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J Biol Chem, Vol. 274, Issue 48, 33905-33912, November 26, 1999


Complementation Analysis in PtdInsP Kinase-deficient Yeast Mutants Demonstrates That Schizosaccharomyces pombe and Murine Fab1p Homologues Are Phosphatidylinositol 3-Phosphate 5-Kinases*

Robert K. McEwenDagger §, Stephen K. DoveDagger parallel , Frank T. Cooke**, Gavin F. PainterDagger Dagger §§, Andrew B. HolmesDagger Dagger §§, Assia Shisheva¶¶, Yoshikuza Ohya||, Peter J. Parker**, and Robert H. MichellDagger

From the Dagger  School of Biochemistry and the Centre for Clinical Research in Immunology and Signalling, University of Birmingham, Birmingham B15 2TT, United Kingdom, the ** Protein Phosphorylation Laboratory, Imperial Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A 3PX, United Kingdom, the Dagger Dagger  Cambridge Centre for Molecular Recognition, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom, the ¶¶ Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, and the || Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is widespread in eukaryotic cells. In Saccharomyces cerevisiae, PtdIns(3,5)P2 synthesis is catalyzed by the PtdIns3P 5-kinase Fab1p, and loss of this activity results in vacuolar morphological defects, indicating that PtdIns(3,5)P2 is essential for vacuole homeostasis. We have therefore suggested that all Fab1p homologues may be PtdIns3P 5-kinases involved in membrane trafficking. It is unclear which phosphatidylinositol phosphate kinases (PIPkins) are responsible for PtdIns(3,5)P2 synthesis in higher eukaryotes. To clarify how PtdIns(3,5)P2 is synthesized in mammalian and other cells, we determined whether yeast and mammalian Fab1p homologues or mammalian Type I PIPkins (PtdIns4P 5-kinases) make PtdIns(3,5)P2 in vivo. The recently cloned murine (p235) and Schizosaccharomyces pombe FAB1 homologues both restored basal PtdIns(3,5)P2 synthesis in Delta fab1 cells and made PtdIns(3,5)P2 in vitro. Only p235 corrected the growth and vacuolar defects of fab1 S. cerevisiae. A mammalian Type I PIPkin supported no PtdIns(3,5)P2 synthesis. Thus, FAB1 and its homologues constitute a distinct class of Type III PIPkins dedicated to PtdIns(3,5)P2 synthesis. The differential abilities of p235 and of SpFab1p to complement the phenotypic defects of Delta fab1 cells suggests that interaction(s) with other protein factors may be important for spatial and/or temporal regulation of PtdIns(3,5)P2 synthesis. These results also suggest that p235 may regulate a step in membrane trafficking in mammalian cells that is analogous to its function in yeast.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In eukaryotes, phosphoinositides play important roles in several cell functions. In particular, they have been implicated in the membrane trafficking events by which membranes and proteins are sorted into vesicles and targeted to various cell compartments (1). Golgi-to-vacuole trafficking of membranes and proteins in Saccharomyces cerevisiae has provided a very informative system for the study of phosphoinositide-dependent membrane trafficking, because it is not essential for growth and so lends itself to mutational analysis. The phosphatidylinositol 3-phosphate (PtdIns3P)1 that is made by the phosphatidylinositol kinase Vps34p is essential for the targeting of proteins from the Golgi to the yeast prevacuolar compartment and also for the prevacuolar compartment-to-vacuole trafficking step (2).

This functional sequence was recently extended by the demonstration that the yeast FAB1 gene, the protein product of which is necessary for membrane efflux from the vacuole, encodes a PtdIns3P 5-kinase (3, 4). Phenotypes caused by mutations in FAB1 (temperature-sensitive growth and massive enlargement of the yeast vacuole, which also fails to acidify) had been identified earlier, but it had been predicted that the Fab1p protein encoded by FAB1 (which we shall term ScFab1p) would be a PtdIns4P 5-kinase (5). Because PtdIns(3,5)P2 has been found in all eukaryote cells so far examined (6, 7), we predicted that ScFab1p would be the first member of a novel family of PtdInsP kinases (PIPkins) that would be dedicated to PtdIns(3,5)P2 synthesis: the PtdIns3P 5-kinases of other organisms would be their Fab1p homologues (3). We suggested that an appropriate general name for this group of enzymes would be "Type III PIPkins," to distinguish them from Type I PIPkins (which are PtdIns4P 5-kinases) and Type II PIPkins (which are PtdIns5P 4-kinases) (for a recent review, see Ref. 8).

Shisheva et al. (9) have recently suggested that p235, a murine protein that displays extensive homology to ScFab1p, has PtdIns 5-kinase activity. Moreover, Tolias et al. (10) observed that a recombinant Type I PIPkin can make PtdIns(3,5)P2 from PtdIns3P in lipid kinase assays and suggested that enzymes of this type may make PtdIns(3,5)P2 in vivo in mammalian cells.

