Chao Zhai

Kuoyu Li

Valentini Markaki

John P Phelan

Katherine Bowers

Frank T Cooke

Barry Panaretou
email: barry.panaretou@kcl.ac.uk

Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane

Keywords: Signalling, phosphoinositide, lipid kinase, Saccharomyces cerevisiae

Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including re-modelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 physically interacts with the plasma-membrane associated phosphatidylinositol 4-kinase Stt4. Here we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphatidylinositol 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4dependant events at the plasma membrane, as opposed to a general role in overall metabolism of phosphatidylinositol 4-phosphate. Use of a pleckstrin homology domain sensor reveals there are substantially less plasma membrane associated 4-phosphorylated phosphoinositides in ypp1 mutants, in comparison to wild-type cells. Furthermore, in vivo labelling with [ 3 H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality at non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphatidylinositol 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of phosphatidylinositol 4-phosphate in sac1 cells is restored in combination with a ypp1 mutant, consistent with the opposing effects these two mutations have on levels of this phosphoinositide.

INTRODUCTION

Polyphosphoinositide second messengers regulate numerous cellular processes in eukaryotes, including organization of the actin cytoskeleton, vesicle trafficking and signalling via MAP kinase cascades. There has been significant progress in understanding how the rapid turnover of polyphosphoinositides (PPIn) is controlled both spatially and temporally (reviewed in [START_REF] Strahl | Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae[END_REF]). Of the seven PPIn species recognized, four have been identified in the budding yeast Saccharomyces cerevisiae. One of these, phosphatidylinositol 4-phosphate (PtdIns4P), is synthesized by three distinct and highly conserved phosphatidylinositol 4-kinases: Pik1 [START_REF] Flanagan | Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability[END_REF][START_REF] Garcia-Bustos | PIK1, an essential phosphatidylinositol 4-kinase associated with the yeast nucleus[END_REF], Stt4 [START_REF] Yoshida | A novel gene, STT4, encodes a phosphatidylinositol 4-kinase in the PKC1 protein kinase pathway of Saccharomyces cerevisiae[END_REF] and Lsb6 [START_REF] Barylko | A novel family of phosphatidylinositol 4-kinases conserved from yeast to humans[END_REF][START_REF] Han | The Saccharomyces cerevisiae LSB6 gene encodes phosphatidylinositol 4-kinase activity[END_REF][START_REF] Shelton | Saccharomyces cerevisiae contains a Type II phosphoinositide 4kinase[END_REF].

Pik1 is found at the Golgi and in the nucleus, Stt4 localizes to the plasma membrane, and Lsb6 is found at the plasma membrane and the vacuolar membrane. Both Stt4 and Pik1 are essential, synthesising a discrete location-specific pool of PtdIns4P. Consequently, deletion of one cannot be compensated by overexpression of the other, even though both proteins catalyse the same biochemical reaction [START_REF] Cutler | STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae[END_REF]. Lsb6, the third phosphatidylinositol 4-kinase, is non-essential; its contribution to total cellular PtdIns4P in vegetative cells being uncertain, given that over 90% of PtdIns4P is lost in a stt4 ts pik1 ts double mutant within one hour of shift to the non-permissive temperature [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF]. Thus far, the only phenotype observed in lsb6 cells is a reduction in endosome motility, an investigation prompted by the interaction of this lipid kinase with Las17 (the yeast orthologue of Wiskott-Aldrich syndrome protein), which is essential for endocytosis. In the lsb6 background, expression of a catalytically inactive Lsb6 mutant could restore wild-type levels of endosome motility as effectively as expressing wild-type Lsb6. Therefore, the role played by Lsb6 in endosome motility is independent of its lipid kinase activity [START_REF] Chang | A WASp-binding type II phosphatidylinositol 4-kinase required for actin polymerization-driven endosome motility[END_REF].

Pik1 localizes to the Golgi, and the PtdIns4P synthesized by this enzyme is essential for Golgi to plasma membrane vesicular trafficking [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF]11,[START_REF] Walch-Solimena | The yeast phosphatidylinositol-4-OH kinase Pik1 regulates secretion at the Golgi[END_REF]. In addition, Pik1 shuttles between the cytoplasm and the nucleus. Mutant alleles of PIK1 that restrict location of the protein to membranes of the secretory pathway, do not sustain viability of a pik1 strain. Therefore, the PtdIns4P generated in the nucleus by Pik1 is essential for cell viability, though the nuclearspecific role of PtdIns4P remains unknown [START_REF] Strahl | Yeast phosphatidylinositol 4kinase, Pik1, has essential roles at the Golgi and in the nucleus[END_REF]. Stt4 localizes to the plasma membrane, in part via an interaction with Sfk1, a membrane spanning protein [14]. The PtdIns4P pool generated by Stt4 is required for aminophospholipid transport from the endoplasmic reticulum to the Golgi [START_REF] Trotter | A genetic screen for aminophospholipid transport mutants identifies the phosphatidylinositol 4kinase, Stt4p, as an essential component in phosphatidylserine metabolism[END_REF], organization of the actin cytoskeleton, maintenance of vacuole morphology [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF] and cell wall integrity [14]. Some of these roles are due to PtdIns4P acting as a precursor for PtdIns(4,5)P 2 synthesis which is catalysed by the plasma-membrane associated phosphatidylinositol 4phosphate 5-kinase Mss4 [START_REF] Desrivieres | MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae[END_REF]. For instance, plasma membrane PtdIns(4,5)P 2 activates the Rho/Pkc1-mediated MAP kinase cascade that maintains cell wall integrity. More specifically, generation of PtdIns(4,5)P 2 recruits the guanine nucleotide exchange factor Rom2 to the plasma membrane via its phosphoinositide PH (pleckstrin homology) binding domain, and is an absolute requirement for activation of the Rho1 GTPase [14].

There has been substantial progress in characterizing mechanisms that terminate PtdIns4P dependent processes, through identification of lipid phosphatases with enzymatic activity towards phosphoinositides [START_REF] Guo | SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases[END_REF], and reviewed in [START_REF] Strahl | Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae[END_REF]). In contrast, mechanisms that regulate the generation of PtdIns4P by Stt4 are poorly defined. Insight into mechanisms that regulate lipid kinases has come from identifying accessory factors. For example, Frq1 and Vps15 are required for both membrane localization and optimal activity of the PtdIns 4-kinase Pik1 and the PtdIns 3-kinase Vps34, respectively [START_REF] Hendricks | Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase[END_REF][START_REF] Stack | Vesicle-mediated protein transport: regulatory interactions between the Vps15 protein kinase and the Vps34 PtdIns 3-kinase essential for protein sorting to the vacuole in yeast[END_REF].

