1

ABSTRACT

Inorganic phosphate is an essential nutrient for all organisms. It is

required for many cellular components as nucleic acids and phospholipids, and as energy-carrying compounds such as ATP. Thus, a regulated uptake of this pivotal nutrient is of outermost importance. Depending of the availability of phosphate in the surroundings the yeast Saccharomyces cerevisiae make use of two different systems for transporting phosphate into the interior of the cell: a low-affinity system that is active during surplus phosphate conditions and a high-affinity system that is active when the availability becomes limited. This thesis focuses on the high-affinity system, which is comprised of the Pho84 and Pho89 transporters. Of the two transporters, Pho84 is the predominant one, responsible for almost all phosphate uptake during low phosphate conditions, and the contribution of Pho89 is of minor importance. Hence Pho84 is by far the most well characterized phosphate transporter. Even though much is known about phosphate transporters in yeast little in known about how phosphate is transported. The work in this thesis aims to broaden the knowledge about the transport mechanism by the means of site-directed mutagenesis and functional characterization. Also the similarity of Pho84 to glucose sensors and the potential role of conserved residues in phosphate signaling are investigated. By the use of a high-affinity system deletion strain (∆Pho84 ∆Pho89), we also managed to investigate the functional importance of well conserved residues in Pho89. In summary: the work presented in this thesis has contributed to increase the knowledge about transport mechanisms in phosphate transporters. Keywords: Phosphate, membrane transport, mutagenesis, Saccharomyces cerevisiae.

2

You should not believe something just because you can explain it.

-Arthur Kornberg

3

POPULÄRVETENSKAPLIG SAMMANFATTNING

Alla celler är omgivna av ett membran. Detta membran skyddar cellens inre mot omgivningen och utan det skulle liv inte vara möjligt. Vissa ämnen kan utan hjälp passera genom membranet utan problem, samtidigt som andra inte kan passera genom membranet. För att dessa ämnen ändå ska komma in eller ut från cellen så har celler utvecklat speciella membran-transportörer för att transportera dessa ämnen genom den barriär som membranet utgör. För att studera hur dessa transportörer fungerar så har vi i den här avhandlingen använt oss av celler av jästen Saccharomyces cerevisiae. Det är en välstuderad modell-organism som ofta används för cellulära studier. System för transport genom membranet är till stor del väl konserverade mellan humana celler och jästceller. Därför kan det vara givande att studera funktionerna hos jästproteinerna för att få information om deras humana motsvarigheter. Det finns ett flertal sjukdomar som t.ex. epilepsi och njursvikt där man har sett förändringar i humana transportörer som har stora likheter med motsvarande proteiner i t.ex. jäst. Oorganiskt fosfat är ett livsviktigt näringsämne för alla organismer. Det krävs för otaliga cellulära processer såsom syntes av DNA och de fosfolipider som utgör cellens membran. Det har också en avgörande betydelse för energi-metabolismen och cellulära signalvägar. Således är ett väl reglerat upptag av detta centrala näringsämne av yttersta betydelse för cellers förmåga att överleva. Beroende av hur mycket fosfat som finns tillgängligt i omgivningen kan jästen S. cerevisiae utnyttja två olika system för att ta upp och transportera in fosfat genom membranet, in i cellen: Ett system med låg affinitet för fosfat och som är aktivt när det finns gott om fosfat, och ett system med hög affinitet som aktiveras när tillgången på fosfat blir begränsad. Denna avhandling fokuserar på hög-affinitets systemet, som består av de två fosfat transportörerna Pho84 och Pho89. Denna avhandling inkluderar studier av vilka delar av transportörerna som är de direkt avgörande för själva förmågan att transportera fosfat. Transportörerna har blivit muterade på gennivå, följt av biokemiska och funktionella analyser av deras transportförmåga. Sammanfattningsvis bidrar studierna i denna avhandling till att öka vår förståelse för hur fosfat-transportörerna transporterar in fosfat i celler.

4

LIST OF PUBLICATIONS The thesis is based on the following papers, referred to in the text by their roman numerals.

I. Mutational analysis of conserved glutamic acids of Pho89, a Saccharomyces cerevisiae high-affinity inorganic phosphate:Na+ symporter.

Andersson M, Samyn D, Persson B.L. Submitted manuscript. II. Mutational analysis of the functional role of conserved arginines in

the Saccharomyces cerevisiae Pho84 phosphate transceptor. Popova Y, Andersson M, Persson B.L., Thevelein J.M. Manuscript. III. Putative role of Asp-76 and Asp-79 in proton-coupled inorganic

phosphate transport of the Saccharomyces cerevisiae Pho84 phosphate:H+ transporter.

Samyn D, Andersson M, Persson B.L. Manuscript.

IV. Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signaling to the PKA and PHO pathways. Samyn D, Ruiz-Pavón L, Andersson M, Popova Y, Thevelein J.M., Persson B.L. Accepted for publication in Biochemical Journal.

5

Additional work outside the scope of this thesis: Characterization and functional activity of recombinant Gtr1 protein – a small GTP binding protein in Saccharomyces cerevisiae. Sengottaiyan P, Spetea C, Lagerstedt J.O., Samyn D, Andersson M, Ruiz-Pavón L, Persson B.L. Revised submitted manuscript

6

ABBREVATIONS ATP Adenosine triphosphate cAMP Cyclic adenosine mono phosphate DNA Deoxyribonucleic acid EmrD E.coli multidrug transporter FucP Fucose transporter GAP1 General amino acid transporter 1 GFP Green fluorescent protein GlpT Glycerol-3-phosphate transporter HXT Hexose transporter IUBMB International Union of Biochemistry and Molecular Biology LacY Lactose transporter MFS Major facilitator superfamily OxlT Oxalate transporter PHS Phosphate proton symporter family Pi Inorganic phosphate PiT Inorganic phosphate transporter family PKA Protein kinase A PolyP Poly phosphate SGD Saccharomyces genome database TC Transporter classification TM Transmembrane TCDB Transporter classification database Ub Ubiquitin UhpC E.coli glycerol-6-phosphate sensor

