosphate (CP) (reviewed by Evans and Guy, 2004). In the subsequent reaction, carbamoyl

23 aspartate (CP Aspartate) is produced from CP and aspartate. The enzyme responsible for synthesis of CP used in the pyrimidine biosynthesis is carbamoyl phosphate synthase CPS II (or CPase P). Formation of CP is not exclusive to the pyrimidine pathway, it is also used as a precursor for arginine biosynthesis, a process in which another CP synthase, CPS I (also known as CPase A), is involved (see the discussion below about arginine biosynthesis). Since CP is synthesized in two distinct cellular processes, the formation of carbamoyl aspartate can be seen as the first unique step in pyrimidine biosynthesis.

Glutamine ATP CPS II - CPS I HCO3 Ura2 Cpa1 Cpa2

CP CP + CP Aspartate Ornithine

Dihydroorotate uracil Citrulline Orotate Fur4 Argininosuccinate OMP

Fur1 UMP Arginine

Figure 5. The pathways for de novo pyrimidine biosynthesis and arginine biosynthesis in yeast. Both pathways start with the synthesis of carbamoyl phosphate (CP) - from glutamine, ATP and bicarbonate (HCO3 ). The first enzyme in the pyrimidine biosynthetic pathway is the bifunctional CPS II or Ura2p, which is responsible for the production of CP and carbamoyl aspartate (CP Aspartate). CP aspartate is further converted into dihydroorotate, orotate, orotidine-5´-phosphate (OMP), and finally uridine monophosphate (UMP). UMP is also generated by the salvage of uracil from the surroundings, in which Fur4p mediates uracil uptake and Fur1p catalyzes the one-step conversion of uracil to UMP. The carbamoyl phosphate synthase CPS I is a complex consisting of two subunits, Cpa1p and Cpa2p, in yeast. After synthesis of CP by Cpa1p and Cpa2p, CP condensates with ornithine to produce citrulline. From citrulline, argininosuccinate and subsequently arginine is produced.

The first two enzymatic conversions of the de novo pathway are catalyzed by the carbamoyl phosphate synthase Ura2p in Saccharomyces cerevisiae (La-

24 croute, 1968). The bifunctional Ura2 protein contains multiple domains, including catalytic domains for both a carbamoyl phosphate synthase (CPase) and an aspartate transcarbamylase (ATCase) activity (Lacroute, 1968; Souciet et al., 1989). A dihydroorotase (DHOase) domain is also present in Ura2p, making this protein a potential catalyst of the third step in pyrimidine biosynthesis, however, this domain is defective in yeast Ura2p (Souciet et al., 1989). In contrast, the mammalian orthologue CAD has functional CPase, ATCase as well as DHOase domains, and is thus able to cata- lyze all three reactions (Coleman et al., 1977). Being the initial and rate-limiting step, carbamoyl phosphate production by Ura2p is subject to strong feedback regulation. Intracellular uridine triphosphate (UTP) thus inhibits the activity of both the CPase and the AT- Case (Lacroute, 1968). UTP appears to downregulate Ura2p activity by binding to the CPase domain which in turn also inhibits ATCase function (Antonelli et al., 1998). ATCase in itself is not susceptible to UTP regulation, suggesting that communication between the two domains is required for full feedback inhibition of Ura2p. The URA2 gene is also subject to transcriptional repression by UTP (Lacroute, 1968; Potier et al., 1990). Carbamoyl aspartate is further converted to dihydroorotate by the DHOase Ura4p in yeast, and in the fourth step, the dihydroorotate dehydrogenase (DHODase) Ura1p catalyzes formation of orotate (Lacroute, 1968). The fifth step consists of formation of orotidine-5´-phosphate (OMP) from orotate and is carried out by either of two orotate phosphoribosyl transferase (OPRTase) enzymes, Ura5p or Ura10p, and in the final reaction of the de novo pathway, the orotidine-5´-phosphate decarboxylase (ODCase) Ura3p forms UMP (de Montiguy et al., 1989; de Montiguy et al., 1990; Lacroute, 1968). From this point, UMP can be converted to UTP and CTP which are incorporated into RNA or used for production of deoxypyrimidines. The second pathway by which pyrimidines are generated is through the salvage of bases or nucleosides, and through uptake from the surrounding environment, a process that is mediated by plasma membrane bound permeases. The FUR4-encoded permease facilitates uptake of uracil bases, whereas Fui1p is a transporter of uridine (Chevallier, 1982: Wagner et al., 1998). It should be noted that Fur4p and Fui1p also are able to take up the toxic analogs of their natural substrates, 5-fluorouracil and 5-fluorouridine, respectively (Jund and Lacroute, 1970). As in the case of the de novo synthesis, the initial step of the salvage pathway is subject to tight regulation in order to maintain a balanced intracellular pool of uracil. Thus, in response to excess uracil, the turnover rate of the FUR4 transcript and the Fur4p permease both increase (Serón et al., 1999). After the uptake of uracil into the cell, UMP is formed by the activity of a phosphoribosyltransferase, Fur1p (Kern et al., 1990). Taking into account that Fur1p recognizes 5-FU as a substrate, which allows for a direct conversion of 5-FU into metabolically