Recently, the protein encoded by the essential ORF YGR198W was identified as a binding partner of Stt4 [START_REF] Hazbun | Assigning function to yeast proteins by integration of technologies[END_REF]. This ORF was recently named YPP1 (sYnuclein-Protective Protein), because it acts as a multi-copy suppressor of the toxic effect of expressing a human alpha-synuclein mutant in yeast [START_REF] Flower | YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation[END_REF]. We have tested the possibility that Ypp1 is an accessory factor for Stt4. We characterize the phenotypes of temperature sensitive (ts) mutants of YPP1, and we show that they are similar to phenotypes associated with stt4 ts mutants. Moreover, overexpression of STT4 suppresses the temperature sensitive growth defect of ypp1 mutants. We go on to show that total levels of PtdIns4P, as well as levels of plasma membrane 4-phosphorylated PtdIns, are low in ypp1 ts mutants. Together, these data show for the first time that Ypp1 makes an essential contribution to Stt4 dependent production of PtdIns4P at the plasma membrane.

MATERIALS AND METHODS Strains, plasmids and media.

Yeast strains used in this study are listed in Table 1. Plasmids are described in the appropriate sections below. Strains were grown in either rich media (YPD) or synthetic minimal media (SD) with appropriate supplements for plasmid maintenance [START_REF] Rose | Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual[END_REF]. Gene deletions were performed by replacing the target ORF with hph (conferring resistance to Hygromycin B), via one step gene replacement, as previously described [23]. Integration at the correct locus was confirmed by PCR using appropriate primers [START_REF] Wach | Yeast Gene Analysis[END_REF].

Isolation of YPP1 conditional alleles.

The heterozygous YPP1/ypp1 diploid Y24828 was obtained from EUROSCARF and transformed with a centromeric URA3 S. cerevisiae/Escherichia coli shuttle vector (pYC3P) bearing the YPP1 promoter (a fragment 0.45kb upstream of the ATG) fused to the YPP1 ORF (a 2.5kb fragment), followed by the ADH1 transcriptional terminator (a 0.4kb fragment). The YPP1 promoter and ORF from this vector were then subcloned into a centromeric L E U 2 S.cerevisiae/Escherichia coli shuttle vector (again bearing the ADH1 terminator), to give pYC1P. Strain Y24828 was transformed with pYC3P and then sporulated, followed by subsequent dissection of tetrads and selection of a ypp1 haploid (strain ZC0) with viability maintained by the presence of the URA3 vector bearing YPP1 (pYC3P). Mutations in YPP1 were generated by error-prone amplification of the YPP1 ORF using pYC3P as template, as previously described [25]. The oligonucleotide primers were designed so that the 5' primer overlaps the 3' end of the YPP1 promoter, and the 3' primer overlaps the 5' end of the ADH1 transcriptional terminator (15bp overlaps in both cases). Vectors bearing the mutant ypp1 alleles were generated by transforming strain ZC0 with a) the mutant ypp1 PCR products plus b) a large linear fragment from pYC1P lacking the wild-type YPP1. The two fragments recombine with each other in vivo, owing to the 15bp overlaps between them. The transformants, selected on SD media lacking both leucine and uracil, possess the pYC3P URA3 vector bearing wild-type YPP1, and a LEU2 vector bearing mutated ypp1. More than 4000 transformants were picked and incubated overnight at 25 0 C in 384 well plates containing SD lacking leucine. Those transformants bearing ts mutants of ypp1 were isolated as described previously [START_REF] Hu | Qri2/Nse4, a component of the essential Smc5/6 DNA repair complex[END_REF]. To ensure these ts phenotypes were attributed to mutated ypp1, plasmids from the strains were recovered, transformed into the ZC0 strain and rescreened for the ts phenotype. In this way, a series of isogenic ypp1 haploid strains were generated, possessing vector-borne copies of wild-type YPP1 (ZC1, Table 1) or ypp1 ts alleles (ZC2-8, Table 1). The coding regions of ypp1 ts mutants were sequenced.

Sensitivity to cell wall damaging agents.

Exponentially growing cultures were adjusted to equal cell density (1 x 10 7 cells/ml) and four successive sixfold serial dilutions were spotted on YPD, or YPD containing the desired concentration of sorbitol, caffeine or SDS. The plates were incubated at the temperatures specified in figure legends. Phosphoinositides were labeled in vivo as previously described [START_REF] Mollapour | Weak acid and alkali stress regulate phosphatidylinositol bisphosphate synthesis in Saccharomyces cerevisiae[END_REF]. Briefly, strains were grown at 25 0 C in SD medium without inositol plus appropriate supplements, in the presence of 15µCi/ml [ 3 H]inositol (GE Healthcare). Once cultures had reached exponential phase, they were split into two aliquots; one was incubated for a further hour at 25 0 C, the other was shifted to 37 0 C for 1 hour. Cells were killed by an equal volume of ice-cold methanol, and sedimented (3000 xg, 5 minutes at 4 0 C). Lipids were extracted, and then deacylated, followed by recovery of the glycerophosphoinositols that were resolved by anion-exchange HPLC on a Partisphere 5-SAX column (Whatman).

Assessing multicopy suppression by selected ORFs.

Candidate ORFs were overexpressed from the vector pY195M, which is a modification of the episomal URA3 vector YEplac195 [28]. The vector bears the MET25 inducible promoter and the transcription terminator from PGK1. Candidate ORFs were amplified from S.cerevisiae genomic DNA and ligated into pY195M. The resulting vectors were transformed into the ZC1 strain, and isogenic strains containing vector borne ypp1 ts alleles. Transformants were transferred to SD plates lacking uracil, leucine and methionine, to induce over-expression of the candidate ORF.

Two-hybrid analysis.

The YPP1 and STT4 ORFs were amplified from yeast genomic DNA, including appropriate restriction sites in the oligonucleotides. The YPP1 PCR product was subcloned into pGAD-C1 [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF] to give pAD-YPP1; STT4 was cloned into pGBDU-C1 [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF] to give pBD-STT4. Vectors pAD-YPP1 and pBD-STT4 were transformed into strains PJ69-4a and PJ69-4 , respectively [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF]. Transformants (a and ) were mated on YPD for 8 h at 30 0 C, and then diploids selected on SD lacking both leucine and uracil. To assess Stt4/Ypp1 interaction, the diploids were innoculated onto SD lacking leucine, uracil and histidine, and in the presence of 1 mM 3-AT (Sigma).

Assessing the activation state of the MAPK cell wall integrity pathway.