7

TABLE OF CONTENT

GENERAL INTRODUCTION ............................................................................ 8 Yeast ............................................................................................................... 8 S. cerevisiae as a model organism ................................................................... 9

MEMBRANES AND TRANSPORTS ............................................................... 10 Cellular membranes ...................................................................................... 10 Plasma membrane transport .......................................................................... 12 Transporter classification .............................................................................. 14

PHOSPHATE ..................................................................................................... 15 Inorganic phosphate ...................................................................................... 15

Inorganic phosphate as nutrient ............................................................... 16 Polyphosphate .......................................................................................... 17

PHOSPHATE ACQUISITION .......................................................................... 18 PHO regulatory pathway .............................................................................. 19

High phosphate conditions ...................................................................... 19 Low phosphate conditions ....................................................................... 20

The low affinity phosphate transport system ................................................ 21 Pho87 and Pho90 ..................................................................................... 22 Pho91 ....................................................................................................... 22

The high affinity phosphate transport system ............................................... 23 Pho89 ....................................................................................................... 23 Pho84 ....................................................................................................... 25 Post translational modification of Pho84 ........................................... 26 Pho84 and protein kinase A................................................................ 27 Pho84 and glucose sensors ................................................................. 28

RESULTS AND DISCUSSION ......................................................................... 30 FUTURE PERSPECTIVES ............................................................................... 33 ACKNOWLEDGEMENTS ............................................................................... 34 REFERENCES ................................................................................................... 35

8

GENERAL INTRODUCTION

Yeast Yeasts have been used by mankind during roughly 9000 years. The

production of fermented beverages has occurred in China since 7000 BC, and evidence has also been found for the production of leavened bread in ancient Egypt around 3100 BC (McGovern et al., 2004; Samuel, 1996). However, it was not until 1860 that Louis Pasteur demonstrated that yeasts plays a direct role in the conversion of glucose into ethanol and carbon dioxide, a process called fermentation (Pasteur, 1860). Yeasts are recognized as unicellular fungi. Classification of yeasts into subdivisions is based on their sexuality or lack of it. Further divisions are based on various physiological, morphological and genetical characteristics (Barnett, 1992). It is only when we reach the taxonomic categories of the species level that we encounter the Saccharomyces cerevisiae, more commonly referred to as baker’s yeast. This is only one of approximately 1,000 species of yeast that have been described to date. This well known and relatively well characterized species has been the host used in all work underlying this thesis. Yeasts in general can be found in a variety of environments, as for instance on plants (Ashya gossypii.), in the intestinal tracts of animals (Candida pintolopesii.), or soil (Schwanniomyces spp.) and water (Debaromyces spp.), but also more extreme environments are known to host yeasts e.g. Antarctic soils (Cryptococcus albidus) (McCarthy et al., 1987; Wendland & Walther, 2005; Capriotti, 1957; Butinar et al., 2007). The yeast S. cerevisiae is believed to be isolated originally from the skin of grapes (Pasteur, 1872; Mortimer & Polsinelli, 1999).

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S. cerevisiae as a model organism S. cerevisiae is one of the best characterized eukaryotic organisms. It is

more complex than bacteria, but less so than higher eukaryotes. As a lower eukaryote it has many crucial features in common with cells of higher eukaryotes: metabolism, cytoskeletal organization and DNA replication exhibit great similarities, and it contain membrane encapsulated organelles. Its ability to grow on defined media confers total control of the environmental parameters to the investigator. Yet, the main reason why S. cerevisiae has become such a widely used model system is the ease with which this organism can be genetically manipulated. This property is mainly exploited using the process of homologous recombination, which occurs at high rate and naturally in yeast. This feature can be put to work for the integration of foreign genes into the yeast genome and/or the modification of endogenous genes (Gietz & Woods, 2002). Such investigations have provided invaluable insights into a large number of cellular functions as signaling pathways, regulation of the cell cycle, organelle biogenesis and transport mechanisms (Tamanoi 2011; Kaizu et al., 2010, Bolotin-Fukuhara & Grivell, 1992). Furthermore, the S. cerevisae genome was the first eukaryotic genome to be completely sequenced. It includes roughly 6,000 genes (Goffeau et al., 1996) and contains a large number of homologues to mammalian proteins with highly similar DNA coding sequences and functions. This makes it feasible to extrapolate information to mammalian cells. To date the most comprehensive database of the S. cerevisiae genes is the Saccharomyces genome database (SGD) freely accessible online at www.yeastgenome.org

Despite all the above mentioned advantages, one should bear in mind that it is not an absolutely ideal eukaryote experimental model system when it concerns studies of certain cytological and growth characteristics. For example, budding is a unique mode of reproduction. The asymmetry of cell division and the presence of the cell wall are not features of higher eukaryotic cells (Hartwell, 1974; Cabib et al., 2001; Negishi & Ohya, 2010).

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MEMBRANES AND TRANSPORT

This section will briefly describe the structure and importance of biological membranes and the systems for transmembrane transport, with a focus on the S. cerevisiae.

Cellular membranes The biological membrane surrounding cells is critical for survival. It

allows the maintenance and separation of the intracellular environment from the external environment, and facilitates the formation of intracellular compartmentalization. Membranes consist of complex compositions of different lipid molecules. Most of the membrane-forming lipids have a common structure, consisting of a hydrophobic hydrocarbon tail and a hydrophilic head group. Hydrophobic forces are the main contributor in shaping the membrane by minimizing the interaction of the hydrophobic tails with water. The hydrophobic properties of the tails cause them to self associate, directing them towards each other and shielded off from the aqueous environment by the hydrophilic head group (Edidin, 2003; van Meer et al., 2008; Koshland, 2002). This arrangement will result in a lipid bi-layer, with a hydrophobic core, that is around 7.5nm wide in S. cerevisiae. This will constitute a hydrophobic barrier which is relatively impermeable for hydrophilic molecules (Figure 1).

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Figure 1. Two layers of lipids with their hydrophobic tails directed towards each other constitute an effective barrier for a wide range of compounds. This barrier defines the boundaries of the cell and the organelles inside of it. Colored dots indicate substrates unable to penetrate the membrane bi-layer.