25 active compounds, this enzyme has gained attention in anticancer research (Erbs et al., 2000). Due to its importance as an early anticancer drug, 5-FU has been studied in several model systems. Palmer et al. (1975) isolated a number of Aspergil- lus nidulans mutants conferring resistance to fluoropyrimidines, including 5- FU. These mutants turned out to be involved in the uptake and conversion of fluoropyrimidines as well as in the regulation of de novo synthesis of pyrimidines. Some of these mutations affected enzymes of the arginine biosynthetic pathway, presumably causing an increase in the pool of CP and subsequently an increased production of uracil analogous to the genes isolated in our overexpression screen (paper III). Furthermore, a 5-FU resistant Salmonella typhimurium mutant has been described, in which the activity of carbamoyl phosphate synthase was elevated, resulting in higher amounts of UTP and CTP (Jensen et al., 1982). Taken together, these previous studies suggest that 5-FU resistance is linked to the intracellular concentration of uracil which in turn is correlated with the production of CP, either from the pyrimidine or the arginine biosynthetic pathway. It is also important to note that the pyrimidine biosynthetic CPase (CPS II) has been shown to be upregulated in several hepatomas and other carcinomas, something which presumably could influence the responsiveness of cancer patients to treatment with fluoropyrimidines (Aoki and Weber, 1981; Denton et al., 1982; Reardon and Weber, 1985; Reardon and Weber, 1986). 5-FU resistant tumours are otherwise commonly associated with high levels of TS (Banerjee et al., 2002). The TS activity is therefore regarded as an important predictive biomarker which can determine whether chemotherapy with 5-FU will be successful. However, TS is not the only determinant for 5-FU resistance and several genome-wide studies have therefore been undertaken in recent years in order to search for novel resistance biomarkers. Interestingly, Schmidt et al. (2004), who did gene expression profiling of 5-FU resistant human colon cancer cells, identified the pyrimidine-specific carbamoyl phosphate, CAD (CPS II) as one of the upregulated genes after looking specifically at genes involved in the pyrimidine biosynthesis. Our work (paper III) suggests that the arginine-specific CPase (CPA1) also could be a potential resistance marker in carcinoma cells.

Arginine biosynthesis Besides the de novo biosynthesis of pyrimidines, carbamoyl phosphate is synthesized and utilized in the arginine biosynthetic pathway. In yeast, the start of arginine production is the mitochondrial conversion of glutamate and acetylcoenzyme A to N-acetylglutamate (Jauniaux et al., 1978). After four additional reactions, ornithine is formed as the end product. At this point, ornithine is condensated with CP in the cytosol, thus forming citrulline, and

26 from citrulline, argininosuccinate and finally arginine are produced in two successive steps (Figure 5). In mammals, the bifunctional urea cycle starts at the condensation of ornithine and CP (Shambaugh, 1977; Morris, 2002). The urea cycle operates primarily in the liver as a supplier of arginine but most importantly, it is responsible for removing toxic ammonia by conversion to urea. In this cycle, arginine is cleaved yielding urea and ornithine. Urea is excreted while ornithine is reused for a new round of ammonia disposal. In contrast, yeast primarily gets rid of excess nitrogen by excreting certain amino acids (Hess et al., 2006). The CP synthase that is specifically involved in the arginine biosynthetic pathway, CPS I, is an oligomeric protein in yeast encoded by the unlinked nonessential CPA1 and CPA2 genes (Lacroute et al., 1965). The subunits of CPS I are of different sizes, the small component corresponds to Cpa1p and the larger to Cpa2p (Piérard and Schröter, 1978). Similar to the de novo pathway for pyrimidine biosynthesis, CP production by CPS I starts with - ATP, glutamine and HCO3 . In this case, Cpa1p is the amidotransferase which makes it capable of hydrolyzing glutamine and transferring an ammonium molecule to Cpa2p (Piérard and Schröter, 1978; Nyunoya and Lusty, - 1984). Cpa2p uses this ammonium together with ATP and HCO3 to make CP (Piérard and Schröter, 1978). The use of glutamine as a nitrogen source by the CPS I complex therefore depends on the initial activity of Cpa1p, whereas the use of NH3, if supplied externally, only requires Cpa2p. CPS I is under negative feedback control by the final product of this pathway, arginine. In the presence of arginine, CPS I is thus repressed, as revealed by studies using a ura2 mutant (Lacroute et al., 1965). Important to note, CPS I is subject to two different regulatory mechanisms. Thus, in media rich in amino acids, there is transcriptional repression resulting in a decrease of CPA1 and CPA2 mRNA (Piérard et al., 1979; Messenguy et al., 1983). This effect is attributed to the general amino acid control machinery, as repression can be abolished by starvation for other amino acids as well as for arginine. Specific control of CPS I is a mediated by arginine alone and only affects the small Cpa1 subunit (Piérard et al., 1979). Thus, in minimal media supplemented with arginine, CPA2 mRNA and Cpa2 protein amounts are at the same levels as in media lacking arginine, suggesting that arginine does not regulate this subunit (Messenguy et al., 1983). In contrast, the Cpa1 protein levels are significantly reduced in response to arginine addition. However, arginine does not seem to control transcription of the CPA1 gene. Instead, there is translational repression of the CPA1 mRNA due to an arginine attenuator peptide (AAP) encoded by an upstream open reading frame (uORF) of the CPA1 mRNA (Werner et al., 1987; Delbecq et al., 1994). In a high arginine environment, APP appears to stall ribosomes thus hindering translation of the CPA1 mRNA (Wang et al., 1999). Exactly how this occurs is, however, still unclear.

27 Intracellular transport The eukaryotic cell is separated from the surrounding environment by a plasma membrane and it is also compartmentalized in that it harbors several different membrane-bound organelles. Transport of proteins between these compartments is a process of fundamental importance, and to ensure that proteins end up in the correct location, intracellular transport must therefore be under tight control. The movement of proteins in the cell follows several trafficking pathways for lipid vesicles, including exocytosis, endocytosis, retrograde transport, cytoplasm to vacuole (Cvt) transport, autophagy, and the vacuolar CPY and ALP pathways (Figure 6).

EXOCYTOSIS ENDOCYTOSIS

CPY NUCLEUS LE/MVB/PVC RETROGRADE ALP

ER GOLGI CVT VACUOLE

AUTOPHAGY

Figure 6. Transport pathways in the yeast cell. Abbreviations: ALP, alkaline phosphatase pathway; CPY, carboxypeptidase Y pathway; CVT, cytoplasm-to-vacuole pathway; EE, early endosome; ER, endoplasmic reticulum; LE, late endosome; MVB, multivesicular body; PVC, prevacuolar compartment.