Exponentially growing cultures were either heat shocked or treated with 10mM caffeine (as specified in figure legends). Preparation of total yeast protein extracts was as described previously [START_REF] Panaretou | ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo[END_REF]. Western blots were probed with either a) anti-(Thr202/Tyr204)-p44/42 MAP kinase antiserum (New England Biolabs) which specifically recognizes the dually Thr190/Tyr192-phosphorylated (activated) Slt2 in yeast, or b) anti-Slt2 (Santa Cruz). Activity of Rlm1 transcription factor activity was assessed using p2-RLM1 a YIL117c promoter-LacZ reporter plasmid [START_REF] Jung | Regulation of the yeast Rlm1 transcription factor by the Mpk1 cell wall integrity MAP kinase[END_REF].

Localization of actin and 4-phosphorylated PtdIns.

Cells were grown to mid-logarithmic phase at 25 0 C, followed by further incubation at 25 0 C or upshift to 37 0 C, then fixed in formaldehyde and stained with rhodamine-phalloidin (Molecular Probes) to visualize actin [START_REF] Benedetti | The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast[END_REF]. The cellular distribution of 4-phosphorylated PtdIns was assessed by transforming strains with pTL511, a centromeric URA3 plasmid expressing a tandem dimer of the PH domain of Osh2 fused to green fluorescent protein (GFP), under control of the constitutively active PHO5 promoter [START_REF] Roy | Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p[END_REF]. Prior to observation by fluorescence microscopy, exponential phase cells were either maintained at 25 0 C or shifted to a strongly non-permissive temperature (39 0 C) for 15 minutes, as described previously [START_REF] Roy | Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p[END_REF].

Endocytosis of Ste3-GFP and Fui1-GFP.

Strains were transformed with pFL38GalFUI1-GFP, a centromeric URA3 vector that enables galactose inducible synthesis of an Fui1-GFP fusion [START_REF] Blondel | Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation[END_REF]. For localization of Fui1-GFP, cells were grown to exponential phase in 2% galactose and 0.02% glucose at 25 0 C, to induce synthesis of Fui1-GFP. This was followed by addition of 2% glucose, to repress Fui1-GFP synthesis. Cells were then maintained at 25 0 C or shifted to 37 0 C for thirty minutes, followed by addition of uridine (80µg/ml) to induce endocytosis. Samples were removed for observation at the intervals described in figure legends, and endocytosis was stopped by adding ice-cold energy poisoning buffer (SD media without carbon source, 10mM NaF, 10mM NaN 3 ) as previously described [35]. GFP fusions were visualized by fluorescence microscopy.

RESULTS

Isolation of ypp1 ts alleles.

To determine the function of Ypp1, we generated conditional lethal alleles by PCR-mediated mutagenesis. Seven ypp1 ts mutants were isolated, three of which exhibited moderate growth at 37 0 C, with the remaining four displaying no growth at all at this temperature (Fig. 1A). Three of the latter, exhibiting a robust ts phenotype, were selected for further work and the Ypp1 coding regions were sequenced. Most error-prone PCR techniques are not truly random as they yield more transitions than transversions. The method we used however, removes the bias [25]. The amino acid substitutions were: ypp1-35 ts G60R, N79I, N102S, E189A, L220P, K329R and S336P; ypp1-39 ts P2A, K197E, F218L, L442S, L453P and C463R; and ypp1-41 ts F343V, V538M, W571R, K577E, I584V, K656E and S678L.

Phenotypes of ypp1 ts mutants are similar to those displayed by stt4 ts and mss4 ts mutants.

The PtdIns(4,5)P 2 generated by Stt4 and Mss4 plays an essential role in the cell wall integrity pathway; inactivation of either kinase leads to cell lysis [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF][START_REF] Desrivieres | MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae[END_REF]. As a result, the lack of growth displayed by stt4 ts cells at 37 0 C is suppressed in the presence of 1M sorbitol [14], a property shared by all seven ypp1 ts mutants (Fig. 1A), albeit to a varying extent. This osmoremedial phenotype is displayed clearly by six of the mutants, and is poorly displayed (but still detectable) by ypp1-35 ts (Fig. 1A).

The ypp1 ts mutants also display other phenotypes characteristic of defects in the cell wall integrity MAP kinase cascade, namely caffeine sensitivity, which can be suppressed in the presence of an osmotic stabilizer such as sorbitol, and sensitivity to low levels of SDS (Fig. 1B). Of the three mutants tested, ypp1-35 ts was the most sensitive to caffeine and SDS. This was expected, because this mutant displayed a robust temperature sensitive phenotype, which was poorly suppressed by osmotic stabilization of media (Fig. 1A).

Plasma membrane phosphoinositides generated by the sequential action of Stt4 and Mss4 have also been implicated in the organization of the actin cytoskeleton. In the wild-type, actin distribution is polarized, as indicated by the concentration of cortical actin in the bud (Fig. 2). A shift from 25 0 C to 37 0 C degrees causes a heat-induced disorganization of the actin cytoskeleton, which is transient in the wild-type, persisting for upto 2 hours, followed by recovery in organization of the cytoskeleton. In contrast to wild-type cells, however, this disturbance in actin organization persisted for at least 3 hours after temperature upshift in ypp1 ts mutants, with random cortical actin patches evident throughout both mother and daughter cells (Fig. 2). Such irregular organization of actin was even noticeable at permissive temperature in the ypp1-35 ts mutant (Fig. 2). An identical phenotype is displayed by both stt4 ts and mss4 ts mutants [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF][START_REF] Desrivieres | MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae[END_REF].

STT4 is a multicopy suppressor of ypp1 ts .

Overexpression of the PtdIns 4-kinase encoded by STT4 is sufficient to restore growth of ypp1-39 ts and ypp1-41 ts at 37 0 C (Fig. 3). This confirms the possibility of biological interaction between these two proteins inferred by both their physical interaction [START_REF] Hazbun | Assigning function to yeast proteins by integration of technologies[END_REF], and the similarities in phenotypes that arise when function of either protein is compromised. Growth of ypp1-35 ts at 37 0 C, however, was not restored. This is probably due to the extent to which Ypp1 function is compromised in this mutant; of the three mutants tested, ypp1-35 ts was the most sensitive to SDS and caffeine (Fig. 1B), and temperature sensitivity was barely suppressed by osmotic stabilization (Fig. 1A). In S.cerevisiae there is one other essential PtdIns 4-kinase, the Golgi-localized Pik1. Even though Stt4 and Pik1 both produce the same second messenger, deletion of one kinase cannot be rescued by overexpression of the other because the enzymes synthesize essential pools of PtdIns4P on different intracellular membranes, and PtdIns4P is not freely diffusible through the cell [START_REF] Cutler | STT4 is an essential phosphatidylinositol 4-kinase that is a target of wortmannin in Saccharomyces cerevisiae[END_REF]. At 37 0 C, the ypp1-39 ts and ypp1-41 ts mutants were rescued by overexpressing STT4, but not by PIK1 (Fig. 3) or by LSB6, the third PtdIns 4-kinase in S. cerevisiae (not shown). This implies a role for Ypp1 in Stt4-dependant events at the plasma membrane, as opposed to a general role in overall metabolism of PtdIns4P. Growth at 37 0 C was not restored in any of the mutants when the phosphatidylinositol 4-phosphate 5-kinase Mss4 was over-expressed (not shown). Rescue by Stt4 overexpression implies that levels of PtdIns4P are too low to support viability of ypp1 ts mutants at 37 0 C.