In order to cross this barrier, there is a need for specialized proteins which will mediate transport of less favorable/hydrophilic solutes at any direction, commonly known as membrane transporters. Membranes contains more proteins than only those involved in transmembrane transport: for instance the plasma membrane harbors proteins involved in transmembrane signal transduction, cytoskeletal anchoring and enzymes involved in cell wall synthesis (Oehlen & Cross, 1994; Cid et al., 1995). The protein and lipid content in each membrane depends on the purpose of that membrane in the cell. In addition to compartmentalization each membrane type may exert several functions but their main functions are summarized in Table 1 below.

Table 1. Different functions of different membranes (Derived from van der Rest, 1995). Membrane type Main function Plasma membrane Separates the interior of the cell from the surrounding

environment Mitochondrial membrane Involved in metabolic energy regeneration Endoplasmic reticulum and Golgi membrane

Involved in protein and lipid sorting and synthesis

Nuclear membrane Encases and protects the DNA Vacuolar and peroxisomal membranes

Compartmentalizes special metabolic and digestive functions

The exact lipid and protein composition of the S. cerevisiae membranes may also be strain dependent and may vary in response to different growth conditions (van der Rest et al., 1995; Blagović et al., 2005).

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Plasma membrane transport Transport across the plasma membrane is mediated in several ways.

First and foremost, transport mechanisms can be divided into passive and active (energy dependent) transport mechanisms.

Passive transport involves carrier-mediated transport or simple diffusion down the concentration or electrochemical gradient, with no energy input needed. Small and non-polar molecules may passively diffuse across the membrane without any aid. This is usually a slow process and an increase in rate is made possible by the assistance of specific proteins. These proteins can be transporters, channels or pores. Since proteins are involved in the diffusion the process is referred to as facilitated diffusion. Since the transport is down the gradient without energy requirements, only one solute is transported at a time, and this is referred to as a uniport mechanism.

When substrates need to be transported against a gradient, active transport is required. This is thermodynamically unfavorable, and transport of these substrates will require transport-proteins that mediate the movement across the membrane in an energy dependent manner. Active transport can be divided into primary and secondary transport. Primary active transport occurs when the transport involves the conversion of light or chemical energy (hydrolysis of ATP) into electrochemical energy, i.e. an electrochemical gradient. Examples of transporters belonging to this group are the Pma1 and Ena1 extruding H+ and Na+, respectively, from the cytoplasmic to the periplasmic side of the plasma membrane at the expense of ATP. (Haro et al., 1991, Serrano et al., 1986). Secondary active transport exploits the free energy stored in this electrochemical energy to move a substrate against its gradient across the membrane. For instance, the energy stored in the gradients of H+

and Na+ can be utilized by the phosphate transporters Pho84 and Pho89, respectively, for energizing the transport of Pi. The different modes of transport are summarized in Figure 2.

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Passive TransportActive Transport

Diffusion Facilitated diffusion

ATPADP + Pi

Primary Secondary

Passive TransportActive Transport

Diffusion Facilitated diffusion

ATPADP + Pi

Primary Secondary

Figure 2. The two different modes of transport across the membrane. The energy requiring active transport against a gradient, and the energy independent passive transport down a gradient. Green dots indicate the main substrate transported.

Secondary active transport occurs when a single substrate is transported against its gradient. For this to occur energy is required. This transport can be powered by coupling the transport of the solute with co-transport of an energetically favored solute, thus these proteins are also referred to as co-transporters. When a substrate is transported coupled to one or more solutes/ions, at the same direction; it is referred to as a symport. If the transport is coupled to a transport of one or more solutes/ions at the opposite direction it is denoted an antiport mechanism (Figure 3). (Van Winkle, 1999).

Figure 3. Three principles of transport. Green dots indicate the main substrate, transported in the direction against the gradient. Red dots indicate coupled solutes/ions, transported in the same direction as their respectively gradient.

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Transporter classification The International Union of Biochemistry and Molecular Biology

(IUBMB) have approved a classification system for membrane transport proteins known as the Transporter Classification (TC) system (freely accessible at http://www.tcdb.org (Saier, 2000). This classification system is generally accepted and the most frequently used for membrane protein research. It incorporates sequence, structural, functional and evolutionary information about transport systems from a variety of living organisms. To date it contains approximately 5,600 transporters and putative transporters which reside in more than 600 families. The transporters are classified on the basis of five criteria.

• Transporter class (i.e., channel, carrier, primary active transporter or group translocator).

• Transporter subclass (which in the case of primary active transporters refers to the energy source used to drive transport).

• Transporter family (sometimes actually a superfamily). • Subfamily in which a transporter is found. • Substrate or range of substrates transported.

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PHOSPHATE

Inorganic phosphate A phosphate ion consists of one central phosphorous atom surrounded

by four oxygen atoms in a tetrahedral arrangement. It has four distinct protonation states (Figure 4), of which the dihydrogen phosphate (H2PO4

-) and monohydrogen phosphate (HPO4

2-) are the predominant forms at physiological pH (pH 4-10).

Figure 4. The four distinct protonation states of inorganic phosphate. Indicated are also the corresponding pKa values. Phosphate is referred to as inorganic phosphate (Pi) when the valences of the central phosphorous atom are occupied with either inorganic ions, e.g. metals, hydrogen atoms or a combination of both.

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Inorganic phosphate as nutrient The intracellular Pi concentration in S. cerevisiae cells, grown under

high levels of Pi, is in general relatively constant at approximately 20-25mM (Auesukaree et al., 2004). As a nutrient, phosphate is essential for all living cells. It is involved in many different aspects of life, being part of nucleic acids and phospholipids. Furthermore, it has been shown to have critical regulatory functions in a large number of cellular processes as signal transduction and metabolic purposes. In most cases phosphate occurs as organophosphate esters, but cells also make us of inorganic phosphate, for instance in the glycolysis when glyceraldehyde-3-phosphate is converted into 1,3-Bisphosphoglycerat (Figure 5), and also in glycogenolysis when glycogen phosphorylase breaks down glycogen into glucose subunits (Livanova et al., 2002; Burnett & Kennedy, 1954; Lehninger, 2000)

Figure 5. Oxidation of glyceraldehyde-3-phosphate to 1,3-Bisphosphoglycerate, catalyzed by glyceraldehyde-3-phosphate dehydrogenase. The reaction requires the presence of Pi.