Taken alone, however, such enzyme assays in vitro do not unambiguously define the biological substrate specificities of inositol lipid kinases. In order to determine what lipids the Fab1p-like kinases make in vivo, we therefore determined in what ways the expression of a mammalian Type Ibeta PIPkin, the murine Fab1p-like protein p235, or the Schizosaccharomyces pombe Fab1p (SpFab1p) would change the endogenously synthesized phosphoinositide complements of yeasts carrying mutants of MSS4 (which encodes PtdIns4P 5-kinase) and of yeasts in which the FAB1 gene was knocked out. The conditional-lethal mss4-1 mutants that we used have a reduced PtdIns(4,5)P2 content (our data); such mutants show defects in their actin cytoskeleton (11, 12), and Delta fab1 cells make no PtdIns(3,5)P2 (3, 4). The utility of this type of approach has already been demonstrated by the finding that expression of a Type I PIPkin, but not of a Type II PIPkin, corrects the temperature-sensitive growth defect of mss4-1 mutant yeast (12).

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

[3H]Inositol was from NEN Life Science Products; [32P]ATP, [14C]inositol, and glutathione-Sepharose 4B were from Amersham Pharmacia Biotech. Silica gel 60-Å TLC plates were purchased from Merck. Triethylammonium bicarbonate and triethylamine were from Fluka. Dowex AG-1 X8 (200-400 mesh) was from Bio-Rad. Protease inhibitors and PtdIns were from Sigma. PtdIns4P was from Calbiochem. PtdIns3P was prepared following our own procedure (13).

Strains and Plasmids-- The plasmids and S. cerevisiae strains used in this study are listed in Table I. Rich YPD and inositol-free synthetic complete (SC) media were prepared according to Sherman (19). SC medium containing 2% (w/v) raffinose contained a full complement of amino acids (3). Strain SDY500 was constructed by mating the 616-3-2 (fab1-1) strain with SEY6210 and sporulating the resulting diploids to recover haploid segregants. The complete upstream and coding sequence of the ScFAB1 gene, as a NotI-XhoI restriction fragment, was excised from pEMY105 and ligated into the NotI-Xho I sites of the single-copy pRS416 vector. The resulting plasmid was designated pFABCEN. Plasmid pRM19 was constructed by ligating the entire SpFAB1 ORF (amplified using Pwo polymerase from genomic S. pombe DNA using primers that inserted BamHI restriction sites at the 5' and 3' ends) into the BglII site of pYO2144. The SpFAB1 ORF was cloned into the BamHI restriction site of pEG-KT to generate plasmid pRM22. The p235s splice variant, which lacks 11 amino acids from a region upstream of the FYVE domain (9) was used for all characterization studies. The 5' and 3' ends of the p235s ORF were polymerase chain reaction-tagged with XbaI and SalI restriction sites, respectively and ligated with XbaI-XhoI-restricted pVT102U to produce plasmid pRM24. For in vitro characterization, XbaI-SalI-tagged p235s was cloned into the XbaI and SalI sites of pEG-KT, producing pRM23. All polymerase chain reaction-amplified clones were confirmed by automated DNA sequencing.

Radiolabeling of Yeast Mutants-- Labeling and analyses of yeast strains employed techniques described in detail by Dove and Michell (20). As appropriate, cells were subjected to hyperosmotic shock by the addition of 1.8 M NaCl to a final concentration of 0.9 M. For comparison between strains, the levels of radioactivity were corrected by standardizing the counts against glycerophosphoinositol (GroPIns), glycero-phosphoinositol 3-phosphate (GroPIns3P), and glycerophosphoinositol 4-phosphate (GroPIns4P), as appropriate. Experiments reported (performed in duplicate) are representative of at least two independent experiments that gave similar results.

For use as standards, [14C]glycerophosphoinositol (3,5)-bisphosphate ([14C]GroPIns(3,5)P2), and [14C]glycerophosphoinositol (4,5)-bisphosphate ([14C]GroPIns(4,5)P2) from [14C]inositol-labeled yeast were isolated and deacylated as described elsewhere (20). Purified [14C]GroPInsPns were stored in buffered solutions (10-15 mM HEPES KOH, pH 7.5) at -80 °C. [32P]GroPIns5P was prepared by dephosphorylating [32P]GroPIns(3,5)P2 using washed erythrocyte ghosts as described previously (21).

Expression of Epitope-tagged p235 and SpFab1p-- Plasmids pRM22 and pRM23 were introduced into strain fab1-Delta 1 using standard techniques and transformants were selected on SC medium lacking uracil (SC-Ura). Liquid cultures were grown overnight in SC-Ura plus 2% (w/v) raffinose, supplemented with all other amino acids. 200 ml of SC-Ura containing 2% (w/v) raffinose was then inoculated to 4 × 105 cells·ml-1 and grown for 12-16 h. Cells were diluted to 4 × 106 cells·ml-1 in SC-Ura/2% (w/v) raffinose/2% (w/v) galactose, grown for a further 5 h, harvested, and lysed using a GlasCol Bio-Nebuliser. GST-fusion proteins were affinity-purified on glutathione-Sepharose as before (3), except that lysis buffers contained 5% (v/v) glycerol.