The physical interaction between Stt4 and Ypp1 was first identified by affinity capture-MS [START_REF] Hazbun | Assigning function to yeast proteins by integration of technologies[END_REF]. We confirmed this by the yeast-two hybrid interaction trap, using a BD-Stt4 fusion as bait and an AD-Ypp1 fusion as prey (Fig. 4A). Furthermore, the interaction between wild-type Ypp1 and Stt4 is weakened at high temperature (Fig. 4A and Fig. 4B), as evidenced by the slower growth rate at 37 0 C versus 25 0 C, on media that select for Ypp1-Stt4 interaction. In addition we went on to show that interaction between Stt4 and the ypp1-35 ts , ypp1-39 ts and ypp1-41 ts mutants, was lost at 37 0 C. Indeed, the interaction between these mutant proteins and Stt4 was so destabilized, that it could not be detected even at 25 0 C (Fig. 4A). We anticipated, however, that interaction at 25 0 C might be displayed between Stt4 and one of the less robust ts mutants, such as ypp1-30 ts (Fig. 1A). This was found to be the case, as a weak interaction between Stt4 and ypp1-30 ts was detectable at 25 0 C (Fig. 4B).

At the restrictive temperature, ypp1 ts cells display diminished levels of plasma membraneassociated 4-phosphorylated PtdIns.

Cells lacking genomic YPP1 but expressing wild-type or mutant ypp1 were transformed with a vector expressing a GFP-PH Osh2 dimer. The pleckstrin homology (PH) domain of Osh2 specifically recognizes PtdIns4P and PtdIns(4,5)P 2 and lacks an accessory site that would lead to association with a specific cellular membrane, allowing it to be exploited as an unbiased reporter for PtdIns4P and PtdIns(4,5)P 2 [START_REF] Roy | Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p[END_REF]. In wild-type cells, at both 25 0 C and 39 0 C, this reporter localized to both punctate internal structures and the plasma membrane (Fig. 5A), in agreement with previously reported localization for this sensor [START_REF] Roy | Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p[END_REF]. In the ypp1-41 ts mutant, however, 15 minutes incubation at 39 0 C led to a rapid loss of plasma membrane localization of the reporter. The effect was even more severe in ypp1-35 ts cells, as GFP-PH Osh2 dimer localization at the plasma membrane was significantly reduced even at permissive temperature (Fig. 5A).

At the restrictive temperature, ypp1-41 ts cells display diminished levels of PtdIns4P.

Wild-type and ypp1-41 ts cells were incubated with [ 3 H]inositol at 25 0 C for upto 6 generation times, to label all inositol-based lipids to near steady-state equilibrium. These exponential-phase cultures were split in two, with one aliquot shifted to 37 0 C for 1 hour, and the other maintained at 25 0 C. Subsequent analysis of labelled lipids by HPLC allowed us to follow the levels of glycerolphosphoinositols derived from all four PPIns made by S.cerevisiae. As expected, levels of PtdIns(3,5)P 2 were almost identical in mutant and wild-type cells (Fig. 5B).

At the permissive temperature, the level of PtdIns4P in ypp1-41 ts cells was almost 60% lower than that seen in wild-type cells (Fig. 5B). A shift to 37 0 C for one hour led to a 50% decline in PtdIns4P in the wild-type. Under these conditions the level of PtdIns4P in ypp1-41 ts did not fall appreciably in comparison to levels in ypp1-41 ts at 25 0 C, though levels of PtdIns4P in this mutant were already low prior to the upshift in temperature.

The changes in PtdIns4P are reflected by the in vivo protein-protein interaction data because the interaction between the Stt4 lipid kinase and wild-type Ypp1 is weaker at high temperature (Fig. 4A and Fig. 4B). If Ypp1 is required for optimal function of Stt4, then an upshift in temperature (diminishing the interaction between the two proteins) would account for the drop in synthesis of PtdIns4P observed at 37 0 C in wild-type cells (Fig. 5B). A consequence of reduced interaction between wild-type Ypp1 and Stt4 would mean that the effect of mutant Ypp1 on Stt4 function would be less noticeable at 37 0 C than at 25 0 C. This explains why, at 37 0 C, levels of PtdIns4P are only slightly lower in the ypp1-41 ts mutant than they are in the wild-type.

Levels of PtdIns(4,5)P 2 were lower in ypp1-41 ts cells relative to the wild-type, at both permissive and non-permissive temperatures, though the decline in levels was not as great as that observed for PtdIns4P. This was not surprising as a similar result was reported when labelled phosphoinositides were assessed in stt4 ts cells, namely a decline in levels of PtdIns(4,5)P 2 that was not as great as the decline in PtdIns4P levels [14].

At the permissive temperature, levels of PtdIns3 P were significantly lower in the ypp1-41 ts mutant when compared to the wild-type; though levels of this phosphoinositide declined to similar levels in wild-type and mutant, following heat shock (Fig. 5B).

Deletion of SAC1 restores viability of ypp1 ts mutants under non-permissive conditions.