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Polyphosphate When Pi is not directly used as building blocks in Pi-rich compounds or

metabolites, a large part will be incorporated into linear, large inorganic polyphosphate polymers, polyP. These polymers are composed of ten to hundreds of molecules of orthophosphate that are linked together by phospho-anhydrous bonds (Kornberg et al., 1999) (Figure 6).

Figure 6. Molecular structure of polyphosphate. n = number of Pi molecules. Polyphosphate plays an important role in maintaining the Pi homeostasis of the cell. It confers a buffer capacity of the intracellular levels of this nutrient to assure the availability of Pi during fluctuating environmental conditions (Kulaev & Kulakovskaya, 2000; Thomas & O'Shea, 2005). Under favorable conditions the concentration of polyP is around 230mM and can constitute up to 20% of the cell dry weight. (Auesukaree et al., 2004; Kornberg et al., 1999).

Despite experimental evidence of several exopolyphosphatases being localized in different organelles for metabolizing polyP (Lichko et al., 2006), to date there are only two genes identified coding for two of these enzymes. PPN1 encodes an exopolyphosphatase which is localized in the nucleus, vacuole and in mitochondria. It displays an enzymatic preference towards long-chain polyP (Kumble & Kornberg, 1996; Lichko et al., 2006). The gene product of the other exopolyphosphatase, PPX1, is localized in the cytosol, the cell envelope and mitochondrial matrix and is more efficient in metabolizing shorter polyP chains (Wurst et al., 1995).

In studies using strains where individual phosphate transporters were deleted, the polyP content was drastically reduced in strains lacking either Pho84 or Pho87. Strains harboring only Pho84 maintained WT levels of polyP content. This indicates that of the five inorganic phosphate transporters, Pho84 has the most important role in maintaining polyP levels (Hurlimann et al., 2007). whether this is due to the intrinsic transport capacity or its role in intracellular signaling is to date not known.

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PHOSPHATE ACQUISITION

S. cerevisiae has five Pi transporters divided into a high-affinity, Pho84 and Pho89, and a low-affinity system, Pho87, Pho90 and Pho91 (Figure 7).

Figure 7. Schematic overview of the two different Pi transport systems that S.cerevisae utilize for Pi transport. Alterations in the usage of high- and low–affinity systems allows for optimal nutrient uptake over a wide range of extracellular concentrations and are commonly used for several nutrients (Ozcan et al., 1996; Auesukaree et al., 2003; Harris et al., 2001). The individual transporters will be discussed in detail in chapter IV.

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PHO regulatory pathway

Many components of the phosphate-responsive signaling pathway, also known as the PHO regulatory pathway, are well characterized (Tohe et al., 1973; Tohe et al., 1988; Oshima, 1997; Mouillon & Persson, 2006). The key components in the PHO regulon are the cyclin-dependent kinase(CDK) complex Pho80-85, the inhibitor of this CDK, Pho81, and the kinase substrate, the transcription factor Pho4. Pho81 is bound to the Pho80-85 complex independently of Pi levels but its activity is regulated in response to the Pi level. High phosphate conditions

The transcription factor Pho4 is associated to the nuclear import receptor Pse1 and imported to the nucleus (Kaffman et al., 1998). At high Pi conditions Pho81 will be inactive, allowing Pho85 to phosphorylate the transcription factor Pho4. This increases the affinity of Pho4 towards the nuclear export receptor Msn5 and causes Pho84 to associate with it and get rapidly exported out of the nucleus (Kaffman et al., 1998). The association of Pho4 with its transcriptional co-activator Pho2 is thus prevented, and consequently the activation of transcription of the PHO-responsive genes Under these conditions the high-affinity Pi-transporters are removed from the plasma-membrane and routed to the vacuole for degradation (Figure 8).

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Vacuole

Nucleus

Phosphate responsive genes

ER

Golgi

P

Pho4P P

P

Pho4

Ub

Ub

Ub

Pho89

Pho84

Pho87

Pho90

Pho91

Pho81

Pho80 Pho85

Msn5

Pho2

Pi

Pho89

Pho84

Pho89

Pho84

Msn5

Vacuole

Nucleus

Phosphate responsive genes

ER

Golgi

PP

Pho4Pho4PP PP

PP

Pho4

Ub

Ub

Ub

Pho89

Pho84

Pho87

Pho90

Pho91

Pho81

Pho80 Pho85

Msn5

Pho2

Pi

Pho89

Pho84

Pho89

Pho84

Msn5

Figure 8. The activity of the PHO-regulon during high Pi conditions. The transcription of the PHO responsive genes are inactivated during high Pi conditions due to the phosphorylation and subsequent nuclear export of the transcription factor Pho4.

Low phosphate conditions

During low Pi conditions (Figure 9) Pho81 is activated in an inositol heptakinase (IP7)-dependent manner. Pho81 will then inactivate Pho85, thus preventing it from phosphorylating Pho4 (Lee et al., 2007). This will result in the accumulation of unphosphorylated Pho4 in the nucleus, resulting in an interaction with the transcriptional co-activator Pho2. Together they will activate the transcription of the PHO-responsive genes. Among the PHO-responsive genes that get up-regulated are (i) the high-affinity phosphate transporters PHO84 and PHO89, (ii) the secreted acid phosphatases PHO5, PHO11 and PHO12, which will hydrolyze organic phosphates and thus releasing Pi into the periplasm, (iii) the vacuole localized alkaline phosphatase PHO8 (Klionsky & Emr, 1989), (iv) PHO86 which is required for the endoplasmatic reticulum exit of Pho84 (Bun-ya et al., 1996) and (v) SPL2 which is a negative regulator of the low affinity phosphate transporter Pho87 and possibly Pho90 (Hurlimann et al., 2009; Ghillebert et al., 2010).

