A Genetic Screen for Aminophospholipid Transport Mutants Identifies the Phosphatidylinositol 4-Kinase, Stt4p, as an Essential Component in Phosphatidylserine Metabolism*

Pamela J. TrotterDagger , Wen-I Wu§, John Pedretti§, Rachel Yates§, and Dennis R. Voelker§

From the Dagger  Division of Nutritional Sciences and Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712 and the § Lord and Taylor Laboratory for Lung Biochemistry and the Anna Perahia Adatto Clinic Research Center, National Jewish Medical and Research Center, Denver, Colorado 80206

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

In an effort to understand molecular mechanisms of intracellular lipid transport, we have focused upon specific events required for de novo aminophospholipid synthesis in the yeast Saccharomyces cerevisiae. A genetic system for examining the steps between phosphatidylserine (PtdSer) synthesis in the endoplasmic reticulum and its transport to and decarboxylation by PtdSer decarboxylase 2 in the Golgi/vacuole has been developed. We have isolated a mutant, denoted pstB1, that accumulates PtdSer and has diminished phosphatidylethanolamine formation despite normal PtdSer decarboxylase 2 activity. The lesion in PtdSer metabolism is consistent with a defect in interorganelle lipid transport. A genomic DNA clone that complements the mutation was isolated, and sequencing revealed that the clone contains the STT4 gene, encoding a phosphatidylinositol 4-kinase. The pstB1 mutant exhibits a defect in Stt4p-type phosphatidylinositol 4-kinase activity, and direct gene replacement indicates that STT4 is the defective gene in the mutant. Creation of an STT4 null allele (stt4Delta ::HIS3) demonstrates the gene is essential. These results provide evidence that implicates phosphoinositides in the regulation of intracellular aminophospholipid transport.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Intracellular transport of membrane proteins and lipids, is vital for cell growth and organelle assembly. Knowledge about the molecular mechanisms of intracellular protein transport has increased considerably in recent years, with the identification of numerous proteins involved in protein secretion and sorting to various intracellular membranes (1, 2). In contrast, our understanding of lipid transport processes in organelle assembly is largely incomplete with respect to the specific mechanisms, genes, and proteins that participate (3, 4). A large body of biochemical studies has led to the proposal of several potential mechanisms that mediate intracellular lipid transport including passive diffusion, lipid transfer proteins, membrane apposition, and vesicle budding and fusion (4). Several lipid transfer proteins have been identified, and their genes and cDNAs cloned, but their importance to general intracellular lipid transport remains unclear (3, 4).

Our approach to understanding the molecular nature of intracellular lipid transport has been to focus upon the specific transport events involved in de novo aminophospholipid biosynthesis using the yeast Saccharomyces cerevisiae (5). An outline of our approach and the relevant metabolic and topologic events are shown in Fig. 1. In the absence of exogenous ethanolamine and choline, phosphatidylserine (PtdSer)1 is synthesized by the action of phosphatidylserine synthase (PSS) in membranes that are part of or closely related to the endoplasmic reticulum (6). Following synthesis, PtdSer is transported to the location of PtdSer decarboxylase 1 (PSD1) at the inner mitochondrial membrane, or PtdSer decarboxylase 2 (PSD2) in membranes with characteristics of Golgi and vacuoles, where phosphatidylethanolamine (PtdEtn) is formed (7, 8). The resultant PtdEtn is then transported back to the endoplasmic reticulum, where it is methylated to produce phosphatidylcholine (PtdCho), which is essential for yeast cell growth (9, 10).

We have previously cloned and created null alleles for the PSD1 and PSD2 genes (8, 10, 11), for our development of a strategy for the isolation of aminophospholipid transport mutants in yeast (Fig. 1). We reason that when cells harboring a null allele for PSD1 (psd1Delta ::TRP1) are grown in the absence of ethanolamine or choline, they must transport nascent PtdSer to the locus of PSD2 (Golgi/vacuole) for PtdEtn synthesis. Conversely, cells with a null allele for PSD2 (psd2Delta ::HIS3) must transport nascent PtdSer to the locus of PSD1 (mitochondrion) for PtdEtn synthesis. In both of the above described genetic backgrounds the PtdEtn formed must be transported from either the Golgi/vacuole or mitochondria back to the endoplasmic reticulum for the synthesis of PtdCho (by PtdEtn methyltransferases), which is required for cell growth and division (9). In addition to the above routes for synthesizing PtdEtn and PtdCho, yeast can synthesize these lipids from free ethanolamine or choline (5). Previous studies with mutants in PSS or both PSD1 and PSD2 clearly demonstrate that choline and ethanolamine can rescue strains unable to synthesize (and thus transport) PtdSer and PtdEtn (8, 9). Thus, mutagenesis of yeast with a psd1Delta ::TRP1 or psd2Delta ::HIS3 background followed by selection for Etn auxotrophs provides the means to isolate new strains with defects in PtdSer and PtdEtn transport. We propose to name mutant strains with defects in PtdSer transport to and PtdEtn export from the mitochondria as pstA and pexA. Likewise, mutant strains with defects in PtdSer transport to and PtdEtn export from the Golgi/vacuole are named pstB and pexB. The goals of the present study were as follows: 1) to develop and carry out a lipid transport mutant selection and initial complementation analysis; 2) to characterize one of the putative lipid transport mutants; and 3) to isolate a gene capable of complementing this phenotypic defect. The results indicate that this selection strategy will, indeed, lead to the identification of strains defective in new aspects of aminophospholipid metabolism. The first of these mutants, denoted pstB1, exhibits a defect in the transport-dependent metabolism of PtdSer at the location of PSD2 in the Golgi/vacuole compartment. A complementing clone containing the STT4 gene, which encodes a phosphatidylinositol 4-kinase (12), was isolated, and the pstB1 mutant demonstrated a defect in Stt4p-type PI-4-K activity. These results provide clear evidence for involvement of phosphoinositides in the regulation of intracellular aminophospholipid metabolism that is coupled to interorganelle transport.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Chemicals-- All simple salts, buffers, amino acids, nutritional supplements, and solvents were purchased from either Sigma or Fisher. Yeast media components (yeast extract, peptone, and nitrogen base without amino acids) were purchased from Difco. Phospholipid standards for thin layer chromatography were obtained from Avanti Polar Lipids and Sigma. Thin layer Silica Gel H plates were purchased from Analtech Corp. The radiochemicals [3-3H]serine, [1-14C]serine, and [gamma -32P]ATP were from Amersham Pharmacia Biotech or ICN. The Ptd [1'-14C]serine was synthesized from DL-[1-14C]serine and CDP-diacylglycerol by the action of PtdSer synthase (13). The glass beads (0.5 mm) used for cell homogenization were purchased from BioSpec. For the protein assays, BCA reagents were purchased from Pierce.