PtdIns4P is essential for cell viability, but an upshift in temperature leads to a decrease in its synthesis (Fig 5B ). If the lower level of PtdIns4P synthesized at 25 0 C in ypp1 ts mutants is the principal cause of lethality on temperature up shift, then limiting the activity of phosphatases that hydrolyse PtdIns4P should raise PtdIns4P levels of this phosphoinositide, thereby restoring viability. The only lipid phosphatases with enzymatic action towards PtdIns4P are those containing the Sac1 homology domain. There are five members of this family in yeast, Sac1, Fig4 and the three synaptojanin-like phosphatases Sjl1/Inp51, Sjl2/Inp52 and Sjl3/Inp53 [START_REF] Guo | SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases[END_REF][START_REF] Hughes | Sac phosphatase domain proteins[END_REF]. Of these, Sac1 is the predominant 4-phosphatase in yeast, as cells lacking the corresponding gene have up to 12 times more PtdIns4P than wild-type cells [START_REF] Guo | SAC1-like domains of yeast SAC1, INP52, and INP53 and of human synaptojanin encode polyphosphoinositide phosphatases[END_REF][START_REF] Foti | Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology[END_REF][START_REF] Konrad | Retention of the yeast Sac1p phosphatase in the endoplasmic reticulum causes distinct changes in cellular phosphoinositide levels and stimulates microsomal ATP transport[END_REF]. Moreover, Stt4 (rather than Pik1 or Lsb6) synthesizes the majority of PtdIns4P that accumulates in sac1 cells [35,[START_REF] Foti | Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology[END_REF]. Loss of Sac1 activity restored viability at 37 0 C to all three ypp1 ts mutants tested (Fig. 6), further confirming that the essential function of Ypp1 is to facilitate PtdIns4P synthesis via activation of Stt4. Sequential action by Stt4 and Mss4 generates PtdIns(4,5)P 2 which tethers the guanine nucleotide exchange factor Rom2 to the plasma membrane, via interaction with the Rom2 PH domain. This activates Rho1, which in turn activates Pkc1, initiating a phosphorylation cascade leading to activation of the MAPK Slt2 (reviewed in [START_REF] Levin | Cell wall integrity signaling in Saccharomyces cerevisiae[END_REF]). Compromising the function of Stt4 causes a decrease in the level of PtdIns4P and in turn, causes destabilization of the cell wall [14]. The same is true when function of Ypp1 is compromised (Fig. 5B and Fig. 1B), so one possibility is that these mutants fail to activate the cell wall integrity MAPK pathway. We investigated whether this was the case. As expected, Slt2 in wild-type cells is unphosphorylated at 25 0 C, but is phosphorylated in the presence of caffeine, which is known to de-stabilize the cell wall (Fig 7A). Of the three mutants tested, ypp1-35 ts was selected for investigating the activity of components involved in the cell wall integrity pathway, because this mutant was the most sensitive to caffeine (Fig. 1B). Slt2 was phosphorylated normally in response to caffeine in ypp1-35 ts (Fig. 7A). Phosphorylated Slt2 activates Rlm1, the major transactivator of genes involved in cell wall maintenance [START_REF] Levin | Cell wall integrity signaling in Saccharomyces cerevisiae[END_REF]. However, a cell wall integrity defect may still result if there is a defect downstream of Slt2, even though signalling through the MAPK cascade itself appears to be normal in ypp1-35 ts shifted to the non-permissive temperature. Given that there are mutants which are defective in Rlm1-mediated transcription, even when Slt2 is activated normally [START_REF] Truman | In the yeast heat shock response, Hsf1-directed induction of Hsp90 facilitates the activation of the Slt2 (Mpk1) mitogen-activated protein kinase required for cell integrity[END_REF], we decided to measure transcription of a LacZ reporter controlled by the Rlm1-dependent promoter of YIL117c. In the presence of caffeine, Rlm1-mediated transcription was stimulated to the same extent in wild-type and ypp1-35 ts cells (Fig. 7B). Consequently, the cell wall integrity defect in ypp1-35 ts is not due to defective signaling downstream of Pkc1. Moreover, overexpression of components of this cascade including the MAPK kinase kinase Bck1, either of the redundant MAPK kinases (Mpk1/2), the MAPK Slt2, as well as Pkc1 itself, all failed to rescue the nogrowth defect at 37 0 C of the three ypp1 ts mutants (not shown).

Endocytosis of plasma membrane proteins is unaffected in the ypp1-35 ts mutant.

Recently, it has been shown that overexpression of YPP1 suppresses the toxicity of the human A30P -synuclein ( -syn) mutant that is associated with early onset Parkinson's disease. Elevated levels of Ypp1 lead to endocytosis and degradation of A30P -syn in the yeast vacuole [START_REF] Flower | YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation[END_REF]. However, -syn is found in the cytosol and is not native to yeast, and as such it is not a typical substrate for the endocytic process. We assessed the possible role Ypp1 might play in endocytosis by following the localization of yeast protein substrates that use the endocytic route for their delivery to the vacuole.

To examine the role of Ypp1 in endocytosis we followed the trafficking of the uridine permease Fui1. The rate of constitutive endocytosis of this transporter is relatively low, which permits visualization of this protein at the plasma membrane (unlike the a factor receptor, Ste3, for example, which undergoes rapid constitutive internalization). However, the rates of endocytosis and subsequent degradation of Fui1 in the vacuole, are increased significantly in the presence of uridine [START_REF] Blondel | Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation[END_REF]. We followed the intracellular trafficking of an Fui1-GFP fusion, synthesis of which was under the control of the inducible GAL10 promoter [START_REF] Blondel | Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation[END_REF]. Cells were grown to exponential phase in galactose to induce synthesis of Fui1-GFP, followed by addition of glucose (to block permease synthesis) and a temperature shift to 37 0 C. Cells were maintained at this temperature for 30 minutes to induce the temperature sensitive phenotype. At this stage, similar levels of Fui1-GFP were seen at the plasma membrane, in both wild-type and ypp1-35 ts mutant cells (Fig. 8). Also, vaculolar fluorescence was clearly observed because of the relative resistance of GFP to vacuolar hydrolases [START_REF] Blondel | Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation[END_REF]. Uridine was then added to the cells, and the temperature was maintained at 37 0 C. As can be seen in Fig. 8, Fui1-GFP staining is lost from the cell surfaces of both wild-type and ypp1-35 ts cells after incubation for 15 or 60 minutes with uridine. Therefore, the ypp1-35 ts mutation has no dramatic affect on the overall endocytosis rate of Fui1-GFP. To confirm that we were following an endocytic process we assessed the localization of the Fui1-GFP fusion in end3 cells treated in the same way. End3 is essential for the internalization step of endocytosis [START_REF] Benedetti | The END3 gene encodes a protein that is required for the internalization step of endocytosis and for actin cytoskeleton organization in yeast[END_REF]. As expected, the Fui1-GFP in the end3 background was not internalized, remaining at the plasma membrane (Fig. 8). Additionally we saw similar results when we followed another endocytic cargo, Ste3-GFP in wild-type and ypp1-35 ts cells (not shown), so our data would suggest that low PtdIns4P does not inhibit endocytosis.

In the wild-type and ypp1-35 ts cells, we noted that levels of internalized Fui1-GFP after the 30 minute heat shock, but prior to addition of uridine, were particularly high (Fig. 8). This was expected, because heat shock decreases the half-life of membrane transporters through stimulation of endocytosis [START_REF] Volland | Endocytosis and degradation of the yeast uracil permease under adverse conditions[END_REF][START_REF] Bultynck | Slm1 and slm2 are novel substrates of the calcineurin phosphatase required for heat stress-induced endocytosis of the yeast uracil permease[END_REF].