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Pho2

Vacuole

Pse1

Nucleus

ER

Golgi

Pho5

Phosphate responsive genes

Pho84

Pho91

Pho89

Pho4

Pho4

Pho4

Pho81Pho80 Pho85

Spl2

Pse1

Pho86

Spl2

Pho87

Pho90Pho87

Pho90Pho87

Pho90

Pho84Pho89

Organic phosphate

PiPho5

Pi

Pho2

Vacuole

Pse1

Nucleus

ER

Golgi

Pho5

Phosphate responsive genes

Pho84

Pho91

Pho89

Pho4

Pho4

Pho4

Pho81Pho80 Pho85

Spl2

Pse1

Pho86

Spl2

Pho87

Pho90Pho87

Pho90Pho87

Pho90

Pho84Pho89

Organic phosphate

PiPho5

Pi

Figure 9. The activity of the PHO regulon during low Pi conditions. By inactivation of the Pho80-Pho85 complex Pho4 is able to localize to the nucleus and activate the transcription of the PHO-responsive genes.

The low-affinity phosphate transport system

As described previously, optimization of phosphate uptake into the cell, during alternating phosphate availability, is ensured by the use of two different transport systems. When there is a surplus of phosphate in the surrounding environment, the low-affinity system saturates the cells requirement for phosphate. The expression of the low-affinity transporters has been shown to be independent of the external phosphate concentration.

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Pho87 and Pho90 The primary sequences of Pho87 and Pho90 are more than 60%

identical on primary amino acid sequence level. This is partly due to the 375 amino acid residues amino terminal SPX domain, harbored by both transporters, while absent in the high-affinity Pi transporters. SPX domains are thought to have regulatory functions and are present in several other eukaryote proteins (Barabote et al., 2006). In Pho87 and Pho90 this domain is involved in the regulation of Pi uptake, but it is not important for the uptake per se since a truncation does not affect protein level or membrane localization at high Pi conditions in neither of the transporters. Moreover, the SPX truncated Pho90 even displayed an increased Pi transport compared to the WT protein (Hurlimann et al., 2009). Starvation for glucose and treatment with rapamycin, a condition known to mimic amino acid and nitrogen starvation, triggers endocytosis and vacuolar degradation of both transporters, a mechanism that was shown to require the SPX domain in both Pho87 and Pho90 (Ghillebert, 2011). Under low Pi conditions both transporters are down-regulated. For the Pho87 down-regulation it has been shown that Spl2 is required, but whether or not Spl2 is also involved in the down-regulation of Pho90 is still under debate (Hurlimann et al., 2009; Ghillebert, 2011). It has previously been shown that Pho87, in a ΔPho84 strain, is able to signal the presence of Pi for activation of the protein kinase A (PKA) pathway, a function that is otherwise carried out by Pho84. (Giots et al., 2003).

Pho91 Pho91 also harbors an N-terminal SPX domain, but the importance of

the domain in this transporter has not been addressed. Pho91 is the least investigated phosphate transporter, and its precise localization is still uncertain. Previously it was shown that Pho91 allows for vigorous growth when over-expressed under the ADH1 promoter in a strain deleted of all the other transporters (Wykoff and O’Shea, 2001). Moreover, expression under its native promoter allowed for minimal growth, implying plasma-membrane localization. In contrast with the above mentioned localization, a more recent study (Hurlimann et al., 2007) showed that the N-terminal GFP-tagged Pho91 was detected in the vacuolar membrane irrespective of if the weak CYC1 promoter or the strong ADH1 promoter was used. Most recently, Popova and co-workers (2010) showed that when over-expressed from the ADH1 promoter Pho91 not only transports Pi, but also supports strong glycerol-3-phosphate uptake, which again implies plasma membrane localization. Because of these discrepancies, further studies are required before the precise localization and role of Pho91 can be established.

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The high-affinity phosphate transport system

Pho89 Pho89 is a member of the inorganic phosphate transporter (PiT) family,

which also contains the well characterized human Pi transporters hPit-1 and hPit-2 (Collins et al., 2004). The contribution to Pi uptake in yeast by Pho89 is very low; it displays a 100-fold lower transport activity than Pho84 (Pattison-Granberg & Persson, 2000). The transport of Pi occurs in a symport manner with cations as co-solutes, preferably Na+ but also with Li+ and K+, and the optimum pH for transport is 9.5 (Zvygailskaya et al., 2008; Martinez & Persson, 1998). The high pH optimum coincides with that Pho89 also show an up-regulation in response to alkaline conditions, partly independent of Pho2/Pho4. The Na+-ATPase ENA1 is also up-regulated in response to alkaline conditions and extruding Na+ out of the cell will contribute to a Na+ motive force across the plasma membrane, which might favor the Na+

dependent Pi transport by Pho89 (Serrano et al., 2002). The topology for hPiT-2 has been addressed experimentally and

confirmed it's predicted 12 transmembrane regions (TM), arranged in seven plus five TM connected with a large intracellular loop (Salaun et al., 2001). Pho89 is also predicted to contain 12 transmembrane (TM) regions arranged in seven plus five TM’s connected with a large intracellular loop, and with both termini on the extracellular side as shown in Figure 10 (Paper I).

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Figure 10. The 12 TM topology predicted by TOPCONS. The residues predicted to constitute the TM domains are shaded grey. Highlighted in red are the conserved GANDVAN sequences in both the N- and C- terminal domains. Highlighted in darker grey are the Glu55 in TM2 and Glu491 in TM10

Saier and co-workers (1999) proposed a signature sequence for the PiT family with the consensus amino acid sequence GANDVANA. This signature sequence has been identified in more than a hundred proteins with homology to the PiT family. The GANDVANA sequence is present in two copies in hPiT2. Mutational analysis of the conserved aspartic acid residue to an asparagine in any of the two sequences confers knockout of Pi transport (Bottger & Pedersen, 2005). Pho89 also harbors two copies of the signature sequence (Figure 10), located in the N- and C-terminal domain at similar positions. Whether these sequences have a similar importance as shown in the hPiT2 is currently unknown.