Fluorescent Labeling with FM4-64-- Yeast cultures were grown for 24-48 h in the appropriate selective medium and labeled with FM4-64 according to Bonangelino et al. (22). Labeled cells were examined by epifluorescence using the rhodamine filter.

Lipid Kinase Assays-- Phosphoinositide kinase assays on recombinant Fab1p, SpFab1p and p235 proteins were performed according to Cooke et al. (3) (and the linked Supplementary Information), except that GST fusion protein-loaded beads were prewashed only once with 1 ml of kinase buffer before assaying.

HPLC Resolution of GroPInsPns-- GroPInsPns were resolved on a Partisphere 5 µm SAX column (Whatman) using gradient 1 (23) or gradient 2 (24). For separating all known isomers of GroPInsP, the novel SD001 gradient based on Buffers A (water) and B (1.25 M (NH4)2HPO4, pH 3.8) was used at a flow rate of 1 ml·min-1: 0 min, 0% B; 5 min, 0% B; 20 min, 2% B; 100 min, 2% B; 120 min, 12% B; 140 min, 12% B; 180 min, 80% B; 200 min, 80% B; 205 min, 0% B. At least one sample from each strain of yeast was spiked with internal [32P]GroPIns5P, [14C]GroPIns(3,5)P2, and [14C]GroPIns(4,5)P2 standards to confirm their retention times. Radioactivity eluting from the column was quantified either by an on-line Radiomatic Flow detector (A520) with a 1 ml flow cell (Packard), or by collecting 30 s fractions, mixing with 3-4 ml of Ultima Flo AP (Packard), and then determining [3H]/[14C] ratios by liquid scintillation spectrophotometry.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Functional Complementation of ScFAB1 Inactivation-- We tested whether expression of the presumptive murine Fab1p-like protein (protein p235 of Shisheva et al. (9); termed PIKfyve in the Mouse Genome DataBase (MGI 1335106) (49)), which is the only mammalian Fab1p homologue so far reported, or of SpFab1p (the Fab1p homologue of S. pombe) would correct some or all of the defects caused by deletion or mutation of ScFAB1 in S. cerevisiae. In pFABCEN cells, which express a single copy of the ScFAB1 gene behind its own promoter, PtdIns(3,5)P2 synthesis is restored (Fig. 1, right), the vacuoles revert to a multilobed morphology similar to that of wild-type cells (Fig. 2a), and the otherwise temperature-sensitive fab1-1 cells grow at their restrictive temperature (Fig. 2b).


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  		Fig. 1.   [3H]PtdIns(3,5)P2 and [3H]PtdIns(4,5)P2 levels in Delta fab1 cells. A fab1::LEU2 strain was transformed with a 2-µm vector containing the gene encoding the Type Ibeta PIPkin (pYO2145). The empty pRS424 2 µm vector was used as a control (fab1::LEU2 + empty plasmid). Wild-type levels of ScFab1p activity were restored by complementation with ScFAB1 on a single-copy plasmid (pFABCEN). Plasmids pRM19 and pRM24 contained SpFAB1 and p235, respectively. As indicated, cells were subjected to hyperosmotic shock (0.9 M NaCl for 10 min). The results have been normalized to a constant level of labeling in monophosphorylated phosphoinositides and are mean ± S.E. for multiple independent duplicated experiments (n = 2 or more). As indicated (n.d., none detected), no [3H]PtdIns(3,5)P2 counts were detected in the Delta fab1 and Delta fab1 + Type Ibeta PIPkin strains.


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  		Fig. 2.   p235, a mammalian Fab1p homologue, complements the phenotypic defects of Delta fab1 cells and catalyzes PtdIns(3,5)P2 synthesis in vivo. a, p235 expression partially suppressed the vacuolar morphological defects of fab1::LEU2 cells. The vacuoles of strains transformed with plasmids pRM24 (fab1::LEU2 + p235) and pFABCEN (fab1::LEU2 + pFABCEN) and with the empty pRS424 vector (fab1::LEU2) were stained with FM4-64 and photographed (rhodamine channel). b, FAB1 homologues were tested for their ability to complement temperature-sensitive fab1-1 mutants at the restrictive temperature. Strains expressing p235 on a multicopy plasmid (pRM24) or FAB1 on a single-copy plasmid (pFABCEN) were streaked on SC-Ura plates and incubated at 37 °C for 3 days; fab1-1 transformed with empty pRS416 and pVT102U plasmids served as controls. c, anion-exchange HPLC analysis of GroPInsPns in a p235-expressing fab1::LEU2 strain. Cells were labeled at 24 °C in inositol-free SC-Ura medium containing 10 µCi·ml-1 [3H]inositol to 106 cells·ml-1. As indicated, cells were subjected to hyperosmotic stress with 0.9 M NaCl for 10 min. Following lysis, lipids were deacylated and resolved by HPLC gradient 1 (as described in Ref. 23) or gradient 2 (as described in Ref. 24). The left panel shows the region in which the three GroPInsP isomers elute. The elution position of co-chromatographed [32P]GroPIns5P is indicated. The center and right panels show the PtdInsP2 isomers of nonshocked and hyperosmotically stressed cells, respectively: elution profiles of co-run [14C]GroPIns(3,5)P2 and [14C]GroPIns(4,5)P2 standards are shown in the right panel (open circles).