Transport to the vacuole is particularly complex in that it involves several different pathways. New proteins destined for the vacuole first use the biosynthetic/secretory pathway, going through the endoplasmic reticulum (ER) and the Golgi, before being diverted to the vacuole, whereas the endocytotic route delivers extracellular material and plasma membrane proteins to the

28 vacuole (Alberts et al., 1994). Cargo from both routes pass through a transi- tional membrane-enclosed compartment variously referred to as the late endosome, the prevacoular compartment (PVC), or the multivesicular body (MVB) (Kucharczyk and Rytka, 2001). In addition, the vacuole receives material directly from the cytosol, intended for degradation and/or biosynthetic reuse, through the autophagy and Cvt pathways. As mentioned above, intracellular trafficking relies on lipid vesicles which bud off from donor membranes and merge with acceptor membranes with the aid of complex protein machinery. Trough a number of genetic screens, a large number of genes that take part in intracellular transport to the vacuole have been identified and many of these genes are named after the screens in which they were discovered, for example the vacuolar protein sorting (VPS) or vacuolar morphology (VAM) genes (Rothman and Stevens, 1986; Bankaitis et al., 1986; Robinson et al., 1988; Rothman et al., 1989; Raymond et al., 1992; Bonan- gelino et al., 2002; Wada et al., 1992; Seeley et al., 2002). By using drugs that interfere with intracellular transport pathways, it is possible to find novel genes involved in trafficking. One such drug is Mo- nensin, which functions as a Na+/H+ ionophore and presumably inhibits transport by neutralizing the trans-Golgi network (Dinter and Berger, 1998). Furthermore, the previously identified VPS genes are frequently recovered in genome-wide screen for sensitivity to various drug compounds, not only drugs affecting intracellular transport (for example see Aouida et al., 2004; Hellauer et al., 2005, Ericson et al., 2008). This is not surprising since the vacuole is the primary detoxification and degradation organelle of the cell (Li and Kane, 2008), and loss of vacuolar transport can therefore result in general drug sensitivity. The properties of the vacuole, the trafficking pathways that lead to the vacuole and the proteins that operate in these routes will be summarized below.

The yeast vacuole The most prominent organelle in the yeast cell is the vacuole, which is the yeast counterpart of the lyzosome in higher eukaryotes (Alberts et al., 1994). Its important role is demonstrated by the many physiological processes in which it plays the starring role, including degradation and macromolecule turnover, pH and ion homeostasis, detoxification and nutrient storage. Vacuolar degradation is needed for constitutive turnover of macromolecules, generation of new building blocks for biosynthetic reactions, removal of aberrant material and regulation of signaling molecules (Ostrowicz et al., 2008). Vacuolar degradation of integral plasma membrane proteins is important in the response to, and adaptation to, changes in the external environment (Smythe and Warren, 1991). For example, the mating pheromone receptor Ste2p is internalized and forwarded to the vacuole upon exposure to

29 extracellular pheromones (Schandel and Jenness, 1994). Removal of plasma membrane proteins from the cell surface is also a way to handle different stress condition. Excess of uracil is toxic to the cell and the plasma membrane uracil permease Fur4p is targeted for vacuolar proteolysis via endocytosis upon exposure to high amounts of uracil (Volland et al., 1994; Séron et al., 1999). Delivery of cytosolic material destined for degradation in the vacuole is mediated by the autophagy pathway (Mijaljica et al., 2007). The primary trigger of autophagy in yeast is nutrient depravation. Autophagy is liable for removal of proteins as well as whole organelles, for instance peroxisomes and mitochondria that are malfunctional or redundant. Since it is the major degradative compartment of the cell, the vacuole harbors a variety of diges- tive enzymes including phosphatases, endo– and exoproteases, ribonucleases and lipases (Kucharczyk and Rytka, 2001). Two of the most prominent resi- dent vacuolar proteins, the carboxypeptidase CPY and the alkaline phosphatase ALP, have given names to the trafficking pathways responsible for their respective delivery from the Golgi to the vacuole (Stevens et al., 1982; Vida et al., 1993; Cowles et al., 1997; Piper et al., 1997). A prerequisite for proper catabolic function of these hydrolases is the acidic internal milieu of the vacuolar lumen. Acidification is carried out by a multiprotein vacuolar ATPase (V-ATPase) which translocates protons across the vacuolar membrane (Graham et al., 2003) (see section below: Organelle acidification and the V-ATPase complex). V-ATPase activity also serves another purpose. Thus, the H+ gradient generated by the V-ATPase is the driving force for many metal ion transporters residing in the vacuolar membrane, which emphasizes another important role of the vacuole, i.e. in ion homeostasis and detoxification. Finally, aside from being an ion reservoir, 2- the vacuole also functions as a storage site for PO4 and amino acids (Klion- sky et al., 1990).

Organelle acidification and the V-ATPase complex Multiple compartments of the eukaryotic cell, including vacuoles, endosomes, the Golgi network and transport vesicles, maintain an acidic interior in comparison to the surrounding cytosol (Kane, 2007). Degradation of macromolecules, receptor-mediated endocytosis, protein sorting and ion homeostasis are some of the processes that rely on proper organelle acidification (Stevens and Forgac, 1997; Kane, 2006). In the secretory and endocytic pathways, the pH of compartments is gradually lowered along the way to the end destination, which in the latter case is the vacuole (Stevens and Forgac, 1997). The key player in acidification is the vacuolar V-ATPase (or H+-ATPase). V-ATPases are evolutionary highly conserved complexes and functions as proton pumps which mediate translocation of H+ over membranes (Graham