Not only does the Pho89 share homology with the hPiT1 and hPiT2 in the GANDVANA sequences. Multiple sequence alignments of PiT family members representing all kingdoms of life show that two glutamic acid residues are highly conserved, corresponding to Glu55 and Glu491 in Pho89 and located in TM2 and TM10, respectively (Bottger & Pedersen, 2002). In hPiT1 and hPit2 these residues were shown to be crucial for Pi transport activity (Bottger & Pedersen, 2002). A replacement of any of these Glu residues in Pho89 with Lys abolishes transport activity. So does a replacement of Glu55 by a Gln, whereas a replacement of Glu491 by a Gln has retained some of its activity (Paper I). The similarity in sequence, transmembrane topology and results upon mutational studies suggests that these glutamic acid

25

residues have a functional role in the transport mechanism, yet their molecular role is yet to be determined.

In contrast to Pho84 and Pho87, Pho89 is not able to sense and signal the availability of Pi for rapid activation of the PKA pathway in Pi starved cells (Zvyagilskaya et al., 2008; Giots et al., 2003).

Pho84 Pho84 symports Pi and H+ with an optimal pH for transport of 4.5

(Bun-ya et al., 1991; Berhe et al., 2001). It is classified as a member of the Phosphate:H+ Symporter (PHS) family belonging to the Major Facilitator Superfamily (MFS). The MFS consists to date of 74 distinct families, which include over 10,000 sequenced members (Saier et al., 2000; Reddy et al., 2012). The members of the superfamily have diverse substrate specificities, acting as transporters for ions, sugars, sugar-phosphates, drugs, neurotransmitters, amino acids, peptides, etc. Topological studies and hydropathy analyses of the protein sequences indicate that almost all members of the MFS possess a uniform topology of 12 TM segments divided in two bundles of six-plus-six helices connected by a large intracellular loop, and both termini orientated towards the cytoplasm (Law et al., 2008). This six-plus-six TM arrangement is believed to have arisen from a gene duplication event in a common ancestor (Rubin et al., 1990). Up to date there are five transporters in the MFS having their crystallized 3D structure solved: the oxalate transporter (OxlT) (Hirai et al., 2002), Lactose/H+ transporter (LacY) (Abramson et al., 2003), Glycerol-3-phosphate/phosphate transporter (GlpT) (Huang et al., 2003), Multidrug transporter (EmrD) (Yin et al., 2006), and recently the Fucose/H+ transporter (FucP)(Dang et al., 2010). The x-ray structures available to date have revealed a conserved fold consisting of a pore located in between the two domains. The two domains rock back and forth against each other, the so-called rocker-switch mechanism, allowing the substrate to have alternating access to each side of the membrane (Huang et al., 2003, Abramson et al., 2003).

The transmembrane topology of Pho84 is predicted to be in agreement with the uniform six-plus-six TM arrangement of most MFS members (Bun-ya et al., 1991), but its crystal structure is yet to be solved. The high structural similarity between the solved MFS crystal structures, together with the common rocker-switch mechanism, makes it feasible to use solved MFS structures as templates for in silico modeling in the absence of a crystal structure. Amongst the solved MFS structures the GlpT fulfils most selection criteria in order to serve as a template for in silico modeling of Pho84 and has thus been used as such (Lagerstedt et al ., 2004) (Figure 11).

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N-terminal C-terminal

Cytoplasm

Periplasm

N-terminal C-terminal

Cytoplasm

Periplasm

Figure 11. Structural overview of the Pho84 structure. Visualized with Pymol software.

Post translational modification of Pho84

During low Pi conditions PHO84 gene expression is highly up-regulated. The transporter resides in the plasma membrane where it constitutes the major contributor to Pi import into the cell. However, the control of Pho84 activity is not limited to transcriptional regulation. During Pi surplus conditions the protein is internalized and degraded in the vacuole (Petersson et al., 1999; Lagerstedt et al., 2002). The mechanism for selective degradation in the vacuole and/or lysosome of plasma membrane proteins is via phosphorylation and ubiquitination events, a process also referred to as the PURE pathway, a multistep process involving Phosphorylation, Ubiquitination, Recognition and Endocytosis (Kelm et al., 2004). For rapid internalization and degradation of Pho84 in response to high Pi, phosphorylation prior to ubiquitination is required. Deletion of residues 304-327 in the central cytoplasmatic loop has been shown to result in a loss of Pi-induced antiphospho-threonine signal and severely delayed internalization

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(Lundh et al., 2009). This stretch of amino acids harbors only one threonine, at position 317 (Thr17), which implies that Pho84 requires phosphorylation of Thr317 prior to ubiquitination and degradation. This residue has been previously predicted to be phosphorylated (Peng et al., 2003). Likewise, the lysine at position 298 (Lys298) was predicted, and also shown, to be a target for ubiquitination. Failure of ubiquitination of Lys298 will result in a major defect in vacuolar sorting but not in the internalization rate of the transporter. This indicates that other lysine residues must be ubiquitinated and are involved in the down-regulation process.

The above mentioned post-translational modifications being responsible for the down-regulation of Pho84 are connected to the PKA pathway, and inhibition of PKA delays the degradation of Pho84 (Mouillon & Persson, 2005). Yet, how PKA is involved in the down-regulation process of Pho84 is currently not known. Moreover, post-translational modifications of Pho84 do not require Pi per se. It has been shown that the non-metabolizable Pi analog methylphosphonate triggers rapid down-regulation of Pho84 without affecting the transcription levels of the transporter, thus its effect is based on post-translational modifications (Pratt et al., 2004). It is currently not known if other phosphate analogs also may trigger the down-regulation of Pho84.