Yeast Strains, Plasmids, and Libraries-- Yeast were cultured in synthetic or YPD media prepared by standard methods (14). Adenine (20 mg/liter), uracil (20 mg/liter), and ethanolamine (2-5 mM) were routinely added to the YPD to give YPDAUE, and ethanolamine and choline supplemented synthetic media were prepared as described previously (15). The parental strain (PTY13; MATa lys2 his leu2 trp1 psd1-Delta 1::TRP1) and the psd2- strain (PTY13-E32; MATalpha lys2 his leu2 trp1 psd1Delta -1::TRP1 psd2) were constructed as described previously (15). The strain carrying a defect in PSS (PTY35; MATalpha his3 ser1 ura3 lys2 thr4 leu2 ade1 cho1-1[pss]) was obtained from a cross of KA101, generously provided by Dr. Susan A. Henry (Carnegie Mellon Univ., Pittsburgh) (9) with Ser1a (Yeast Genetic Stock Center). The original pstB1 mutant (PTY13-E5; MATa lys2 his leu2 trp1 psd1-Delta 1::TRP1 pstB1) was obtained from the same mutagenesis of the parental strain PTY13 and isolation of ethanolamine auxotrophs as described previously for psd2 mutants (15). The outcrossed pstB1 strain (PTY47; MATa ura3 met14 lys2 trp1 psd1::TRP1 or psd1Delta -1::TRP1 pstB1) was obtained by crossing PTY13-E5 with PTY40 (MATa ura3 met14 his3 ade suc2 trp1 psd1::TRP1). The SEY diploid strain was obtained by crossing SEY6210 (MATalpha ura3 leu2 his3 trp1 suc2 lys2) and SEY6211 (MATa ura 3 leu2 his3 trp1 suc2 ade2), generously provided by Dr. Mike Yaffe (University of California, San Diego).

The YCp50 yeast genomic library was generously provided by Dr. Vytas Bankaitis (University of Alabama, Birmingham). The pCRII plasmid was obtained from Invitrogen, and the plasmid YEp352 was obtained from Dr. Alex Franzushoff (University of Colorado Health Sciences Center, Denver, CO). The pGEM4Z vector was from Promega, and the pUC18 containing the HIS3 was generously provided by Dr. Rodney Rothstein (Columbia University, NY). All primers were synthesized by the Molecular Resources Center at the National Jewish Medical and Research Center (Denver, CO).

Assay of Phosphatidylserine Decarboxylase (PSD) Activity-- PSD activity was assayed with Ptd[1'-14C]Ser as the substrate using a 14CO2 trapping on 2M KOH-impregnated filter paper (8, 16). Briefly, cells were grown to log phase, harvested, and washed. Cell pellets were resuspended at 0.1 g wet weight/ml in PSD homogenate buffer containing 0.25 M sucrose, 10 mM KPO4 at pH 6.8, 3 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 10 mM 2-mercaptoethanol. Homogenates were prepared by agitating the cell suspension at 4 °C with 0.5-mm glass beads in a Biospec Mini-Beadbeater (Bartlesville, OK). Separation of the beads from the homogenate was achieved by centrifuging the homogenate through a hole punctured with a 27-gauge needle at the bottom of a microcentrifuge tube. The 400-µl reactions contained 50-150 µg of homogenate protein, 1 mg/ml Triton X-100, 25 mM KH2PO4 at pH 6.8, 0.125 M sucrose, 1 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride, and 1 mM Ptd[1'-14C]Ser (~0.1-0.2 µCi/µmol). After 45 min at 36 °C the reaction was terminated by the addition of 0.5 ml of 0.5 N H2SO4. The filter containing 14CO2 was then placed in 0.5 ml of H2O plus 4.5 ml of ScintiSafe Plus (Fisher) liquid scintillation mixture, and radioactivity was quantified using a Beckman LS-6500 liquid scintillation spectrometer. When only PSD2 activity was measured, the assay differed by containing 4-10 µg of homogenate protein, no Triton X-100, and 1-acyl, 2-(N-4-nitrobenzo-2-oxa-1,3-diazole)-aminocaproyl-Ptd [1'14C]serine (3 × 104 cpm assay, 50 µCi/µmol) as the substrate (15). PSD activity is reported as nanomoles of PtdSer decarboxylated per mg of protein/45 min.

Assay of Phosphatidylserine Synthase (PSS) Activity-- PSS activity was determined by monitoring the incorporation of [3H]serine into lipid-soluble products (17). Cell homogenates were prepared as described above. Reactions (100 µl) contained 50 mM Tris-HCl at pH 8.0, 0.6 mM MnCl2, 0.2 mM CDP-diacylglycerol, 4 mM Triton X-100, 0.5 mM L-[3-3H]serine at a specific activity of 10,000 cpm/nmol, and 50-200 µg of homogenate protein. Samples were incubated 20 min at 30 or 36 °C, after which the reaction was terminated by the addition of 0.5 ml of 0.1 N HCl in methanol. The lipid-soluble products were then extracted by adding 1 ml of chloroform and 1.5 ml of M MgCl2, mixing, and separating the phases by centrifugation. The lower chloroform phase was then washed once with acidic synthetic upper (1 N HCl:methanol, 9:10 v/v saturated with chloroform). A sample of the resulting chloroform phase was then removed to a scintillation vial, dried under nitrogen, and radioactivity determined as described above. Data are expressed as nanomoles of [3H]serine incorporated into lipid-soluble products/mg of protein/20 min.