As well as stimulating endocytosis of Fui1, uridine also stimulates exocytic trafficking of this permease by inducing direct traffic from the Golgi to the endosomal system, bypassing the plasma membrane. This type of traffic, however, did not contribute to the changes in localization of Fui1-GFP we observed, because we repressed Fui1-GFP synthesis by addition of glucose, prior to heat shock and subsequent addition of uridine. Therefore, any changes in localization of Fui1-GFP caused by heat shock or uridine are only attributable to endocytic trafficking. This is confirmed by the lack of vacuolar staining in the end3 background, which is blocked for the early stages of endocytosis, but not deficient in exocytic traffic [START_REF] Blondel | Direct sorting of the yeast uracil permease to the endosomal system is controlled by uracil binding and Rsp5p-dependent ubiquitylation[END_REF].

The induction of endocytosis by heat shock meant that subtle changes caused by compromised Ypp1 function may not be observed at 37 0 C. For this reason, we assessed localization of Fui1-GFP at 25 0 C. As expected, the rate of endocytosis in response to addition of uridine was much slower at 25 0 C than at 37 0 C. In wild-type cells, there was little change in levels of plasma membrane-associated Fui-GFP by fifteen minutes after addition of uridine (Fig. 9), in contrast to a complete lack of plasma membrane-associated transporter at the same stage at 37 0 C (Fig. 8). At 25 0 C, by sixty minutes after addition of uridine, all of the Fui1-GFP had been internalized. However, there was no difference in Fui1-GFP internalization between the wild-type and ypp1-35 ts (Fig. 9). Even though 25 0 C is a permissive temperature for ypp1 ts mutants, the levels of PtdIns(4)P are significantly lower in these mutants in comparison to levels in wild-type cells (Fig. 5B). On the other hand, it has been reported that elevated levels of PtdIns(4)P which are found in sac1 cells, lead to impaired rates of endocytosis [35].This was true for endocytosis of Fui1-GFP, with plasma-membrane associated transporter clearly visible in sac1 cells even after sixty minutes of exposure to uridine, in contrast to the complete internalization of the transporter in wild-type cells (Fig. 9). Combining the sac1 with ypp1-35 ts recapitulated the wildtype kinetics of Fui1-GFP internalization (Fig. 9), which is consistent with our data showing the opposing effects of sac1 and ypp1-35 ts on PtdIns(4)P levels. In agreement with this is the compensation of the endocytic defect in sac1 cells in a background that is compromised for function of the PtdIns 4-kinase encoded by STT4 [35]. Not surprisingly, levels of PtdIns(4)P are restored to near wild-type levels in an stt4 ts sac1 ts double mutant [START_REF] Foti | Sac1 lipid phosphatase and Stt4 phosphatidylinositol 4-kinase regulate a pool of phosphatidylinositol 4-phosphate that functions in the control of the actin cytoskeleton and vacuole morphology[END_REF].

DISCUSSION

Biological interaction between Stt4 and Ypp1.

A PSI-blast search revealed six putative orthologues of Ypp1 in humans; with one of them, TTC7B (Q86TV6), sharing 15% amino acid sequence identity with Ypp1 [START_REF] Flower | YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation[END_REF]. Use of algorithms such as Prosite and Pfam, does not identify any recognizable domains in Ypp1. However, use of a recently developed search tool [START_REF] Karpenahalli | TPRpred: a tool for prediction of TPR-, PPR-and SEL1-like repeats from protein sequences[END_REF] identifies two putative tetratricopeptide repeat (TPR) protein-protein interaction domains, one towards the N terminus and the other towards the C terminus. We characterized the role played by Ypp1 in cell physiology by generating conditional lethal alleles of the corresponding gene.

A Tandem Affinity purification (TAP) tag screen revealed a physical interaction between the PtdIns 4-kinase Stt4 and Ypp1 [START_REF] Hazbun | Assigning function to yeast proteins by integration of technologies[END_REF], which we confirmed using a two-hybrid assay. Stt4 is found at the plasma membrane [14]. Ypp1 localises nearby, in a punctate pattern around the periphery of the cell [START_REF] Flower | YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation[END_REF]44]. Until now, there have been no reports describing a functional interaction between these proteins. Our study is the first to demonstrate a functional interaction between the PtdIns 4-kinase Stt4 and Ypp1. We show the phenotypes of stt4 ts mutants are similar to those of our ypp1 ts mutants, namely a reversal of the temperature sensitivity by osmotic stabilization of media, sensitivity to agents that destabilize the cell wall, such as caffeine (also rescued by osmotic stabilization), and defects in actin organization. In addition we show that overexpression of Stt4 suppressess the growth defect of two ypp1 ts mutants. Overexpression of the other PtdIns 4-kinases in yeast, Pik1 and Lsb6, does not suppress the temperature sensitive growth defect of ypp1 ts mutants at 37 0 C. This implies a specific role for Ypp1 in Stt4-dependant events at the plasma membrane, as opposed to a general role connected with the function of all three PtdIns 4-kinases in S.cerevisiae. In agreement with this, we see reduced binding at the cell periphery by a GFP-PH Osh2 sensor which is specific for PtdIns4P and PtdIns(4,5)P 2 . This is more evident in the ypp1-35 ts mutant, which shows clear reduction of staining even at the permissive as well as the non-permissive temperature. Staining at the plasma membrane by this sensor is directly related to Stt4 activity, as plasma membrane localization of GFP-PH Osh2 is lost in an stt4 ts mutant shifted to the non-permisive temperature [START_REF] Roy | Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p[END_REF]. The clear decrease in PH domain staining at the cell membrane in ypp1 ts mutants is accompanied by an increase in the intensity of punctate intracellular staining, especially in the ypp1-35 ts mutant at 25 0 C and the ypp1-41 ts mutant at 37 0 C. In addition, this intracellular punctate staining is entirely due to PtdIns4P generated by Pik1, as GFP-PH Osh2 staining is lost completely when Pik1 activity is depleted, but retained when Stt4 is inactivated [START_REF] Roy | Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p[END_REF]. The increase in intensity of punctate intracellular staining in the ypp1-35 ts mutant at 25 0 C and the ypp1-41 ts mutant at 37 0 C could be due to a compensatory increase of PtdIns4P by Pik1. Alternatively, levels of the PH sensor may not be saturating, so a decrease in plasma membrane associated 4-phosphorylated PtdIns means that more sensor is free to bind this lipid at intracellular locations.

Audhya et al. report an increase in the levels of PtdIns4P in wild-type cells, following a 1 hour upshift to 37 0 C [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF]. This is contrary to our observation of heat shock leading to a reduction in levels of this phosphoinositide. The difference may be due to the method used for radiolabelling phosphoinositides. Audhya et al. labelled cells by incubation with [ 3 H]inositol for 10 minutes [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF], so measurable levels of phosphoinositides could be influenced by differences in the rate of incorporation of label. We labelled to near-steady state equilibrium so any changes seen in phosphoinositide levels relate solely to their mass.