Expression of p235 rescued several of the phenotypic defects of fab1 mutants. p235-expressing fab1-1 cells grew at the restrictive temperature (Fig. 2b), and p235 suppressed the vacuolar defects of Delta fab1 cells to a substantial degree (Fig. 2a). Moreover, p235 restored the ability of Delta fab1 cells, which make no PtdIns(3,5)P2 (3), to make a basal concentration of PtdIns(3,5)P2 similar to that present in wild-type cells (Fig. 1, right; Fig. 2c, center panel). However, hyperosmotic stress (0.9 M NaCl for 1-10 min) provoked no increase in PtdIns(3,5)P2 synthesis in the p235-expressing cells (Fig. 1, right; Fig. 2c, right panel): this type of stress provokes rapid PtdIns(3,5)P2 synthesis in S. cerevisiae and S. pombe (6) and also in fab1 cells that overexpress exogenous ScFab1p (3). The negative result obtained with p235 is consistent with the fact that none of the mammalian cell lines we have tested have shown enhanced PtdIns(3,5)P2 synthesis when hyperosmotically stressed (6).2

Under some experimental conditions in vitro, p235 synthesizes a phosphoinositide that appears to be PtdIns5P (9, 49). However, we never detected any PtdIns5P in yeast lipids that were analyzed by an HPLC method that resolves deacylated PtdIns3P, PtdIns4P and PtdIns5P, even in the p235-expressing cells (Fig. 2c, left panel). Moreover in vitro, recombinant p235 phosphorylated PtdIns3P much more readily than PtdIns or PtdIns4P under the experimental conditions under which we previously defined the PtdIns3P 5-kinase activity of ScFab1p (Fig. 3a). When PtdInsP2 spots from such PtdIns3P kinase assays were deacylated, the resulting GroPInsP2 co-chromatographed with GroPIns(3,5)P2, confirming that PtdIns(3,5)P2 was synthesized (Fig. 3c). p235 preferentially synthesized PtdIns(3,5)P2, even when the only PtdIns3P present was a contaminant present in commercially available PtdIns and PtdIns4P preparations. We conclude that p235 serves solely as a PtdIns3P 5-kinase in vivo and that its main activity in kinase assays in vitro is the 5-phosphorylation of PtdIns3P.


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  		Fig. 3.   Substrate specificities of Fab1p homologues. a, TLC analysis of the products of lipid kinase assays using GST-fusion proteins of Fab1p, SpFab1p, and p235 (as indicated above the autoradiograph). Assays were as described under "Experimental Procedures," using the substrates indicated below the autoradiogram. The positions of migration of known products of kinase assays using GST-Fab1p are shown on the right. b and c, confirmation that PtdIns(3,5)P2 is the product of the kinase reaction when PtdIns3P is used as a substrate with SpFab1p (b) and with p235 (c). The spots corresponding to the major product of these reactions were recovered, deacylated, and analyzed as described previously (20). 32P-Labeled products were co-chromatographed with 3H-labeled standards (broken curves). The slower-migrating spot from the products of the assay of p235 kinase activity, with PtdIns as substrate, was also analyzed (not shown). It was PtdIns(3,5)P2, presumably formed from PtdIns3P, that contaminated the PtdIns substrate.

Expression of SpFab1p also restored the ability of Delta fab1 cells to make a basal concentration of PtdIns(3,5)P2 similar to that present in wild-type cells (~5% of the concentration of PtdIns(4,5)P2) and about half of that present in pFABCEN cells (Fig. 1, right; Fig. 4a, left panel). SpFab1p-expressing cells showed a modest increase in PtdIns(3,5)P2 synthesis in response to hyperosmotic stress (Fig. 4a, right panel). There was an approximately 2-fold elevation in PtdIns(3,5)P2 concentration, as compared with a 14-fold increase in cells expressing ScFAB1 from a single-copy plasmid (Fig. 1, right). This behavior is similar to that of strains carrying the fab1-2 temperature-sensitive allele of ScFAB1, which at restrictive temperatures show only a 2-fold stimulation in PtdIns(3,5)P2 in response to hyperosmotic shock (3). In contrast to the results with p235, expression of SpFab1p neither complemented the temperature-sensitive growth phenotype of fab1-1 cells (data not shown) nor corrected the vacuolar morphology of fab1::LEU2 cells (Fig. 4b). However, when recombinant SpFab1p was assayed for lipid kinase activity in vitro, it was an active PtdInsP kinase that showed a marked preference for PtdIns3P as a substrate (Fig. 3a). Deacylation of the product and co-chromatography with a GroPIns(3,5)P2 standard confirmed that the PtdInsP2 synthesized was PtdIns(3,5)P2 (Fig. 3b).