Phospholipid Analysis-- Radiolabeled [3H]serine incorporation into phospholipids was determined in strains grown in synthetic medium containing 2 mM each of ethanolamine and choline. Initially, cultures were grown to late log phase at 30 °C in minimal medium containing auxotrophic requirements plus ethanolamine and choline. Then, the cells were diluted to approximately 2 × 106 ml in the same media with the addition of 20 µCi/ml L-[3-3H]serine. Cells (1 ml) were harvested after 6 h of log phase growth at 30 °C and combined with 10 mg of carrier cells on ice. Pellets were washed twice with 0 °C water, and the lipids were extracted and analyzed by thin layer chromatography as described previously (10). Radioactivity associated with the different lipid classes was determined by thin layer chromatography (8) and liquid scintillation spectrometry as described above.

Isolation of the PSTB1 Gene-- The PSTB1 gene was isolated by complementation of the ethanolamine auxotrophy of the PTY47 strain described above. DNA from a yeast genomic library, in the vector YCp50-URA3, was transformed into cells using the YEASTMAKER transformation system available from CLONTECH. Cells harboring plasmids were first selected by growth on synthetic medium lacking uracil. The >10,000 transformants were then screened for ethanolamine prototrophic colonies. The complementing plasmid was recovered from the yeast and amplified in Escherichia coli as described previously (18). The plasmid isolated from the E. coli was then re-transformed into the PTY47 mutant to confirm its complementing activity. Plasmid loss experiments were carried out by growing the plasmid-containing strain under non-selective conditions for greater than 30 generations and monitoring the coincidence of loss of uracil and ethanolamine prototrophy.

DNA Sequencing and Plasmid Constructs-- Sequencing was performed utilizing the ABI Prism Ready Dye Deoxy Terminator Cycle Sequencing Kit. Cycle sequencing products were purified from unincorporated dyes using Centricep Spin Columns (Princeton Separations, Adelphia, NJ). These products were then analyzed at the National Jewish Center Molecular Resource Center on an ABI 377 automated sequencer. The complementing plasmid originally isolated (YCp50-pstB20) contained an insert of 10 kb. Deletion of a 4-kb HindIII fragment at one end of the insert abolished complementing activity, indicating that the HindIII site in the insert was within the gene of interest. The YCp50-pstB20 from which the HindIII fragment had been deleted (YCp50-pstB20-Delta HindIII) was sequenced from the insert HindIII site into the gene using the M13 forward primer. Alternatively, the HindIII fragment was cloned into the pCRII vector and sequence obtained with the T7 primer. Sequence analysis of PSTB1 across the missing XbaI restriction site was carried out utilizing the YCp50-pstB20-Delta HindIII plasmid and an 18-base pair primer corresponding to base pairs 4668 to 4651 of the published STT4 sequence (5'-TACGGTCAATGAAGCAGC-3'). Sequence data obtained were analyzed using the MacVector sequence analysis program and BLAST comparison (19) to internet sequence data bases. The pstB20 insert was subcloned into the multiple copy yeast vector YEp352 between the PvuI and SphI sites.

Assay of Phosphatidylinositol 4-Kinase (PI-4-K) Activity-- PI-4-K activity was determined using a protocol based on that reported by Yoshida et al. (12). Cells were grown overnight in rich medium to a density of 1-2 × 106/ml, washed, and resuspended at 100 mg wet weight per ml in a homogenization buffer containing 20 mM Tris-HCl at pH 7.4, 1 mM EGTA, 2 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each of leupeptin, antipain, and aprotinin. Homogenates were prepared as described above, and cell-free supernatants were obtained by centrifuging at 1500 × g for 5 min and retaining the supernatant. The 50-µl reactions were performed with between 1 and 7 µg of cell-free supernatants protein in a mixture containing 50 mM Tris-HCl at pH 7.4, 5 mM MgCl2, 0.5 mM EGTA, 50 mM NaCl, 0.4 mM phosphatidylinositol, and 0.16 mM phosphatidylserine. Reactions were started by adding [gamma -32P]ATP (5 µCi/reaction) to a final concentration of 10 µM, incubated for 10 min at 30 °C, and terminated by the addition of 2 ml of chloroform:methanol (2:1, v/v) and 0.5 ml of 1 N HCl. After mixing and centrifuging, the upper phase was removed. The lower phase was washed twice with acidic synthetic upper phase (1 N HCl:methanol, 9:10 v/v saturated with chloroform). The resultant lower phase was then removed and split in two. One-half was transferred into a scintillation vial, dried under nitrogen, redissolved in scintillation mixture, and total radioactivity quantified as above.

The other half of the sample was analyzed by thin layer chromatography. 32P-Labeled lipids (primarily PtdIns-3-phosphate (PtdIns3P), PtdIns-4-phosphate (PtdIns4P), and PtdIns-4,5-bisphosphate (PtdInsP2)) were then separated on Silica gel 60 plates treated with trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid and developed in a solvent system containing 100 ml of methanol, 80 ml of chloroform, 60 ml of pyridine, 60 ml of H2O, 4 ml of 90% formic acid, 16 g of boric acid, 0.5 g of 2,6-di-tert-4-methylphenol, and 100 µl of ethoxyquin as described by Walsh et al. (20). Alternatively, despite the inability to separate PtdIns4P from PtdIns3P, comparable PI-4-K activities were obtained using untreated plates and a solvent system of chloroform, methanol, 4.2 N ammonium hydroxide (45:35:10) as described by McKenzie and Carman (21). The 32P-labeled lipids were then detected and quantitatively analyzed by PhosphorImager analysis. The identity of the lipids was determined by comparison with authentic lipid standards and published Rf values (20). Data obtained from the PhosphorImager analysis were used to calculate the percent 32P label incorporated into each of the labeled products, which was used along with the previously determined total 32P label incorporated and the specific activity of the label to determine the picomoles of PtdIns4P produced. Enzyme activities are reported as pmol of PtdIns4P formed per mg of cell protein/h.