Consequently, our results are in agreement with another study which also used equilibrium labelling [START_REF] Desrivieres | MSS4, a phosphatidylinositol-4-phosphate 5-kinase required for organization of the actin cytoskeleton in Saccharomyces cerevisiae[END_REF]. In any case, our data thus far suggest the primary defect responsible for phenotypes exhibited by ypp1 ts mutants is a reduction in levels of PtdIns4P. This was confirmed by loss of the Sac1 PtdIns 4-phosphatase restoring growth to all three ypp1 ts mutants at 37 0 C.

The Pkc1-dependent MAPK cascade is not defective in the ypp1-35 ts mutant.

Optimal activity of Stt4 is needed for cell wall integrity. The PtdIns(4,5)P 2 , synthesized at the plasma membrane by sequential action of Stt4 and Mss4, is bound by the PH domain of Rom2, which in turn localizes Rom2 to the plasma membrane where it activates Rho1. This leads to activation of the Rho1/Pkc1-mediated MAPK cascade. Inactivation of Stt4 leads to mislocalization of Rom2, and the activation of the cascade is severely compromised, leading to limited phosphorylation of the Slt2 MAP kinase [14]. The ypp1 ts mutants were sensitive to agents that destabilize the cell wall, so we anticipated that activation of Slt2 and subsequent activation of the Rlm1 transcription factor would also be abrogated in these strains, but this was not the case. In ypp1-35 ts , Slt2 is phosphorylated and Rlm1 mediated transcription is stimulated in response to an external stimulus known to activate the cell wall integrity MAPK cascade, namely incubation with caffeine. Accordingly, though the level of plasma membrane associated PtdIns4P in ypp1-35 ts may be low (Fig. 5A), it is sufficient to allow adequate PtdIns(4,5)P 2 synthesis for proper activation of the cell-wall integrity cascade.

Compromise of Ypp1 function does not lead to a defect in internalization of endocytic cargo proteins.

Phosphoinoisitides play key roles in endosomal trafficking. For example, the 3phosphorylated derivatives of PtdIns play roles throughout the endocytic pathway (reviewed in [START_REF] Strahl | Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae[END_REF]). In addition, a number of recent studies have indicated that 4-phosphorylated derivatives of PtdIns are also required for endocytosis. Defects in the endocytic pathway occur in the sac1 background, in which levels of PtdIns4P are elevated by up to 10 fold [35]. However, we show that the drop in PtdIns4P observed in the ypp ts mutants does not affect internalization of Fui1 and Ste3, two well-established transmembrane substrates of the endocytic machinery. This suggests that intracellular levels of PtdIns4P can fall significantly, without a major effect on endocytosis. In agreement with this are data from a similar approach using cells compromised for function of the Stt4 PtdIns 4-kinase itself. When incubated at non-permissive temperature, Ste6 (a plasma membrane transporter subjected to constitutive endocytosis), is efficiently internalized and targeted to the vacuole in both stt4 ts and wild-type cells [START_REF] Audhya | Distinct roles for the yeast phosphatidylinositol 4-kinases, Stt4p and Pik1p, in secretion, cell growth, and organelle membrane dynamics[END_REF]. Given that endocytosis is particularly sensitive to elevated PtdIns4P, it follows that that the defect in endocytosis we observed in sac1 cells was rescued by combination with a ypp1 ts mutation that restores cellular levels of this phosphoinositide.

Some components of the clathrin mediated endocytic machinery are PtdIns(4,5)P 2binding proteins [45,[START_REF] Jost | Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation[END_REF]. Furthermore, PtdIns(4,5)P 2 is also involved in re-modelling of the actin cytoskeleton, which in turn facilitates endocytosis [START_REF] Perrais | Dynamics of endocytic vesicle creation[END_REF][START_REF] Kaksonen | Harnessing actin dynamics for clathrin-mediated endocytosis[END_REF]. PtdIns(4,5)P 2 is synthesized by Mss4, the only PtdIns 4-phosphate 5-kinase in yeast. As expected, a reduction in levels of PtdIns(4,5)P 2 caused by loss of Mss4 function, leads to a defect in endocytosis [49]. Levels of PtdIns4P, the precursor of PtdIns(4,5)P 2 synthesis, fall in the ypp1 ts mutants. However, this is not accompanied by a significant fall in PtdIns(4,5)P 2 synthesis. This may explain why endocytosis of Fui-GFP remained unaffected, given that activity of the PtdIns(4,5)P 2 effectors involved in endocytosis do not change significantly.

Expression of the Parkinson's disease A30P -syn mutant is lethal in yeast. This is suppressed by raising the levels of Ypp1, which elevates endocytosis and degradation of A30Psyn [START_REF] Flower | YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation[END_REF]. However, this may not be due to a stimulation of endocytosis itself. Instead, the enhanced rate of A30P -syn degradation may be due to recruitment of A30P -syn to the plasma membrane, where it becomes assimilated into the naturally occurring endocytic processes operating in yeast. Three observations are in agreement with this. Firstly, Ypp1 is localized in a punctate pattern in close proximity to the plasma membrane. Secondly, Ypp1 binds to A30Psyn but not to the wild-type or A53T -syn mutant. This explains why A30P -syn is degraded, while wild-type or A53T -syn remain intact [START_REF] Flower | YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation[END_REF]. Thirdly, loss of Ypp1 function does not affect the endocytosis of yeast membrane proteins that are endocytic substrates (Figs. 8 and9).

We investigated the phenotypes displayed by three ypp1 ts alleles. Multiple amino acid substitutions were found in the corresponding mutant proteins, with seven substitutions found in ypp1-35 ts and ypp1-41 ts and six substitutions in ypp1-39 ts . Further work will be required to determine which mutations are responsible for the ts phenotypes. We have shown that Ypp1 contributes to generation of PtdIns4P by the lipid kinase Stt4. Some of the mutations fall within the putative TPR protein-protein interaction domains of Ypp1, which may explain the loss of interaction with the Stt4 lipid kinase. The precise role played by Ypp1 in function of Stt4 remains to be determined, and may involve delivery of PtdIns substrate to Stt4, or a direct effect on the catalytic action of Stt4 mediated by interaction with Ypp1.