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  		Fig. 4.   SpFab1p catalyzes PtdIns(3,5)P2 synthesis in vivo but does not suppress all Delta fab1 defects. a, GroPInsP2 levels from shocked (right) and nonshocked (left) fab1::LEU2 cells expressing plasmid pRM19. Cells were labeled at 24 °C in inositol-free SC-Trp medium containing 10 µCi·ml-1 [3H]inositol for five cell divisions. They were challenged with medium (basal) or 0.9 M NaCl for 10 min before being killed. Deacylated lipid samples were spiked with [14C]GroPIns(3,5)P2 and [14C]GroPIns(4,5)P2 standards (open circles) and were separated using gradient 2 (24). b, SpFAB1 did not suppress the mutant vacuolar phenotype of fab1 cells. Fluorescence images (rhodamine channel) are shown of FM4-64-stained fab1::LEU2 strains transformed with pYO2144 (fab1::LEU2) or pRM19 (fab1::LEU2 + SpFab1p).

                              
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Table I
Plasmids and S. cerevisiae strains used in this study (for further details, see under "Experimental Procedures")

PIPkin-Ibeta Restores PtdIns(4,5)P2 Synthesis in mss4 Mutants-- We examined the effect of expressing a Type I PIPkin and the Fab1p proteins in strain YOC808, which is wild-type for the FAB1 gene but harbors the temperature-sensitive MSS4 allele mss4-1. YOC808 cells fail to grow at 37 °C, at least partly because of defects in their actin cytoskeleton, and this fault is overcome by overexpression of a mammalian Type I PIPkin (12). To confirm that the mss4-1 lesion limits PtdIns(4,5)P2 synthesis, the PtdIns(4,5)P2 content of YOC808 cells was analyzed at 23 °C and after 2 h at the restrictive temperature (38 °C). Even at the lower temperature, the mss4-1 cells contained about one-quarter of the [3H]PtdIns(4,5)P2 of wild-type cells (Fig. 5, a versus b; Fig. 6, left). When the temperature was raised, the wild-type cells increased their PtdIns(4,5)P2 complement but PtdIns(4,5)P2 declined further in the mss4-1 cells (Fig. 6, left). Expression of Type Ibeta PIPkin in these cells restored their PtdIns(4,5)P2 content to a level somewhat higher than that of wild-type cells, confirming that the Type Ibeta PIPkin is an active PtdIns4P 5-kinase in yeast (Fig. 5c). Expression of a mammalian Type II PIPkin, described as a PtdIns5P 4-kinase (25), had no effect on the PtdIns(4,5)P2 complement (not shown). We have never detected PtdIns5P in yeast (see above), so this was to be expected. Overexpression of ScFab1p, SpFab1p or p235 also had no major effect on the PtdIns(4,5)P2 content of mss4-1 cells (Fig. 5, d-f, respectively; Fig. 6, left), indicating that these enzymes make no contribution to PtdIns(4,5)P2 synthesis.


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  		Fig. 5.   ScFab1p, SpFab1p, and p235 do not enhance PtdIns(4,5)P2 levels in mss4-1 cells. mss4-1 strains expressing the mammalian Type Ibeta PIPkin, ScFab1p, SpFab1p, and p235 were grown at 23 °C in selective inositol-free SC medium containing 10 µCi·ml-1 [3H]inositol. Cultures were grown to a cell density of 1 × 106 cells·ml-1 and then split into four samples. Two samples were grown at 23 °C for a further 2 h, and the other two were grown at the restrictive temperature for 2 h, after which their lipids were extracted and analyzed. HPLC chromatograms are shown of the GroPInsP2s at 23 °C of wild-type (YPH499) (a), mss4-1 (YOC808) (b), mss4-1 expressing Type Ibeta PIPkin (c), mss4-1 expressing ScFab1p (d), mss4-1 expressing SpFab1p (e), and mss4-1 expressing p235 (f).


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  		Fig. 6.   PtdIns(3,5)P2 and PtdIns(4,5)P2 in mss4-1 temperature-sensitive cells. The strain used was YOC808 carrying the mss4-1 temperature-sensitive allele and, as indicated, transformed with 2-µm plasmids containing the Type Ibeta PIPkin (pYO2145), ScFAB1, SpFAB1, or p235 ORFs. S. cerevisiae YPH499 was the parental strain of YOC808. Cells were incubated at the restrictive (38 °C) or nonrestrictive (23 °C) temperature for 2 h before lipid extraction. Values, calculated as in Fig. 1, are representative of multiple independent experiments performed in duplicate (mean ± S.E.; n = >= 2).