Disruption/Deletion and Replacement of the STT4/PSTB1 Allele-- A 5.0-kb XhoI/SacII fragment was removed from STT4 and replaced with a HIS3 marker gene in three steps. The STT4 gene in YCp 50 was first cleaved by XhoI, BamHI digestion, and a HIS3 gene flanked by XhoI and BamHI was inserted over the deletion. Subsequently, this construct was cleaved with BamHI and SacII to create a larger deletion in the gene that was replaced with an 18-bp BamHI/SacII adaptor. The final deletion construct was excised using EcoRI and SphI cleavage, and this stt4Delta 1::HIS3 DNA was ligated into the appropriate sites of pGEM4Z. The chromosomal copy of STT4 was disrupted by one-step gene replacement (22). The stt4Delta 1::HIS3 sequence was linearized with EcoRI and SphI (3.3 kb) or EcoRI and HincII (2.5 kb) and transformed into a diploid strain (SEY diploid) auxotrophic for histidine (his3) using the YEASTMAKER system (CLONTECH). Recombinants were selected by acquisition of histidine prototrophy.

The presence of a disrupted chromosomal STT4 gene was confirmed by polymerase chain reaction (23). Primers flanking the deletion/disruption were constructed such that one primer annealed to the 5' end of the STT4 gene and the other to the complementary strand within the HIS3 marker gene, yielding a 1.0-kb fragment for the wild type allele and a 0.5-kb band for the disrupted/deleted allele. The transforming DNA did not contain the sequence corresponding to the 5' PCR primer, ensuring that the appropriate PCR products could only be generated after integration into the STT4 locus. Genomic DNA was prepared from strains by standard methods (24), and the PCR reaction was performed using the GeneAmp PCR kit from Perkin-Elmer in a Perkin-Elmer DNA Thermalcycler. Amplified fragments were visualized by agarose gel electrophoresis and staining with ethidium bromide (25).

Direct allelic replacement was also carried out by one-step gene replacement (22). For replacement of the stt4 allele, the STT4 gene was linearized from YCp50-pstB20 (STT4) using PvuI and NdeI, which also cuts the YCp50 vector twice. For the control allele replacement, the PSD1 gene was excised from YCp50-PSD1 with HindIII. The linearized DNA was transformed into a pstB1 strain (for STT4) or a PSD double mutant (for PSD1), and ethanolamine prototrophs were selected. Transformants were routinely screened for uracil prototrophy to check for the presence of any intact vector that may have contaminated the linear DNA preparations. None of the ethanolamine prototrophs were uracil prototrophs.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Mutant Selection Strategy and Complementation Analysis-- Yeast express two phosphatidylserine decarboxylase (PSD) enzymes, PSD1 which resides in the inner mitochondrial membrane (7) and PSD2 which localizes to cellular compartments with characteristics of vacuoles and Golgi (15). Strains containing a psd1-Delta 1::TRP1 or the psd2-Delta 1::HIS3 null allele are completely dependent upon the remaining PSD activity for growth in the absence of exogenous ethanolamine (or choline) (8). We hypothesized that strains unable to transport PtdSer from the endoplasmic reticulum to the location of either PSD enzyme or unable to export PtdEtn back to the ER for PtdCho synthesis would also present as ethanolamine auxotrophs (Fig. 1). Strains containing either the psd1-Delta 1::TRP1 or the psd2-Delta 1::HIS3 null alleles were subjected to mutagenesis, and ethanolamine auxotrophic strains were isolated. Since defects in the remaining PSD or PSS would also result in ethanolamine auxotrophy, all mutants were assayed for these activities. Those mutants exhibiting no enzyme defects in PSD or PSS are provisionally classified as having a defect in PtdSer transport (denoted pst) to or PtdEtn export (denoted pex) from the location of the PSD enzyme. Mutants with potential defects in transport to or export from PSD1 in the mitochondria (PSD1, psd2-Delta 1::HIS3) are referred to as pstA and pexA, respectively. Likewise, those with potential defects in transport to or export from the Golgi/vacuole are referred to as pstB and pexB. Incorporation of [3H]serine into the aminophospholipids is utilized to classify mutants as pst or pex: the former exhibiting accumulation of label in PtdSer due to impaired transport to PSD, and the latter showing accumulation of label in PtdEtn due to a defect in export of PtdEtn to the ER for methylation to PtdCho (data not shown). Examination of the pstB/pexB group has yielded 19 complementation groups. Results with the pstA/pexA group are incomplete. This report will focus on a mutant designated pstB1.


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Fig. 1.   Schematic diagram of aminophospholipid biosynthesis and transport pathways in yeast. In the absence of exogenous ethanolamine and choline, aminophospholipids are synthesized via the de novo pathway. Serine (Ser) is incorporated into phosphatidylserine (PtdSer) by PtdSer synthase (pss) in the endoplasmic reticulum (ER). The resultant PtdSer is then transported to the location of PtdSer decarboxylase 1 (psd1) at the inner mitochondrial membrane or PtdSer decarboxylase 2 (psd2) in the Golgi/vacuole where it is converted to phosphatidylethanolamine (PtdEtn). Transport to the PSDs is hypothesized to be mediated by proteins encoded by pstA or pstB genes, respectively. PtdEtn produced by PSD1 or PSD2 is then transported from the mitochondria or Golgi/vacuole back to the ER by postulated proteins encoded by pexA or pexB genes, respectively. Within the ER, PtdEtn is methylated to phosphatidylcholine (PtdCho) by the sequential action of PtdEtn methyltransferases 1 and 2 (pem1 and pem2). Alternatively, when ethanolamine (Etn) and choline (Cho) are present, PtdEtn and PtdCho are synthesized via the CDP-dependent Kennedy pathway (not shown in detail).