30 et al., 2003). Proton translocation is accomplished by utilization of energy derived from hydrolysis of cytosolic ATP. The yeast V-ATPase is a 14 subunit enzyme complex composed of a peripheral part,V1, attached to a membrane-bound part, V0. The V1 subcomplex is responsible for the ATP hydrolysis and is composed of the Vma1p, Vma2p, Vma4p, Vma5p, Vma7p, Vma8p, Vma10p and Vma13p proteins in yeast (Kane, 2007). The remaining six subunits, Vma3p, Vma6, Vma9, Vma11p, Vma16p, and Vph1p/Stv1p constitute the integral proton-translocating V0 subcomplex. In yeast, there are two isoforms of one of the V0 subunits, Vph1p and Stv1p (Manolson et al., 1992; Manolson et al., 1994). Although they share 54% amino acid identity, there are some important differences between Stv1p and Vph1p. Thus, Vph1p localizes to the vacuole whereas Stv1p is proposed to reside in the Golgi and/or the endosome (Manolson et al., 1992; Manolson et al., 1994; Perzov et al., 2002). However, Stv1p is also to some extent present in the vacuolar membrane and can partially compensate for loss of VPH1 (Perzov et al., 2002). Loss of V-ATPase activity in higher eukaryotes is lethal but vma mutants of fungi remain viable (Kane, 2006). Disruption of any of the VMA genes in S. cerevisiae (with the exception of STV1 and VPH1 where both isoforms must be deleted) leads to a distinct growth phenotype (Kane, 2006). More specifically, the characteristic of the vma mutants is the inability of cells to grow at pH 7.5 (Nelson and Nelson, 1990). Yeast vma mutants are, however, able to survive at a lower extracellular pH of around 5.5, which suggests that alternative ways to acidify intracellular compartments must exist. Since growth at a low pH in the absence of a functional V-ATPase relies on the endocytotic pathway, it was proposed that fluid phase endocytosis may de- liver protons from the surrounding media to the cell (Munn and Riezmann, 1994). Alternatively, diffusion or uptake of weak acids can restore vacuolar acidification in absence of the V-ATPase, a mechanism which is independent of endocytosis (Plant et al., 1999).

The CPY pathway The carboxypeptidase Y (CPY) pathway is one of the best characterized transport pathways. In this pathway, proteins travel from the ER, traverse the late Golgi, pass the late endosome and then finally reach the vacuole (Mul- lins and Bonafacino, 2001). The CPY passageway has lent its name from one of its most studied passenger, the soluble carboxypeptidase Y protein. Upon entry of the precursor form of CPY into the interior of the ER, the signal peptide of CPY is removed and the protein is then glycosylated, which is followed by addition of more oligosaccharides in the Golgi (Bryant and Stevens, 1998). The resulting form of CPY is then forwarded to the vacuole where it is converted into mature CPY by proteolytic cleavage.

31 Coating of vesicles Vesicle formation is driven by the recruitment of cytosolic coat proteins to the emerging vesicles. Assembly of the coat onto the membrane leads to deformation and shaping of a spherical bud (Bonifacino and Glick, 2004). Specifically, vesicles coated with the multimeric protein clathrin are active in trafficking beyond the Golgi. The clathrin coat is composed of a heavy chain encoded by the CHC1 gene in yeast and a light chain encoded by the CLC1 gene (Payne and Schekman, 1985; Silveira et al., 1990). Unassembled clathrin exist as a triskelion of three heavy chains and three light chains which oligomerize on the membrane and form a basket-like arrangement (Lafer, 2002). A major constituent of the clathrin-coat is the heterotetrameric adaptor protein (AP) complex. The immediate role of the AP complex is to interact with clathrin and cargo proteins which in turn promotes recruitment of these constituents to membranes where vesicle formation takes place (Lafer, 2002; Puertollano, 2004). There are four basic AP complexes, AP-1, AP-2, AP-3 and AP-4 and each AP complex is restricted to a defined intracellular trafficking route (Hinners and Tooze, 2003). In recent years it has also become evident that another type of clathrin-linked adaptor protein exists. These GGA (Golgi-localized, gamma-ear-containing, ARF-binding) proteins are monomeric units and yeast have two isofoms, Gga1p and Gga2p (Nakayama and Wakatsuki, 2003; Hirst et al., 2000). Two non-clathrin coats are responsible for the bidirectional vesicle flow between ER and Golgi, COPI and COPII (coat protein) (Barlowe, 2000). COPI coated vesicles are responsible for trafficking in the retrograde direc- tion through the Golgi apparatus and from the Golgi to the ER and whereas anterograde transport from the ER to the Golgi apparatus is facilitated by COPII vesicles.

Tethering of vesicles Tethering represents the initial and reversible contact between donor (vesicles) and acceptor (target) membranes. Tethering occurs after shedding the protein coat from the vesicle but prior to fusion and docking. The Class C Vps complex is a tethering complex acting in vesicle fusion at the vacuole, but also in transport from the Golgi to the endosome (Srivastava et al., 2000; Peterson and Emr, 2001). The core Class C complex comprises four proteins, Pep3p/Vps18p, Pep5p/Vps11p, Vps16 and Vps33 (Rieder and Emr, 1997). Two additional proteins, Vam2p/Vps41 and Vam6p/Vps39p, are obligatory at the vacuolar membrane and this six-component complex is called the HOPS complex (homotypic fusion and vacuole protein sorting) (Seals et al., 2000: Wurmser et al., 2000). Most recently, Peplowska and colleagues (2007) identified a novel variant of the HOPS complex located at the endosome. The CORVET (class C core vacuole/endosome tethering)

32 hexameric complex contains Vps8 and Vps3 in addition to the core Class C Vps complex. Vam6p/Vps39p, which is part of the HOPS complex, physically interacts with a small Rab GTPase Ypt7p (Wurmser et al., 2000). Rab GTPases are key regulators in vesicular transport and functions as signalling molecules in tethering (Cai et al., 2007). By switching between a guanosine triphosphate (GTP)-bound form and a guanosine diphosphate (GDP)-bound form, the Rab protein is active or inactive. As Rab GTPases are stimulated to an active state by GDP/GTP exchange factors (GEFs), they are able to mediate tethering. The role of Vam6p/Vps39p is to act as a GEF for Ypt7p (Wurmser et al., 2000). The endosome-located CORVET complex cooperates with another Rab GTPase, namely Vps21p, via the binding of the Vps3p subunit (Peplowska et al., 2007). Since Vps21p physically interacts with Vps3p, it has been proposed that Vps3p is the GEF for Vps21p but there is still some uncertainties regarding the exact role of Vps3p. Less is known about the Mon1p-Ccz1p complex, but is seems to have a role in fusion of vesicles to the vacuolar membrane. Vacuolar localization of the Mon1p-Ccz1p complex is relying on the HOPS complex during homotypic vacuole fusion and Czz1p is functionally associated with the Rab GTPase Ypt7p which is known to function in fusion at the vacuolar membrane (Wang et al., 2002; Wang et al., 2003; Kucharczyk et al., 2001).