Type I PIPkins Do Not Make PtdIns(3,5)P2 in Vivo and Do Not Phenotypically Rescue Delta fab1 Cells-- The fab1::LEU2 strain, which contains an inactivated FAB1 allele, has a wild-type MSS4 gene and hence a normal PtdIns(4,5)P2 complement. However, it makes no PtdIns(3,5)P2 either under basal conditions or when osmotically stressed (Fig. 1, right; Fig. 7a, left panel). These cells have enlarged vacuoles that occupy much of the cell and fail to correctly acidify (3-5). When expressing a mammalian Type Ibeta PIPkin, they still made no PtdIns(3,5)P2 (even when hyperosmotically stressed; Fig. 1 and Fig. 7a, right panel), and their vacuolar phenotype remained abnormal (Fig. 7b). Moreover, the Type I PIPkin did not correct the temperature-sensitive growth phenotype of fab1-1 cells (data not shown).


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  		Fig. 7.   The mammalian Type Ibeta PIPkin does not suppress the defects associated with FAB1 inactivation. a, anion-exchange HPLC analysis of basal GroPInsP2 levels in fab1 cells. Plasmids carrying the genes encoding ScFab1p (fab1::LEU2 + pFABCEN) or the mammalian Type I PIPkin (fab1::LEU2 + PIPkin-Ibeta ) were expressed in a fab1::LEU2 strain background. All strains were labeled in inositol-free SC media containing 10 µCi·ml-1 [3H]inositol for five cell divisions at 25 °C until the cell density of the culture reached 1 × 106 cells·ml-1. Cells expressing the mammalian PIPkin were labeled in SC-Trp, whereas the pFABCEN strain and a control pRS424 strain were cultured on SC-Ura media. Cells were killed and lysed, and the lipids were extracted and deacylated. GroPInsP2 levels in fab1 cells expressing the control plasmid, ScFAB1, and the Type I PIPkin are shown in the chromatograms from left to right, respectively. The positions of [14C]GroPIns(3,5)P2 and [14C]GroPIns(4,5)P2 internal standards are shown as open circles in the control and Type I PIPkin traces. b, vacuolar morphology of a FM4-64 labeled fab1 strain transformed with mammalian Type Ibeta PIPkin (plasmid pYO2145). This strain was grown to midexponential phase at 23 °C in inositol-free SC-Trp, and the vacuoles were labeled with FM4-64 according to Bonangelino et al. (22). The vacuoles were visualized by fluorescence microscopy (rhodamine channel). A strain expressing ScFAB1 from a CEN ARS plasmid (pFABCEN) that displayed wild-type vacuolar structures is shown in the left panel.

ScFab1p and Its Homologues Are Very Similar Both in Overall Organization and in Amino Acid Sequence, Thus Defining a Family of Closely Related Proteins-- Having suggested that ScFab1p and its orthologues in other organisms will make up a "Type III" family of PIPkins that specifically 5-phosphorylate PtdIns3P (3), we compared the molecular organizations and amino acid sequences of the available full-length ScFab1p-like proteins. The sequences of five close relatives of ScFab1p have been reported so far: from S. pombe (SpFab1p; the two overlapping clones AL023534 and AL021838), Caenorhabditis elegans (CeFab1p; AL023817), Drosophila melanogaster (DmFab1p; AL035311), mouse (p235; AF102777), and Arabidopsis thaliana (AtFab1p; AL035525).

Fig. 8a compares the overall domain arrangements of these proteins, and Fig. 8, b and c, shows detailed alignments of their FYVE and kinase domains, respectively. All of the sequences include a C-terminal PtdInsP kinase domain, an N-terminal FYVE zinc finger domain, and a central domain with similarities to a conserved sequence motif present in Cct1p and its homologues (26): sequence conservation is most striking in the FYVE and kinase domains. FYVE domains, at least some of which specifically bind the ScFab1p substrate PtdIns3P (27-30), contain the highly conserved signature motif (R/K)(R/K)HHCR (see Ref. 31) (Fig. 8b, asterisks) within a region rich in Cys and His residues. The Cct1p-like domain resembles a domain present in chaperone complexes implicated in actin and tubulin folding (26). The kinase domains of all the Type III PIPkins identified so far include three invariant catalytic residues that are shared with protein kinases, phosphoinositide 3-kinases, and other PIPkins (32-36). In ScFab1p, these are Lys-2059, Asp-2196, and Asp-2216. In protein kinase A, the Lys residue equivalent to Lys-2059 of ScFab1p interacts with the alpha -phosphate group of ATP (37) and is essential for kinase activity (32). Substitution of Lys-2059 with Met abolishes ScFab1p PtdIns3P 5-kinase activity (data not shown).