The pstB1 Mutant-- The first screening of approximately 10,000 colonies for ethanolamine auxotrophs in a strain carrying the psd1-Delta 1::TRP1 null allele (PTY13) yielded 12 ethanolamine auxotrophic strains, of which four were psd2- mutants (15) and two were pss- mutants. Complementation analysis of the remaining six mutants indicated that as many as three different mutations were present. One of the complementation groups contained two strains denoted PTY13-E2 and PTY13-E5, which were unable to complement one another, and the PTY13-E5 mutant was chosen for further analysis. The mutation carried by this strain is denoted pstB1. The pstB1 allele in PTY13-E5 was crossed out of the mutagenized background by mating with a psd1-Delta 1::TRP1, PSTB1 strain (PTY40). Tetrad analysis demonstrated a 2:2 segregation of the ethanolamine auxotrophy consistent with a mutation at a single locus.

Fig. 2A shows the growth of the pstB1 mutant and its parental strain in the presence and absence of ethanolamine. In the presence of ethanolamine the pstB1 strain grows as well as the parental strain. In the absence of ethanolamine the pstB1 strain shows little evidence for growth. Without ethanolamine, the pstB1 strain begins to lose viability after 6 h, or about 2 doublings (Fig. 2B). By 25 h of culture, the viability of the strain grown without ethanolamine is about 3 orders of magnitude lower than that found when the nutrient is present. Among the criteria used for defining strains as defective in steps between PtdSer synthesis and decarboxylation are the retention of parental PSD and PSS activities. Fig. 3, A and B, shows PSS and PSD enzyme activities measured in the parental psd1-Delta 1::TRP1 PSD2 strain (PTY13), the original pstB1 mutant (PTY13-E5), a pss- control strain (PTY35), and a psd1-Delta 1::TRP1 psd2- control strain (PTY13-E32). The data demonstrate that the pstB1 mutant has normal PSS and PSD2 activities as compared with the parental strain. The relatively high PSD activity in the pss- strain is a consequence of the presence of both PSD1 and PSD2 alleles, whereas the parental and pstB1 strains only contain a functional PSD2 allele.


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Fig. 2.   The pstB1 mutant requires ethanolamine for growth and viability. A, log phase parental (PTY13) and mutant, pstB1 cells (PTY47), grown in synthetic medium containing ethanolamine (2 mM) were harvested by centrifugation, washed three times in minimal medium, and re-inoculated to give an absorbance of 0.05 in synthetic medium plus (black-square, bullet ) or minus (square , open circle ) ethanolamine. Cell growth was monitored by culture absorbance at 550 nm. B, the viability of PTY47 was measured as a function of time in the presence (bullet ) or absence (square ) of 2 mM ethanolamine. Log phase cells were processed as described in A and inoculated at approximately 1.5 × 105/ml. At the indicated times, duplicate aliquots were removed from the cultures and diluted, and samples were spread onto YPD plates. Surviving colonies were counted after 3 days of incubation at 30 °C. Data are from duplicates in a representative one of two experiments.


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Fig. 3.   The pstB1 strain exhibits normal PtdSer synthase and decarboxylase activity. A, PtdSer synthase activity was measured in the parental strain used for mutagenesis (PTY13), a pss- (cho1) strain (PTY35), a psd- strain (PTY13-E32), and pstB1 strain (PTY13-E5). B, PtdSer decarboxylase activity was measured in the same strains described for A. The parental and pstB1 strains contain the psd1Delta ::TRP1 allele. The psd- strain contains the psd1Delta ::TRP1 and psd2 alleles. The pss- strain contains PSD1, PSD2 and cho1 alleles. Data are the mean of duplicates from a representative one of two experiments.

Although the in vitro PSD2 activity of the parental and pstB1 strains is comparable, it remained possible that the defect in the mutant was in the PSD2 gene and related to in vivo PSD2 function (i.e. mislocalization), rather than a mutation in a separate PSTB1 gene. A genetic experiment was performed to test the activity of the PSD2 allele present in the pstB1 mutant. The mutant (psd1-Delta 1::TRP1, PSD2, pstB1) strain was crossed with a strain lacking PSD activity (psd1-Delta 1::TRP1, psd2-Delta 1::HIS3, PSTB1). The resultant diploid (psd1-Delta 1::TRP1/psd1-Delta 1::TRP1, PSD2/psd2-Delta 1::HIS3, pstB1/PSTB1) which contains only the PSD2 allele from the pstB1 mutant, is prototrophic for ethanolamine. This result demonstrates that the PSD2 allele in the pstB1 mutant is fully functional. Furthermore, these data demonstrate that the pstB1 mutation is recessive.

In order to determine whether this mutant has a defect in steps between PtdSer synthesis and PtdEtn formation, the incorporation of [3H]serine into the aminophospholipids was analyzed. Fig. 4A compares the profile of [3H]serine incorporation by the parental psd1-Delta 1::TRP1 strain (PTY13) and a pstB1 mutant (PTY47) expressed as the percent of total radiolabel observed in PtdSer, PtdEtn, PtdCho, and PtdIns and as the ratio of label observed in PtdEtn:PtdSer. Serine labeling of PtdSer in the mutant is increased by 26%, whereas subsequent labeling of PtdEtn is decreased 37%. As depicted in Fig. 4B, this results in a ratio of PtdEtn:PtdSer labeling in the mutant (0.46) that is 50% lower than the parent strain (0.93). These differences in serine metabolism, although small, are consistent with the conclusion that the lesion in the pstB1 mutant is at the transport step between the site of PtdSer synthesis in the ER and its decarboxylation by PSD2 in the vacuole/Golgi compartment.