Pho84 and protein kinase A

Activation of the PHO pathway in response to changing Pi concentration levels in the surrounding environment implies that the S. cerevisiae cell possesses a sensor mechanism which is able to detect these variations. A deletion of the PHO84 gene results in a constitutive PHO pathway activation, even under high Pi conditions. This suggests that Pho84 acts as a phosphate sensor, relaying a signal upstream of Pho81, thus being involved in the regulation of the PHO pathway. Yet, over-expression of unrelated phosphate transporters (Wykoff and O’Shea, 2001) seems to suppress its constitutive phenotype. So a direct role for sensing and transcriptional repression of the PHO-responsive genes has been contradicted. Even though Pho84 does not seem to be directly involved in sensing and signaling the presence of phosphate for repression of the PHO regulatory pathway it is capable to act as a nutrient sensor activating the protein kinase A (PKA) pathway in response to phosphate (Giots et al., 2003). PKA is a key player in the nutritional control of metabolism, stress resistance, cell cycle and growth, and is thus of pivotal importance for adaption to changing environmental conditions (Rubio-Texeira et al., 2010).

Depending on which nutrient that is sensed, PKA signaling and subsequent activation occurs via different signaling pathways. Glucose

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sensing by the G protein-coupled receptor, Gpr1, involves cAMP as second messenger (Thevelein & de Winde, 1999), whereas sensing and signaling of phosphate by Pho84 (Giots et al., 2003), ammonium by Mep2 (van Nuland et al., 2006) and amino acids by Gap1 (Donaton et al., 2003) is independent of cAMP. However, for phosphate-, amino acid- and ammonium-signaling to the PKA, the presence of rapidly fermentable sugar (e.g. glucose) is required.

The ability of Pho84 to convey a Pi-dependent signal to the PKA is independent of its function as a Pi transporter (Paper IV). By replacing an aspartic acid at position 358 by asparagine or glutamic acid the transport was severely decreased or abolished, respectively, yet with only a minor change in its signaling activity. This finding correlates well with the previous finding (Popova et al., 2010) of non-transported phosphate containing compounds that can activate PKA signaling. Similar findings with non-transported signaling agonists have been obtained for the general amino acid transceptor Gap1 (Van Zeebroeck et al., 2009). However a deletion of three residues of the large central cytosolic loop, lysine 283, isoleucine 284 and glutamic acid 285 (Δ283-285) did not affect transport activity of Pho84 but almost abolished signaling to PKA (Paper II). Moreover, it was shown that despite the absence of its substrate Pho84 does sustain a basal level of signaling to PKA. This was shown when a replacement of an arginine at position 275 to a lysine yielded a non- transporting protein still capable of basal signaling (Paper II).

Pho84 and glucose sensors

Pho84 shows similarity at sequence level with the glucose sensors Snf3 and Rgt2 of S. cerevisiae (Gancedo, 2008). On a primary amino acid level it is more related to these sensors than other inorganic phosphate transporters in S.cerevisae (Paper II). These are both members of the MFS and have a proposed transmembrane topology in common with Pho84. These sensors convey a signal required for transcriptional activation of hexose transporter (HXT) genes in response to the level of available glucose. Snf3 conveys the signal for low glucose availability, inducing the transcription of the high affinity transporters HXT2, HXT6 and HXT7; whereas Rgt2 conveys the signal for high availability of glucose inducing the HXT1 expression. An arginine to lysine replacement in these sensors resulted in a constitutively signaling phenotype (Ozcan et al., 1996). A constitutively signaling phenotype was also shown when substituting the corresponding arginine residue in the Escherichia coli external glucose-6-phosphate sensor (UhpC) (Schwoppe et al., 2003). The UhpC has the ability to both sense and signal the presence and transport its substrate, which seems to be similar in the case of the Pho84 permease.

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Despite similarity at sequence level, topology and signaling function of Pho84, compared to that of Snf3, Rgt2 and UhpC, any replacement of the corresponding conserved residue, R195 of Pho84, abolished membrane localization of the protein (Paper II). Apparently the mechanism of conveying a signal by Pho84 seems not to be the same as in the glucose and glucose-6-phosphate sensors.

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RESULTS AND DISCUSSION

In this chapter the main findings in the studies are briefly presented. Mutational analysis of conserved glutamic acids of Pho89, a Saccharomyces cerevisiae high- affinity inorganic phosphate:Na+ symporter. (Paper I)

Pho89 belongs to the inorganic phosphate transporter (PiT) family, and there is almost nothing known about the structure and function of Pho89. In a previous alignment of the PiT family members, two conserved glutamic acids were identified, corresponding to Glu55 and Glu491 in Pho89. The Glu55 equivalent in human hPiT1 and hPiT2 was investigated for its involvement in Pi uptake and found to be of crucial importance in both transporters. The equivalent of Glu491 was also investigated in PiT2 with a similar result. We aimed to address if these residues do have a conserved function in Pho89 or if the function is specific for the hPit proteins. Topology predictions were undertaken. These showed that the glutamic acid residues are located in the same TM regions as in PiT2, TM2 and TM10, respectively. Alignments of these TMs revealed very high similarity between these proteins and subsequent mutational analysis of these residues on transport activity revealed highly similar results. Taken together, based on the similar topology, the highly conserved TM regions and similar impact of transport activity it is very likely that the glutamic acid residues have a conserved function in these transporters. We show here for the first time residues in Pho89 directly involved in Pi transport. Mutational analysis of the functional role of conserved arginines and the central cytoplasmic loop in the Saccharomyces cerevisiae Pho84 phosphate transceptor. (Paper II)

In Paper II we could establish that the main phosphate provider in S. cerevisiae, Pho84, exhibits a higher degree of sequence similarity to yeast glucose transporters and sensors, organo-phosphate transporters from E.coli

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and members of the human SPX (sugar/phosphate exchanger) subfamily than to the other endogenous inorganic phosphate transporters.

Substitution of arginine residues conserved through the entire sugar porter family, and Pho84, conferred constitutive signaling alleles of the yeast glucose sensors. But in contrast, any replacement of the corresponding conserved arginine in Pho84, at positions l95, prevented membrane localization of the transporter. Despite the conservation it clearly has other, structural features, in Pho84 as compared to the glucose sensors.