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  		Fig. 8.   Multiple sequence alignment showing the similarities between the FYVE motifs and catalytic domains of ScFab1p and its proposed orthologues. Amino acid sequences were aligned using the Pileup program in the GCG Wisconsin software package. Identical residues are shaded black, whereas similar residues are shaded gray. Numbers to the left and right show the position of the sequences within the deduced primary sequences. a, diagram showing the relative positions of the domains shared by ScFab1p (GenBankTM accession number P34756), SpFab1p (AL023534 and AL021838), p235 (AF102777), CeFab1p (AL023817), DmFab1p (AL035311), and AtFab1p (AL035525). b, alignment of the FYVE domains. Asterisks identify the signature FYVE motif. c, alignment of the catalytic domains. +, residues proposed to form an activation loop for substrate binding; dagger , residues thought to be involved in ATP binding (Lys-2059, Asp-2196, and Asp-2216 in ScFab1p). The sequences of this activation loop are very different in each of the three PIPkin families but are well conserved throughout any one family. To illustrate this, the relevant sequences of the catalytic domains of Mss4p (GenBankTM accession number P38994) and of one representative each of the mammalian Type I and II PIPkin families (U78575 and U85245, respectively) are shown below the six aligned Fab1p-related sequences.

Although ScFab1p was initially designated as a likely Type I PIPkin (38), the kinase domains of all of the Type III PIPkins proteins lack an "insert" region that is present in the Type I and Type II PIPkins (38, 39). At least in the recombinant Type II PIPkin, this insert is structurally disordered (35). A second region of divergent sequence in the kinase domains of the PIPkins is again disordered in the Type II PIPkin (35). By analogy with protein kinases (32, 40, 41), this may be a substrate-binding activation loop that is involved in defining the substrate specificity of the Type II PIPkin (35). The relevant sequence in the Type III PIPkins (Thr-2200 to Gly-2238 in ScFab1p) is indicated by the plus signs in Fig. 8c. Alongside that region of the sequence comparison of the different Type III PIPkins, we also show the relevant parts of a mammalian Type I PIPkin, of Mss4p (functionally equivalent to a Type I PIPkin in S. cerevisiae), and of a mammalian Type II PIPkin. The sequences of this "activation loop" are very different in each PIPkin family but are well conserved throughout any one family. For the Type III PIPkins, the consensus sequence is T(F/Y)T(W/L)DKKLE(S/T/M)WVKXXG(I/L)(V/L)G: this motif has a well maintained pattern of hydrophobicity and of charge (including three conserved basic residues) in all Type III PIPkins. This motif may be involved in defining PtdIns3P as the substrate for 5-phosphorylation.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Recent studies suggest that mammalian Type I PIPkins can act as PtdIns3P 5-kinases in vitro (10, 25, 42). On the basis of these experiments, Tolias et al. (10) have suggested that the Type I PIPkins are responsible for the synthesis of PtdIns(3,5)P2 that has been observed to occur in mammalian cells (6, 7). We directly addressed this substrate specificity issue by expressing the murine Type Ibeta PIPkin in Delta fab1 MSS4 and FAB1 mss4-1 yeast. Our data show that the murine Type Ibeta PIPkin is expressed in yeast in a functional state because this enzyme restores wild-type levels of PtdIns(4,5)P2 to FAB1 mss4-1 yeast as well as complementing the mss4-1 phenotype (12). In contrast, the murine Type Ibeta PIPkin is unable to synthesize any PtdIns(3,5)P2 when expressed in Delta fab1 MSS4 yeast and failed to complement the Delta fab1 phenotype. This suggests that the ability of Type I PIPkins to synthesize PtdIns(3,5)P2 in vitro is not a biologically relevant activity: although there appears to be a modest increase in PtdIns(3,5)P2 levels in FAB1 mss4-1 cells expressing the murine Type Ibeta PIPkin relative to FAB1 mss4-1 cells containing an empty vector, this ability to increase the steady-state levels of PtdIns(3,5)P2 requires that the cells also express a wild-type FAB1 gene, i.e. no increase occurs in Delta fab1 MSS4 cells. The increase in the steady-state levels of PtdIns(3,5)P2 in the FAB1 mss4-1 cells is therefore unlikely to result from direct synthesis of PtdIns(3,5)P2 by the murine Type 1beta PIPkin.

Because the Type I PIPkins do not synthesize PtdIns(3,5)P2 in vivo, the identity of the mammalian PtdIns3P 5-kinase appeared unresolved. The similarity of a cDNA encoding p235, a PIPkin homologue, to the yeast FAB1 gene suggested to us that the protein product of this gene might fulfil this function. Other studies have shown that this enzyme can act as a PtdIns 5-kinase in vitro (9, 49), but our results indicate that p235 does not serve as a PtdIns 5-kinase in vivo in yeast: neither p235 nor any other Type III PIPkin caused any detectable PtdIns5P synthesis. We suspect that the p235-catalyzed 5-phosphorylation of PtdIns that is seen in vitro does not reflect its normal biological function. Such observations re-emphasize the conclusion that attempts to define the substrate specificities of phospholipid-directed enzymes solely on the basis of enzymological assays in vitro, which do not reproduce the conditions of substrate presentation that prevail in vivo, must be interpreted with caution. Complementation studies similar to those described here may in future prove valuable for analyzing the substrate specificities of other lipid-metabolizing enzymes.