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Fig. 4.   The PSTB1 strain exhibits abnormal lipid labeling corrected by PSTB1-20. Incorporation of L-[3-3H]serine into phospholipids of the parental strain (PTY13, black bars); a pstB1 mutant (PTY47, hatched bars); a mutant strain harboring a complementing single copy plasmid (PTY47-YCp50-PTSB1-20, stippled bars) and a mutant strain harboring a multicopy complementing plasmid (PTY47-YEP352-PTSB1-20, open bars). Labeled phospholipids were extracted from log phase cells grown for 6 h in synthetic medium containing 2 mM ethanolamine and choline and 20 µCi/ml L-[3-3H]serine. The lipid classes were resolved by thin layer chromatography, and radioactivity was quantified by liquid scintillation spectrometry. Data are the mean ± S.E. from seven total determinations performed in four separate experiments expressed as percent of total radiolabel incorporated into each phospholipid (A) and the ratio of incorporation into PtdEtn:PtdSer (B).

Isolation of a Gene That Complements the pstB1 Mutant-- The outcrossed pstB1 strain (PTY47) was utilized to screen a YCp50 yeast genomic library for clones capable of complementing the pstB1 mutation by restoring ethanolamine prototrophy. Of approximately 30,000 Ura+ YCp50-library transformants of the pstB1 mutant, 7 showed robust growth in the absence of ethanolamine. The strains were then tested for simultaneous loss of plasmid (i.e. conversion to ura- phenotype) and ethanolamine prototrophy, after 30-40 generations of growth under non-selective conditions. Three of the strains lost plasmid at an unusually high rate (>50%), much higher than normal rates of plasmid loss expected for a centromeric plasmid such as YCp50 (<5%). One of these transformants was further examined, and it was demonstrated that loss of the plasmid (scored as ura-) also resulted in loss of ethanolamine prototrophy, indicating that the plasmid, denoted YCp50-PTSB1-20, contained an insert responsible for complementing the mutation. The YCp50-PTSB1-20 plasmid was rescued from yeast into E. coli, purified, and re-transformed into the pstB1 mutant. The YCp50-PTSB1-20 plasmid complemented the ethanolamine auxotrophy of the pstB1 mutant and restored normal growth in liquid media.

Next it was of interest to determine if the YCp50-PTSB1-20 clone, which is capable of complementing the growth phenotype of the pstB1 mutant, also corrects the observed biochemical phenotype (see Fig. 4). Serine incorporation was monitored in an outcrossed pstB1 strain (PTY47), the pstB1 mutant carrying the originally isolated, low copy YCp50-pstB20 plasmid and the pstB1 mutant carrying the PTSB1-20 insert subcloned into the high copy plasmid YEp352 (see "Experimental Procedures"). As shown in Fig. 4A, the presence of the PTSB1-20 insert has a profound effect on serine incorporation into the aminophospholipids. As described above, the pstB1 mutant has a decreased PtdEtn:PtdSer ratio of [3H]serine incorporation when compared with the parental strain (Fig. 4B). When the mutant carries the low copy YCp50-PTSB1-20 plasmid, serine accumulation into PtdSer is decreased and conversion to PtdEtn is increased (51%) to give a PtdEtn:PtdSer ratio of 0.84, restoring serine incorporation to a pattern indistinguishable from the parental strain (Fig. 4B). In the mutant containing the high copy YEp352-pstB20 plasmid a 32% decrease in PtdSer labeling and a 79% increase in conversion to PtdEtn results in an increase in the PtdEtn:PtdSer ratio of more than 2.5-fold. This profile indicates correction of the defect to a level slightly above that found for the parental strain (Fig. 4B). These data demonstrate that the sequence present in the PTSB1-20 insert not only complements the growth defect of pstB1 but also the measurable biochemical phenotype.

Analysis of the Complementing Insert-- Initial examination of the genomic DNA capable of complementing pstB1 mutation demonstrated the presence of a 10-kb insert in a YCp50 vector. When a 4-kb HindIII fragment was removed from the insert, complementation was lost, indicating that this site was within the gene of interest, denoted PSTB1. The 4-kb HindIII fragment was subcloned into a YEp352 yeast vector and sequenced from the internal HindIII site. The resulting DNA sequence was compared with the genome sequence in the S. cerevisiae Genome Data base.2 The complementing sequence was found to be identical to the STT4 gene, previously reported as encoding a phosphatidylinositol 4-kinase presumably involved in the protein kinase C signaling pathway (12). To compare the PSTB1 insert to STT4 further, a restriction map was generated and compared with the predicted map based on the published sequence for STT4. The maps generated for PSTB1 and STT4 were indistinguishable for the endonucleases HindIII, PstI, BamHI, and NdeI. The patterns obtained utilizing XbaI, however, indicated that one of these sites is not present in the PSTB1 gene. In order to determine the basis of this differing restriction map, a sequencing primer corresponding to base pairs 4668-4651 of STT4, about 70 bases downstream from the missing XbaI site, was designed. The PSTB1 insert was then sequenced upstream through the site. The DNA sequence obtained corresponded to base pairs 4644-4268 of STT4 (12). However, within the XbaI recognition/cleavage site there was a single C right-arrow T base pair change from TCTAGA to TTTAGA. This substitution resulted in loss of the XbaI site but retention of the predicted amino acid sequence for STT4. From these data, we conclude that the PSTB1 gene is essentially identical to the previously described STT4 gene and that the difference in the restriction map is due to a base pair substitution that does not alter protein structure.

The pstB1 Defect Involves a PI-4-K-- Given the previously described function of the STT4 gene product as a PI-4-K, we next examined this enzyme activity in the pstB1 mutant. When PI-4-K activity was measured as described by Yoshida et al. (12), which differs from standard methods (21, 27) primarily in the use of 10 µM rather than >100 µM ATP, omission of detergents, and the inclusion of high levels of PtdSer, the pstB1 strains exhibited catalytic activity that was 10% of the parental strain value (Fig. 5). PI-4-K activity was restored in strains harboring either the low copy (YCp) or the high copy (YEp) plasmids carrying the PSTB1/STT4 insert, which possess activities 70% and 30% higher than the parental strain, respectively. These data clearly implicate an involvement of this PI-4-K activity in the defect observed in the pstB1 mutant.