Docking and fusion via SNAREs As membranes are drawn together by the tethering complex, SNARE proteins step in to complete docking and fusion. SNAREs (soluble N- ethylmaleimide-sensitive factor attachment protein receptor) act as trans- membrane anchors on donor and acceptor membranes (Cai et al., 2007). As a result of tethering, membranes are drawn together to such proximity that SNAREs on opposed membranes are able to pair. SNAREs are categorized based on their subcellular localization, t-SNAREs are confined to target membranes and v-SNARES to the transport vesicle membranes. Alterna- tively, SNAREs can be divided into Q-SNAREs and R-SNAREs distin- guished by a glutamine or arginine residue in a conserved position (Fasshauer et al., 1998). Exactly how SNARE assemble depends on the tethering complexes, and is still to be unraveled, but Stroupe et al. (2006) demonstrated that the vacuolar HOPS complex connects to a phox homology (PX) domain in the t-SNARE Vam7p.

The ALP pathway The ALP pathway circumvents the MVB and thus forms a direct route from the Golgi to the vacuole (Cowles et al., 1997; Piper et al., 1997). As indi- cated by the name, the gene product of PHO8, alkaline phosphatase (ALP) is a prominent cargo of this route and in similarity to CPY, ALP starts off as a

33 proenzyme (Klionsky and Emr, 1989). ALP vesicles in yeast do not seem to be clathrin-coat dependent, but formation of an oligomeric complex of Vam2p/Vps41p is a prerequisite for ALP vesicle formation and the Vam2p/Vps41p protein physically binds to the adaptor complex AP-3 (Dar- sow et al., 2001; Rehling et al., 1999).

Endocytosis Endocytosis represents a ubiquitous recycling and degradation pathway which goes from the plasma membrane into the cell where extracellular material and plasma membrane components (receptors, ligands, permeases) are internalized and frequently degraded. Endocytosis starts at immobile plasma membrane protein structures called eisosomes which marks the endocytotic initiation site (Walther et al., 2006). Invagination of the plasma membrane creates endocytotic vesicles which travel to the first compartment along the endo-lysosomal pathway, the early endosome (EE) (Shaw et al., 2001). Cargo is then selectively sorted for either recycling or forwarding via the late endosome to the vacuolar compartment. Proteins that are destined for vacuolar degradation are singled out by a monoubiquitin moiety which targets protein for the MVB (Umebayashi, 2003). Key determinants in recognition of ubiquitinated cargo and formation of the MVB are four ESCRT complexes (Endosomal Sorting Complexes Re- quired for Transport) (Hurley and Emr, 2006). The ESCRT 0 complex (Vps27p and Hse1p) establishes contact with the cargo upon binding to ubiquitin (Bilodeau et al., 2002). ESCRT 0 recruits ESCRT I (Vps23p, Vps28p, Vps37p) which in turn recruits both ESCRT II (Vps22p/Snf8, Vps25p, Vps36p) and ESCRT III (Vps2p, Vps20p, Vps24p, Vps32p/Snf7p) (Hurley and Emr, 2006).

The Cvt and autophagy pathways The cytoplasm-to-vacuole (Cvt) pathway and the autophagy pathway are two separate but related ways of supplying the vacuole with cytosolic material. The Cvt pathway is active under normal conditions while autophagy is primarily a starvation-induced transport route for delivering proteins and organelles to the vacuole (Mijaljica et al., 2007). The Cvt and autophagy pathway both sequester the cargo within unique double-membrane vesicles, but of different sizes (Teter and Klionsky, 2000).

Retrograde transport The retrograde traffic from the late endosome back to the late Golgi is important for recycling of transport molecules. The GARP/VFT (Golgi Associ- ated Retrograde Protein/Vps Fifty-Three) complex represents a tethering unit

34 specifically dedicated to this inter-compartmental transport system (Conibear et al., 2003). It was first identified as a stoichiometric three-part complex consisting of Vps52p, Vps53p and Vps54p but it was later found that also Vps51p belongs to the GARP/VFT complex (Conibear and Stevens, 2000; Conibear et al., 2003; Reggiori et al., 2003). The GARP/VFT complex is recruited by the Golgi-located Rab GTPase, Ypt6p, which initiates membrane tethering and SNARE pairing (Siniossoglou and Pelham, 2001).

35 Present Investigations

Aim The aim of this thesis work has been to investigate drug action by using genome wide screening methods in the yeast Saccharomyces cerevisiae. More specifically, this study was undertaken to gain a deeper insight into the mechanisms for intracellular transport and to identify novel drug targets and explore the underlying mechanisms for susceptibility and resistance of the anticancer drug 5-fluorouracil.