Table 1. The yeast strains used in this study

Y24828 BY4743; Mat a/ ; his3 1/his3 1; leu2 0/leu2 0; lys2 0/LYS2; EUROSCARF* MET15/met15 0; ura3 0/ura3 0; YPP1/ypp1::kanMX4 ZC0 MATa, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC3P (YPP1/URA3) ZC1

MATa, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC1P (YPP1/LEU2) ZC2

MATa, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC1P2 (ypp1-2 ts /LEU2) ZC3

MATa, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC1P6 (ypp1-6 ts /LEU2) ZC4

MAT a, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1 :: kanMX4, this study bearing pYC1P30 (ypp1-30 ts /LEU2) ZC5

MAT a, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC1P34 (ypp1-34 ts /LEU2) ZC6

MATa, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC1P35 (ypp1-35 ts /LEU2) ZC7

MATa, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC1P39 (ypp1-39 ts /LEU2) ZC8

MAT a, leu2 0, ura3 0, his3 1, lys2 0, MET15, ypp1:: kanMX4, this study bearing pYC1P41 (ypp1-41 ts YPP1 (ZC1) and ypp1 ts mutants (ZC6 and 8) were grown to exponential phase at 25 0 C and then shifted to 37 0 C for 3 hours. Cells were fixed with formaldehyde. Actin cables and cortical actin patches were stained with rhodamine-phalloidin (A), corresponding phase images (P) are also presented. The YPP1 wild-type and ypp1 ts mutants (ZC1 and ZC6-8) were transformed with multicopy vectors bearing the ORFs indicated in the central panel under the control of the MET25 promoter. Gene expression was induced by inoculating on selective media lacking methionine followed by incubation for 3 days at 25 0 C or 37 0 C. Two-hybrid interaction in diploid his3-200/his3-200 auxotrophs expressing BD-Stt4 plus AD-Ypp1 or the indicated AD-ypp1 ts mutants. Cells were incubated at 25 0 C or 37 0 C on SD media lacking histidine. Interaction between AD-and BD-fusions is scored by growth, as this leads to expression of a HIS3 reporter. All plates contained 1mM 3-AT, which inhibits basal activity of the HIS3 reporter product. YPP1 wild-type (ZC1), ypp1 ts mutants (ZC6-8) and corresponding isogenic sac1 strains (ZC21-24) were grown at 25 0 C to exponential phase, and diluted to equal cell density. Six-fold serial dilutions were spotted across YPD followed by incubation for 3 days, at the temperatures indicated. Cells producing Fui1-GFP under control of the GAL10 promoter were grown at 25 0 C in standard defined media with galactose, to exponential phase. Glucose (2%) was added to repress synthesis of the fusion, and cells were shifted to 37 0 C. After 30 minutes at 37 0 C, uridine was added to induce endocytosis. Cells were visualized prior to addition of uridine, and at the intervals indicated after addition of uridine. Identical fields are shown under fluorescence (Fui1-GFP) and DIC optics.

[ 3 H

 3 ]inositol labeling of S.cerevisiae and extraction of labeled phosphoinositides.

7

  Biochemical Journal Immediate Publication. Published on 04 Jul 2008 as manuscript BJ20080209 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080209 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society The MAPK cell wall integrity pathway operates normally in the ypp1-35 ts mutant.

Figure legends Figure 1

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Cells lacking Ypp1 activity fail to reorganize the actin cytoskeleton during heat shock.

Figure 3 .

 3 Figure 3. STT4 is a multicopy suppressor of ypp1 ts .

Figure 4 .

 4 Figure 4. Interaction with Stt4 is abrogated by ypp1 ts mutations.

Figure 5

 5 Figure 5 At restrictive temperature, ypp1 ts mutants display a diminished level of plasma membrane associated 4-phosphorylated PtdIns.(A) Using a Pleckstrin homology (PH) domain sensor. YPP1 wild-type and ypp1 ts mutants (ZC1, ZC6 and ZC8) were transformed with a vector bearing a tandemly repeated dimer of PH Osh2 bracketed by GFP. Strains were grown at 25 0 C to exponential phase. Prior to imaging, an aliquot of each culture was shifted to a strongly non-permissive temperature (39 0 C) for 15 minutes. (B) [ 3 H]inositol labelling. YPP1 wild-type (ZC1) and the ypp1-41 ts mutant (ZC8) were incubated with [ 3 H]inositol at 25 0 C to label all inositol-based lipids to near steady-state equilibrium. Subsequently, cultures were split in two, with one aliquot maintained at 25 0 C and the other shifted to 37 0 C for 1 hour. [ 3 H]inositol-containing lipids were extracted and deacylated. The glycerophosphoinositols were separated by HPLC and quantified by scintillation counting. All

Figure 6 .

 6 Figure 6. Viability of ypp1 ts mutants at non-permissive conditions is restored in a sac1 background.

Figure 7 .

 7 Figure 7. The MAP kinase cell wall integrity pathway is activated in the ypp1-35 ts mutant, in response to caffeine.(A) YPP1 (ZC1) and the ypp1-35 ts mutant (ZC6) were grown to exponential phase at 25 0 C and then incubated for 1 hour in the presence or absence of 10mM caffeine. Cells were harvested and levels of dually phosphorylated Slt2 (indicated as Slt2-P) and total Slt2, were detected on western blots using appropriate antisera. (B) The same strains were transformed with a vector bearing a YIL117c-promoter LacZ fusion. LacZ activity was assayed following 1 hour after the addition of 10mM caffeine. YIL117c-LacZ is a reporter for Rlm1 dependent transcription (data on Y axis are presented as LacZ Miller units, error bars represent ± s.e.m. (n=3).

Figure 8 .

 8 Figure 8. Endocytosis of Fui1-GFP promoted by uridine at 37 0 C.

Figure 9 .

 9 Figure 9. Endocytosis of Fui1-GFP promoted by uridine at 25 0 C.Cells producing Fui1-GFP under control of the GAL10 promoter were grown at 25 0 C in standard defined media with galactose to exponential phase. Glucose (2%) was added to repress synthesis of the fusion. After 30 minutes at 25 0 C, uridine was added to induce endocytosis. Cells were visualized prior to addition of uridine (0 min), and at the intervals indicated after addition of uridine. Identical fields are shown under fluorescence (Fui1-GFP) and DIC optics.

S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 04 Jul 2008 as manuscript BJ20080209 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080209 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Biochemical Journal Immediate Publication. Published on 04 Jul 2008 as manuscript BJ20080209 THIS IS NOT THE FINAL VERSION -see doi:10.1042/BJ20080209 S t a g e 2 ( a ) P O S T -P R I N T Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society

ACKNOWLEDGEMENTS

This work was supported by grant BB/E020550/1 from the Biotechnology and Biological Research Council (BBSRC) to B.P. C.Z. was a recipient of a scholarship from the KC Wong Foundation and the China Scholarship Council. We thank Tim Levine (Institute of Opthalmology, University College London) for providing the pTL511 vector, Rob Piper (Department of Physiology and Biophysics, University of Iowa) for providing the pJLU34 vector, and Rosine Haguenauer-Tsapis for providing the pFL38GalFUI1-GFP vector (Institut Jacob Monod, Paris).