In addition to restoring basal PtdIns(3,5)P2 levels, p235 rescued the temperature-sensitive growth phenotype and the vacuolar defects of fab1-1 and Delta fab1 cells, respectively. The complementation of these phenotypic defects suggests that p235 (but not SpFab1p) can functionally interact with the proteins that regulate the spatial and/or temporal synthesis of PtdIns(3,5)P2: expression of both enzymes in Delta fab1 yeast results in a similar basal level of PtdIns(3,5)P2 synthesis. These results make it likely that Fab1p-like proteins fulfill similar roles both in making PtdIns(3,5)P2 and in regulating membrane traffic (probably in the Golgi/lysosome/endosome continuum) in most or all eukaryotes, including mammals. This would be consonant with the fact that synthesis of PtdIns3P, the substrate of the Type III PIPkins, by Vps34p and other Type III phosphoinositide 3-kinases is essential for protein trafficking to vacuolar/lysosomal compartments both in yeast (43-45) and in mammalian cells (46-48).

The absence of a hyperosmotic stress-induced increase in PtdIns(3,5)P2 in p235-expressing cells suggests that this enzyme is not involved in the stress response in mammalian cells and also demonstrates that basal PtdIns(3,5)P2 synthesis is sufficient to rescue all the known phenotypic defects of Delta fab1 yeast. In contrast, SpFab1p partly restored the hyperosmotic activation of PtdIns(3,5)P2 synthesis, but it neither complemented the temperature-sensitive growth of fab1-1 cells nor restored the morphology of Delta fab1 cells, suggesting that an additional regulatory interaction (separate from its PtdIns3P 5-kinase activity and involved in the response of the cell to stress) is conserved between S. cerevisiae and S. pombe: the different regulatory properties seen with SpFab1p and p235 might merely have reflected a difference in their expression levels or rates of degradation. However, although expression of FAB1 from a single-copy plasmid was sufficient to complement the phenotypic defects of Delta fab1 and fab1-1 cells (see Fig. 2, a and b), expression of the SpFAB1 ORF from constitutively active promoters of the high copy plasmids pYO2144 (Table I) and pVT102U (data not shown) and from the galactose regulated promoter of pEGKT in 2% (w/v) galactose (data not shown) failed to rescue these defects. Thus, because both enzymes restored basal PtdIns(3,5)P2 synthesis to fab1 cells and complemented at least one of the other functions of ScFab1p, this trivial explanation seems much less likely than a real difference in the functional properties of the two enzymes.

In support of our experimental data, the results of the sequence comparison establish that the six known proteins of the Fab1p family that includes the experimentally untested proteins of Arabidopsis, Caenorhabditis, and Drosophila are very closely related, consistent with the idea that they fulfil the same function in each of their host organisms. This conclusion, combined with the earlier demonstrations of the specific PtdIns3P 5-kinase activity of ScFab1p (3), makes it clear that ScFab1p and its homologues do indeed make up a new family of Type III PIPkins that synthesize PtdIns(3,5)P2: we suggest that this could be abbreviated to PIPkin-III, with an appropriate prefix to indicate species (e.g. ScPIPkin-III for Fab1p).

    FOOTNOTES

* This work was supported by grants from the Medical Research Council, the Royal Society and the University of Birmingham Medical School.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to Phyllis Boath upon the occasion of her retirement after 10 years of excellent technical assistance to all at Centre for Clinical Research in Immunology and Signalling.

§ The first two authors contributed equally to this work.

To whom correspondence should be addressed. Tel.: 44-121-414-5415; Fax 44-121-414-6840; E-mail: R.K.McEwen@bham.ac.uk.

parallel Recipient of Beit and Medical Research Council Fellowships.

§§ Supported by a BBSRC grant.

2 S. K. Dove, F. T. Cooke, P. J. Parker, and R. H. Michell, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PtdIns3P, phosphatidylinositol 3-phosphate; PtdIns(3, 5)P2, phosphatidylinositol (3,5)-bisphosphate; PtdIns3P 5-kinase, phosphatidylinositol 3-phosphate 5-kinase; PtdIns(4, 5)P2, phosphatidylinositol (4,5)-bisphosphate; PIPkins, phosphatidylinositol phosphate kinases; GroPIns, glycerophosphoinositol; GroPIns3P, glycerophosphoinositol 3-phosphate; GroPIns4P, glycerophosphoinositol 4-phosphate; GroPIns(3, 5)P2, glycerophosphoinositol (3,5)-bisphosphate; HPLC, high pressure liquid chromatography; SC, synthetic complete; ORF, open reading frame.

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