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Fig. 5.   The pstB1 mutant exhibits reduced STT4-type PI-4-K activity that is recovered by transformation with plasmids carrying the complementing STT4/PSTB1 insert. The [gamma -32P]ATP-dependent phosphorylation of PtdIns was measured at 30 °C in cell-free supernatants prepared from cells grown in rich medium. A, autoradiogram of phosphorylated lipids separated on a thin layer chromatography plate developed in a boric acid-containing solvent system as described under "Experimental Procedures." PI-3-P, phosphatidylinositol 3-phosphate; PI-4-P, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate. B, PI-4-K activity was determined by quantitation of PtdIns phosphorylation to produce PI-4-P using PhosphorImager analysis as described under "Experimental Procedures." Data are the mean ± S.E. from six total determinations performed in three separate experiments. Strains are as follows: parental (PTY13); mutant A, pstB1, (PTY13-E5); mutant B, pstB1, (PTY47); pstB1 + YCp50-STT4 (PTY47 carrying the STT4/PSTB1 gene in the low copy vector), pstB1 + YEp352-STT4 (PTY47 carrying the STT4/PSTB1 gene in the high copy YEp352 vector).

Since the defect in PI-4-K activity could result from a mutation in either the structural gene for the enzyme or a gene product that regulates the structural gene, we examined the consequences of creating a null allele. The null allele for STT4 (designated stt4Delta 1::HIS3) was created by deleting 5 kb of the gene and replacing it with the HIS3 gene (Fig. 6A). The stt4Delta 1::HIS3 allele was introduced into a diploid yeast strain by single step gene disruption (22). The presence of the null allele was confirmed by PCR analysis as shown in Fig. 6B. Diploids, heterozygous for the null and wild type alleles at the STT4 locus, were induced to sporulate and the resulting tetrads were examined for viability and His+ prototrophy. Thirteen tetrads gave identical results segregating His+:His- progeny in a ratio of 0:2. This result indicates that STT4 is an essential gene. Yoshida and co-workers (12) have reported that strains harboring a null allele for STT4 can be rescued by high osmotic strength (i.e.M sorbitol) medium. In the genetic background used in these studies, the osmoremedial phenotype was not observed.


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Fig. 6.   Disruption/deletion of the STT4/PSTB1 gene. A, physical map of the construct used to disrupt the STT4/PSTB1 gene. A 5-kb fragment was removed from the STT4/PSTB1 sequence by digestion with XhoI and SacII and replaced with a 1.3-kb XhoI/BamHI fragment containing the HIS3 marker gene using an 18-bp BamHI/SacII adaptor. The resultant stt4Delta ::HIS3 construct was linearized from the bacterial pGEM4Z vector by cleavage with EcoRI and SphI and EcoRI and HincII. The numbers indicate the size of restriction fragments in kilobases (kbp). The primers used for PCR analysis are indicated by arrows B, BamHI; E, EcoRI; S2, SacII; Sp, SphI; X, XhoI. B, disruption/deletion of the chromosomal STT4/PSTB1 gene is confirmed by PCR analysis. Genomic DNA was prepared from wild type and transformed strains and subjected to PCR amplification with all three primers as described under "Experimental Procedures." Presence of a wild type allele is indicated by a 1.0-kb fragment and the disrupted/deleted allele by a 0.5-kb fragment. Lane a, wild type STT4 plasmid; lane b, stt4Delta ::HIS3 plasmid; lane c, transformed diploid (STT4/stt4Delta ::HIS3); lane d, wild type haploid.

Another approach to elucidating whether the pstB1 strain contains a mutation in STT4 or a regulatory gene is by using direct allelic replacement. The linearized STT4 gene was recovered from the YCp50-URA3 plasmid by PvuI/NdeI digestion. The gene was purified by agarose gel electrophoresis and used to transform the pstB1 strain. This approach yielded ethanolamine prototrophs of pstB1 with the same frequency as obtained by direct allelic replacement with the PSD1 gene. From this result we conclude that the mutation present in the pstB1 strain is within the STT4 gene.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the past decade information about the molecular mechanisms of intracellular protein sorting and transport has increased significantly, although details about the specific gene products and mechanisms involved in intracellular lipid transport has lagged far behind (1, 3). The goal of our ongoing research is to identify genes and gene products responsible for mediating intracellular lipid transport functions using the yeast, S. cerevisiae. The current results describe an experimental system for the isolation of strains defective in aminophospholipid transport and identify the first gene specifically implicated in steps between PtdSer synthesis and decarboxylation.

The approach utilized for the isolation of aminophospholipid transport mutants depends upon the introduction of a null allele for either PSD1 or PSD2 (8, 10), which renders the strain entirely dependent upon PtdSer transport to and decarboxylation by the remaining PSD enzyme, for PtdEtn and PtdCho synthesis, in the absence of exogenous ethanolamine and/or choline (Fig. 1). Thus, mutagenesis of strains carrying a null allele for one PSD and isolation of ethanolamine auxotrophs should yield not only mutants in the remaining PSD or PSS but also in genes encoding for mediators of interorganelle lipid transport.

The present studies used a selection protocol designed to find strains defective in lipid transport to PSD2 in the Golgi/vacuole which will also result in the isolation of psd2 and pss mutants. As expected, secondary screening of mutants by measuring activities of these two enzymes revealed three classes of ethanolamine auxotrophs as follows: 1) strains lacking PSD activity but retaining parental PSS activity; 2) strains retaining parental PSD activity (PSD2) but lacking PSS activity; and 3) strains retaining parental PSD and PSS activities. Those strains in the latter group were classified as likely aminophospholipid transport mutants. Initial selection, screening, and complementation analysis has suggested that events occurring between PtdSer synthesis and PtdEtn availability for methylation enzymes along the PSD2 (Golgi/vacuole) pathway is quite complex, perhaps involving as many as 19 genes. For the current study, we have focused upon a mutant possessing a strong requirement for ethanolamine (Fig. 2A), while retaining parental PSD and PSS activities (Fig. 2, B and C).