Results Paper I Functional genomics of monensin sensitivity in yeast: implications for post-Golgi traffic and vacuolar H+-ATPase function. Intracellular protein transport has been investigated for decades, but many of the factors contributing to protein movement within the cell are still unknown. In a previous study conducted in this lab, 620 non-essential haploid yeast deletion mutants (the EUROFAN collection) were screened for monensin sensitivity in order to identify novel genes involved in protein trafficking (Murén et al., 2001). Monensin is an H+/Na+ exchanger that interferes with intracellular transport by causing neutralization of the trans-Golgi compartments (Dinter and Berger, 1998). The screen was designed in such a way that the monensin concentration used would partially reduce the growth of the wild type, through a partial inhibition of the essential exocytotic pathway. It was expected that mutants with a partial defect in the same pathway would reveal themselves as growing much slower than the wild type on the drug, due to further impairment of the exocytotic transport route. Several novel genes affecting intracellular transport were found in that screen, but the fact that the initial study was not comprehensive, prompted us to screen the complete EUROSCARF collection of approximately 5000 yeast deletion strain for sensitivity to monensin. By screening yeast cells robotically pinned onto growth media containing monensin, 63 deletion mutants were identified with decreased growth on monensin, as compared to the wild type strain. The majority of the mutants

36 recovered in the screen were deleted for genes with known functions in intracellular trafficking and were primarily involved in post-Golgi transport. We proceeded to investigate the specificity of our screen. The result from the monensin screen was compared to two other genome-wide screens that aimed at identifying novel VPS (vacuolar protein sorting) or VAM (vacuolar morphology) genes by CPY secretion assays and vacuolar morphology studies, respectively (Bonangelino et al., 2002; Seeley et al., 2002). In addition, we also choose to compare our result to a screen for salt sensitivity, something which is not obviously related to vacuolar function (Warringer et al., 2003). 2 analysis revealed a high correlation between the sets of genes found in our monensin screen and the VPS and VAM screens, and this correlation was much stronger than with the sets of genes found in the salt sensitivity screen. Surprisingly, none of the 14 vacuolar ATPase subunits appeared in our screen, even though they were found in the VPS and VAM screens. A possible explanation for this is that vacuolar ATPase mutations and monensin produce similar effects, i.e a loss of acidification in post-Golgi compartments. This hypothesis was tested by constructing double mutants of V- ATPase subunits and monensin-sensitive genes. Mutants that block endocytosis or retrograde transport showed synthetic lethality with a vma1 mutant, which lacks the catalytic subunit of the V-ATPase. This is consistent with previous findings and supports the notion that proton uptake through endocytosis may compensate for reduced intracellular acidification due to lack of V-ATPase activity (Munn and Riezmann, 1994). It has further been suggested that a low pH in the trans-Golgi is particularly important for maintenance of the exocytotic pathway, and thus for viability. The yeast V-ATPase contains either of two subunits depending on its location; V-ATPase with Vph1p resides in the vacuole and V-ATPase with Stv1p is found primarily in the Golgi/endosome (Forgac, 1999). To find out which V-ATPase is more important for viability in the absence of endocytosis, we made double mutants between endocytosis mutants and either vph1 or stv1. However, we found that double mutants with either of the two V-ATPase subunits showed only weak interactions, unlike the synthetic lethality observed with vma1. We concluded that Vph1p and Stv1p have largely redundant functions, at least with respect to these genetic interactions. We further found that a deletion of VMA1 is epistatic over the monensin sensitivity of mutants involved in vacuolar transport, suggesting that Vam1p is required for monensin toxicity in these mutants. In a ypt7 mutant, a deletion of vph1 was also epistatic over monensin sensitivity, but the opposite effect was seen when stv1 was deleted. One possible interpretation is that the absence of stv1 may enhance the effect of monensin by influencing on the activity or localization of the Vph1-containing V-ATPases.

37 Finally, the effect of monensin on CPY secretion was examined in all 63 mutants by Western Blots. Several mutants affecting post-Golgi transport had a more severe defect in CPY secretion in the presence of monensin.

Paper II Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast The anti-cancer agent 5-fluorouracil (5-FU) was developed more than 50 years ago (Heidelberger et al., 1957) but still, our understanding of how 5- FU acts in the eukaryotic cell is not comprehensive. The mechanism of action of 5-FU is complex since 5-FU is converted to metabolites interfering with both DNA and RNA metabolism. It is generally accepted that one of the primary targets of 5-FU is thymidylate synthase (TS), the enzyme responsible for converting dUMP to dTMP (Longley et al., 2003). However, in recent years a growing body of evidence has caused a revision of the tradi- tional view on 5-FU action (Giaever, et al., 2004; Lum et al., 2004). In this paper, we set out to study the action of 5-FU on a global scale in order to gain a better understanding of its mechanism of action. We used the same approach and methodology as in Paper I in that we screened a haploid yeast knockout strain collection for increased sensitivity to the drug. Out of 4676 mutants, a total of 138 strains were found to be sensitive to 5-FU. The corresponding genes were classified into several functional groups including DNA damage response, ergosterol biosynthesis, nucleocytoplasmic transport, protein modification and degradation, intracellular transport, transcription, rRNA assembly and processing and tRNA modification. Intriguingly, several of the most sensitive mutants were involved in tRNA maturation. The most sensitive mutant in the entire screen, trm10, belongs to this group. Trm10p is a methyltransferase that is responsible for m1G formation at position 9 in tRNAs (Jackman et al., 2003). It has recently been shown that double mutants for non-essential tRNA modification enzymes that act outside the anticodon loop are temperature sensitive, due to an increased instability of hypomodified tRNAs (Alexandrov et al., 2006). It is possible that the combined loss of tRNA modifications, by deletion of a gene coding for one modification enzyme and inhibition of another enzyme by binding to 5-FU-containing tRNA could produce a similar effect, i.e. destabilization at an elevated temperature. This prediction was confirmed by a phenotypic characterization of the tRNA modification mutants, several of which showed increased temperature sensitivity in the presence of 5-FU, indicating that the tRNAs may have been destabilized under these conditions. Thus, the trm1, trm8, trm82, trm10, pus1 and tan1 mutants, all of which affect modifications outside the anticodon loop, showed elevated sensitivity to 5-FU at 38 ºC, whereas mutants affecting modifications in the

38 anticodon loop or tRNA transport, were only slightly affected by the temperature. To find further support for an effect of 5-FU on tRNA stability, the pair- wise genetic interactions between our tRNA modification mutants were monitored on 5-FU containing media and at 38 ºC, as well as under stress conditions which do not affect tRNA stability. We found that the mutants showed similar synthetic interactions on 5-FU and at 38 ºC, whereas they did not interact in the same way at 16 °C or in the presence of 1M NaCl. Finally, we found that a wild type strain also is temperature sensitive in the presence of 5-FU, but only at higher concentrations. This suggests that tRNAs may be destabilized by 5-FU also in the wild type cells. Altogether, our results support the notion that the effect of 5-FU on tRNA modification mutants may involve tRNA destabilization due to hypomodification of tRNA.