Arginine 275 in Pho84 corresponds to another conserved arginine where a replacement caused an increase in the affinity for the substrate glucose-6-phosphate in the UhpC. However, in Pho84 removal of the side chain by an alanine replacement was shown to abolish membrane insertion. On the other hand, a conservation of the charge by a lysine replacement at this position abolished the transport capacity but conferred constitutive signaling to the PKA. Our work has identified the first transport-defective allele with maintained basal signaling of the Pho84 thereby demonstrating that transport and signaling can be uncoupled.

The arginine at position 418 in Pho84 is a part of the conserved MFS motif-RKGRR. Conservation of the charge by replacement into a lysine resulted in a slight reduction of transport activity but unperturbed signaling capacity. However removal of the positive charge by a cysteine replacement prevented Pho84 to correctly insert in the membrane, the need of the positive charge at this position was thus demonstrated.

In alignments with phosphate transporters Lys283, Leu284 and Glu285, located in the large central cytosolic loop were found to be conserved, although not in comparison with sugar transporters and sensors. Interestingly, a deletion of these residues in Pho84, 283-285Δ, did not significantly decrease the transport activity but almost abolished signaling to the PKA. This mutant allele is the first reported with maintained transport activity but almost abolished signaling capacity. This clearly demonstrates that transport and signaling of Pho84 are two different functions and can be uncoupled.

Two other deletion mutants of the large cytosolic loop, 311-328Δ and 309-327Δ were created. They were both expressed and targeted to the plasma membrane as in the WT. However, even though both mutants showed a decrease in signaling, 311-328Δ was not affected in transport activity, whereas 309-327Δ showed an increase in transport activity. Putative role of Asp-76 and Asp-79 in proton-coupled inorganic phosphate transport of the Saccharomyces cerevisiae Pho84 phosphate:H+ transporter. (Paper III)

A general model on the transmembrane topology for MFS members have been proposed. This suggests that the translocation pore is constituted of transmembrane domain 1, 2, 4, 5, 7 and 8, while the remaining domains

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perform a scaffolding function. Thus, residues involved in the translocation of either substrate or co-substrate would be located in channel lining domains. Previous studies have identified a number of functional important residues in these domains that upon mutation exert an impact on the transport function. By the means of site directed mutagenesis and transport activity assays we have addressed the functional importance of two conserved residues, Asp79 and Asp76, that are located directly above the conserved Asp179 in the translocation pore. Replacement of Asp76 with either Glu or Asn resulted in a significant increase in Km and the Asn replacement also conferred an significant increase in Vmax. Thus this residue do have a functional importance for transport activity. However, replacement of Asp79 to Glu or Asn did not have an significant impact on transport activity, hence this residue seems not be of importance for transport activity. This was somewhat surprising since Asp79 was predicted to form an N-O-bridge with Arg168. In a previous study (Paper IV) Arg168 mutations conferred a decrease in transport activity and if there was a N-O bridge between these residues one would expect similar results. Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signaling to the PKA and PHO pathways. (Paper IV)

Despite the wealth of information available for the function and regulation of Pho84, knowledge of the specific residues directly involved in transport and sensing is still lacking. In Paper IV we used multiple sequence alignments and in silico modeling as a basis for a mutagenic study of the phosphate transport and sensing mechanism of the Pho84. This allowed, for the first time, individual residues involved in these mechanisms to be identified.

We showed that the ability of Pho84 to convey a Pi-dependent signal to the PKA is independent of its function as a Pi transporter. By replacing an aspartic acid at position 358 by asparagine or glutamic acid the transport was severely decreased or abolished, respectively, yet with only a minor change in it signaling activity. Also a lysine at position 492 was identified as important for the affinity towards phosphate as shown by the change in the transport kinetics upon mutagenesis of this residue.

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FUTURE PERSPECTIVES

The mechanism and function of many nutrient transporters have been extensively characterized during the last decades but many of the questions raised remain to be answered. A detailed understanding of the entire transport cycle could facilitate the understanding of various diseases where mutations in nutrient transporter are involved and could have therapeutical implications. Thus, it is of outermost importance that we strive towards to fully understand the transport of nutrients on a molecular level. The crystallized 3D structures of membrane transporters which have been solved recent years have made big contributions to our understanding of their transport mechanisms, not only by providing us with direct information but also helping us develop new tools such as molecular dynamics and docking studies which can be very informative in combination with mutagenic studies. Solving more crystal structures of nutrient transporters, and the use of in silico models based on previous solved structures, will allow for more information to be added to our present knowledge. The aim with the work of this thesis have been to shed some light over the transport mechanisms of two phosphate transporters in yeast, and the work presented here have contributed to increasing this knowledge. But it is still only a tip of an iceberg since there are numerous of transporters that could be beneficial, in one way or another, to characterize.

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ACKNOWLEDGEMENTS

In a published paper usually much more work has been done then the results shown in the paper. So is also the case with this thesis. Despite that only my name is shown on the cover, all this work could not have been done without support. I would like to thank all the people supporting me in various ways during these years. I would like to thank former and present members of the yeast research group. Bengt, my main supervisor, for giving me the opportunity to work here at the Linnaeus University. Also my co-supervisor Johan Thevelein in Belgium for some nice collaborations, and having far too many ideas of what we could do. Fredrik, you finished up soon after I started so I didn’t get enough time to harass you with stupid questions. Siamak, without your support in the very beginning I would really have been lost in the lab. Palani your work has been the main reason for most of the meetings since you started, and it is always welcome with a break in the daily work. Lorena, the most recent member of the group. Dieter for being supportive during times when things did not work out as planned, and despite being a weird Belgian guy you made me laugh and forget reality for a while. I am also grateful for the pedagogical input in writing this thesis from Håkan. My friends Johan, Pernilla, and Peter. No one makes it without friends. Madeleine, your own thesis work was always a source of inspiration and without it I would never had made it. I would also like to thank my family; even though you did not understand what I did you never gave up hope that I would make it some day. In particular I would like to thank my mother, Margaretha, and my nephew, Wilhelm, who did not make it to see me graduate, I wish you could have been here. Finally, Susanne, thank you for everything.

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