Paper III Identification of genes whose overexpression confer resistance to the anticancer drug 5-fluorouracil in yeast In order to identify possible resistance mechanisms against 5-FU, we performed a screen using a yeast multicopy plasmid library. We recovered five genes that conferred resistance to 5-FU: CPA1, CPA2, HMS1, YAE1 and YJL055W. Cpa1p and Cpa2p together constitute the carbamoyl phosphate synthase isozyme CPS I which normally functions in the arginine biosynthetic pathway (Piérard and Schröter, 1978). HMS1 codes for a Myc-related transcription factor which is required for filamentous growth (Robinson and Lopez, 2000; Lorenz and Heitman, 1998), whereas the functions of YAE1 and YJL055W are still unknown. We proceeded to test our five 5-FU resistant overexpression strains on other drugs in an attempt to determine the specificity of the resistance to 5- FU. YAE1, CPA1 and YJL055W overexpressing strains showed resistance to 5-fluoroorotic acid and CPA1 also to 6-azauracil. In contrast, none of the overexpression strains was more resistant to monensin or methotrexate, two drugs affecting other metabolic pathways. These results suggest that the overexpression strains somehow affect the pyrimidine biosynthetic pathway, rather than having a general effect on drug uptake. To further investigate how the overexpressed genes confer resistance to 5- FU we carried out a cross-dependency analysis. Deletion mutants of the overexpressor genes were first tested for sensitivity to 5-FU and the mutants displayed only a slight growth defect in response to 5-FU. YAE1 was not tested since it is an essential gene. The cross-dependency experiment showed that HMS1, YAE1 and YJL055W all require CPA1 and/or CPA2 in order to mediate 5-FU tolerance. We conclude that 5-FU tolerance mediated by the

39 overexpressor plasmids involves arginine metabolism and most likely carbamoyl phosphate production. CPS I is subject to feedback inhibition by arginine at the translational level, and this effect is mediated by an arginine attenuator peptide in the 5´untranslated region of the CPA1 mRNA (Werner et al., 1987; Delbecq et al., 1994). We therefore also examined the effect of excess arginine. We found that strains harbouring the HMS1 and YAE1 plasmids still showed resistance to 5-FU in the presence of high concentrations of arginine. To check if expression of the CPA1 gene was affected in the presence of any of the other four overexpressor genes, we used RT-PCR but the CPA1 transcript seemed to remain at the same level in the presence of all four plasmids. Upregulation of the de novo pyrimidine biosynthesis results in elevated pools of UTP, which in turn can cause 5-FU resistance (Jensen et al., 1982). Only YJL055W conferred resistance to 5-FU in a ura3 mutant, in which the pyrimidine biosynthetic pathway is shut down. From this we could conclude that the 5-FU resistance of the overexpressor genes is dependent on the pyrimidine biosynthetic pathway with the possible exception of YJL055W. Finally, in vivo labeling experiments demonstrated that incorporation of [14C]5-FU into tRNAs is reduced in the presence of the overexpressor plasmids. This is consistent with the idea that de novo pyrimidine biosynthesis in these cells is stimulated by a flow of excess CP from the arginine pathway into pyrimidine biosynthesis.

40 Conclusions and Future perspectives

The results in paper I provide a starting point for elucidating how intracellular transport depends on the acidification of different intracellular compartments. Absence of a functional V-ATPase caused synthetic lethality with monensin-sensitive mutants in the retrograde and endocytotic pathways, but not with mutants involved in vacuolar trafficking. These results are consistent with the notion that a low pH in the trans-Golgi, rather than vacuolar acidification, is required for cell viability. The data presented in paper I further suggest the possibility that the pH in the Golgi could be regulated via endocytic uptake of protons to compensate for the loss of a functional V- ATPase. This hypothesis could be tested by measuring the pH in the trans- Golgi under different conditions and in different mutants, by using fluores- cent dyes. It should be noted, however, that the Golgi complex is notoriously difficult to identify in yeast, and it is still unclear to what extent specific proteins, such as V-ATPase subunits, localize to the trans-Golgi (Perzov et al., 2002). Our result also shed some light on previous observations that low pH are required for survival of V-ATPase mutants in yeast (Nelson and Nelson, 1990; Munn and Riezman, 1994). In paper I, a model is discussed in which the V-ATPase isozyme Vph1p, which normally resides at the vacuole, is mislocalized to the plasma membrane thereby enhancing the effect of monensin. Localization studies using Vph1-GFP could reveal if Vph1p ends up in the plasma membrane under certain conditions. One could also test if the monensin-sensitive phenotype of mutants involved in vacuolar transport could be reversed by addition of either bafilomycin or concanamycin, drugs which inhibit the V-ATPases (Bowman et al., 2004). Finally, one could test if overexpression of endocytosis-related genes can override the effects of monensin stress. This would provide further support for the role of endocytosis in acidification of the Golgi. Although 5-FU is commonly administrated to cancer patients, the overall responsiveness to this drug is frequently unsatisfactory and to increase the success rate in 5-FU chemotherapy, it is therefore crucial to establish exactly how 5-FU works. We have found that tRNA modification mutants are highly sensitive to 5-FU, as shown in paper II. When interpreting these results one should keep in mind that several tRNA modification enzymes are known to be inhibited by 5-FU (Santi and Hardy, 1987; Samuelsson, 1991). It is therefore likely that the increased 5-FU sensitivity of these mutants is a conse-

41 quence of lowering the modification status on tRNA, through the combined effects of these mutants and the inhibitory effects of 5-FU in other tRNA- modifying enzymes. HPLC analysis of tRNA modifications in yeast cells exposed to 5-FU can establish to what extent tRNAs are hypomodified in response to 5-FU. Further investigations are also needed to characterize the effects of tRNA hypomodification. In this context, it should be noted that combined loss of more than one modification outside the anticodon loop has been shown to cause tRNA destabilization, which can be detected as enhanced tRNA at elevated temperatures (Alexandrov et al., 2006). Northern blot analysis of individual tRNAs and studies of their aminoacylation status could help to establish whether there is increased tRNA degradation also in 5-FU treated cells. The fact that loss of tRNA modifications enhance cellular sensitivity to 5- FU, opens up the possibility of developing new anticancer drugs which can act in conjunction with 5-FU in order to achieve better cancer treatment. Furthermore, we found that wild type yeast cells are sensitized to 5-FU at higher temperatures. This effect was even more pronounced in tRNA modifications mutants, presumably due to further tRNA instability. Hyperthermia in combination with 5-FU has been shown to increase cytotoxicity (Kido et al., 1991) and the combined use of heat treatment and 5-FU has been clini- cally tested (Kouloulias et al., 2005). Our data may provide an explanation for this effect. Thermochemotherapy might be even more successful if drugs that interfere with tRNA modifications can be developed. CPA1 and CPA2 both confer resistance to 5-FU when overexpressed. This is interesting considering the fact that they are involved in the production of carbamoyl phosphate. CP which is produced in the arginine biosynthetic pathway could be shuttled into de novo synthesis pathway for pyrimidines, thus causing 5-FU resistance (Jaques and Sung, 1981). A rea- sonable explanation is that excess CP would increase uracil synthesis, thus competitively weakening the effect of 5-FU. Consistent with this we found that the specific incorporation of [14C]5-FU into tRNA is diminished in cells overexpressing CPA1 and CPA2. It is not yet known, however, whether CPS I can mediate 5-FU resistance in human tumours. More work need to be done to resolve this question but in a first step, human CPS I could be overexpressed in yeast and tested for its effect on 5-FU resistance. Putting our results into a larger perspective, it is important to consider that modulation of CPS I may not be sufficient to overcome resistance to 5-FU in tumour cells with increased pyrimidine biosynthesis. A combined inhibition or downregulation of both CPS I and CPS II might be necessary to achieve this goal.

42 Acknowledgements

I would like to express my deepest gratitude and appreciation to all the people who have contributed to this thesis and helped me along the way.

First of all, my supervisor Hans Ronne, for giving me this chance and for believing in me. For being so enthusiastic about science and for understanding the true value of “Diablo”.

Leif Andersson for valuable “examinatorssamtal”.

Eva, for being such an encouraging person, for shared love of silence and solitude, for the small gifts of eatable nature, but above all, for your endless support throughout all the battles. Tina, the funniest “dokrotand” around. For dancing with hobbits, idiotic walks in snow storms and for really caring. And thank you for sharing your real-life work soap, it’s been a blast! Zhen for your kindness and all the help in the lab. Gunilla, for a good collabora- tion and fun times in “Big cookie land”. Niklas, Mattias C, Anders for always being so nice, helpful and cheerful. Mattias Ö, Mattias T, Jimmy, Carolina, Frida, Jenny, Monika, Pernilla, Jenni, Simon M, Micke and past and present members of the lab for providing a nice work atmosphere. Colleagues at IMBIM and SLU, especially Kristina Lundberg for your help during teaching. Rahma for our loooong and enjoyable lunches. I am so glad that you stayed at IMBIM!

Anders Byström and Kerstin Stråby for advice and expertise regarding tRNA.

A special thanks to all the excellent teachers and supervisors at Göteborgs Universitet and Mälardalens Högskola; Stefan Hohmann and the Göteborg yeast crew, Carl Påhlson and Mats Lindén, who inspired me to go into the field of research and for always treating your students as equals.

All släkt och alla vänner, speciellt min fina faster Gullvi som alltid är så glad och omtänksam, samt Alli, ditt intresse för mikrober är beundransvärt och jag är så glad att du finns där för mig och min familj.

43 Peter Dominant, Ylva, Emma, Jacob, Rebecka, Max, Michelle, Celine. Härliga familjen Taikon som förgyller mina dagar med mycket skratt, bus och värme. Hos er hittar jag alltid glädje och avkoppling.

Min älskade Jånas. För att du kör Porschen som en galning, för att du är min privata livvakt när prinsessan ska shoppa och för att du får mig att kikna av skratt när du cyklar som en gammal gubbe. Det finns ingen som utmanar mig som du gör, du är min Manny (utan humlorna…). Du lyfter upp mig när jag faller och du tröstar mig när det stormar. Ditt lugn, din kreativitet och ditt stöd är ovärderligt och utan dig hade detta aldrig varit möjligt.

Mina fantastiska föräldrar. Pappa för att du är så otroligt och obegripligt uppfinningsrik, för att du alltid ställer upp och fixar åt mig och för att du så snällt låter mig ro båten när bensinen har tagit slut. Mamma för att du är den mest empatiska och omtänksamma människa som finns på denna jord, för att jag får köra över dina prydnadssaker och för att du övertygade din dotter till att köra ned i diket. Utan er är jag ingenting och det finns inte ord stora nog för att beskriva vad ni betyder för mig. Jag älskar er för allt ni är, för vad ni ger och för att ni alltid finns där för mig.

44 References

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Acta Universitatis Upsaliensis Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 402

Editor: The Dean of the Faculty of Medicine

A doctoral dissertation from the Faculty of Medicine, Uppsala University, is usually a summary of a number of papers. A few copies of the complete dissertation are kept at major Swedish research libraries, while the summary alone is distributed internationally through the series Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine. (Prior to January, 2005, the series was published under the title “Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine”.)

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