Despite the clear ethanolamine requirement of the pstB1 mutant (Fig. 2A), the biochemical consequence as measured by serine labeling of aminophospholipids was "leaky." Accumulation of label in PtdSer and decreased formation of PtdEtn was modest, albeit significantly different from the parental strain (Fig. 4). The growth defect of the pstB1 mutant was not corrected by transformation with a low copy plasmid containing the PSD2 gene, but overexpression of PSD2 from a high copy plasmid permits the strain to grow in the absence of ethanolamine (data not shown). These data suggest that in the pstB1 mutant, the percentage of PtdSer that reaches the location of PSD2 is insufficient to provide the necessary PtdEtn and ultimately PtdCho. Overexpression of PSD2 likely allows increased levels of enzyme to be localized not only within the Golgi/vacuole but also within the ER, which could completely bypass any need for PtdSer transport. Taken together, these data suggest that PtdSer transport may be rate-limiting for the synthesis of PtdEtn, and the function carried out by a transport component defective in the pstB1 is likely vital for cell viability, such that a greater defect may be lethal. We have tried to measure the accumulation of PtdSer within the endoplasmic reticulum of the pstB1 strain but have thus far been unable to do so. The principal difficulty has been technical in nature since the endoplasmic reticulum fraction does not resolve sufficiently well on density gradients when the cells are grown under the labeling conditions described in Fig. 4.

A clone from a low copy YCp50 yeast genomic library, initially denoted YCp50-PTSB1-20, which was isolated based on complementation of the ethanolamine-requiring growth defect of pstB1 also corrected the biochemical defect in conversion of PtdSer to PtdEtn (Fig. 4). Expression of the PTSB1-20 insert from the multicopy YEp352 vector increased the conversion of PtdSer to PtdEtn to a level slightly exceeding that in the parental strain (Fig. 4B). Thus, the clone corrected both the growth and biochemical phenotype.

Sequencing and restriction analysis identified the gene responsible for complementation of the pstB1 mutant as a previously described gene, called STT4, that encodes a phosphatidylinositol 4-kinase (PI-4-K) and exhibits genetic interactions within the protein kinase C (PKC) signaling pathway (12, 28, 29). Independent evidence that Stt4p is a PI-4-K has been obtained by Cutler and colleagues (30) who have recently demonstrated that anti-Stt4p immunoprecipitates the enzyme activity. The STT4 gene was originally isolated by its ability to complement a staurosporine-sensitive mutant. Staurosporine is an inhibitor specific for PKC, and mutants in yeast PKC1 are sensitive to this agent (28). Overexpression of PKC1 suppresses the stt4 mutation but does not alleviate all of the stt4 mutant phenotypes, indicating that the Stt4p may be involved upstream in the Pkc1p signaling pathway, and leaves open the possibility that Stt4p is involved in more than one pathway (29). Finally, it has also been observed that stt4 mutants exhibit a defect in the progression from the G2 to M phase of the cell cycle, exemplified by duplication of DNA but no chromosomal segregation (29). This is particularly interesting, since another protein with associated PI-4-K activity, TOR2, has been implicated in G1 to S phase cell cycle progression (31).

Based on the discovery that the STT4 gene, encoding a PI-4-K, complemented the growth and biochemical phenotypes of the pstB1 mutant, PI-4-K activity was analyzed as described previously for the Stt4p (12) and shown to be deficient in the pstB1 mutant. Parental levels of PI-4-K activity were restored by transformation of the mutant with either centromeric or episomal plasmids containing STT4/PSTB1-20. It is interesting to note that PI-4-K activity in cells containing either the low or high copy STT4/PSTB1 was indistinguishable (Fig. 6). Yoshida et al. (12) also found that STT4 expressed on a multicopy plasmid did not result in PI-4-K activity greater than in wild type cells. Moreover, excess Stt4p was degraded in cells harboring the multicopy construct. The authors suggested that there may be another factor, with which the Stt4p must interact, that is required to stabilize and/or activate the PI-4-K. Although our experiments do not address this latter point directly, we observed that yeast are very intolerant of excess copies of STT4 and presumably Stt4p. In the presence of ethanolamine, cells lose YCp50-STT4 at the remarkably high rate of 50% in 24 h. Such a rate of loss would be consistent with the capacity of Stt4p to titrate out a limiting factor required for essential cellular processes. Consistent with the above observation, Cutler et al. (30) have recently reported that high level expression of Stt4p under GAL regulation is lethal. These same authors have also found that STT4 is an essential gene.

Phosphoinositides have recently become recognized as regulators in several aspects of membrane transport, particularly in vesicle-mediated trafficking (32-35). In mammalian cells, a PI-4-K activity associated with the adrenal chromaffin granule membrane has been implicated in priming for stimulated exocytosis (36), and mammalian PI-3-K has been suggested to regulate the intracellular trafficking of cell-surface receptors (34, 37). The mammalian PtdIns transfer protein and the PtdIns-4P-5 kinase have been identified as necessary components in exocytosis of secretory granules (38, 39). A yeast PI-3-K encoded by the VPS34 gene is required for sorting of vacuolar proteins (34). In addition, yeast possessing a defect in the SEC14 gene, encoding a phospholipid transfer protein that exchanges PtdIns and PtdCho, has implicated membrane composition as an important variable for proper Golgi function (40). The mechanism by which such phosphoinositides mediate their function in membrane transport has been hypothesized to involve possibilities as diverse as membrane receptors that recognize specific PtdIns phosphates (35), alteration of membrane curvature by addition of phosphates to PtdIns leading to budding (26), and clustering of membrane proteins by associations with and among PtdIns phosphate molecules (32, 35).

The current experiments are the first demonstration that a PtdIns kinase may be involved in regulating some aspect of membrane lipid transport. The role of the STT4/PSTB1 gene product is unlikely to be a direct regulation of PSD2 enzyme activity, as neither the pstB1 mutation nor overexpression of STT4/PSTB1 on either a low or high copy plasmid has a discernible effect on overall PSD2 enzyme activity (Fig. 3B and data not shown). We have ruled out any direct involvement of phosphatidylinositol 4-phosphate in the catalysis of PSD2. Direct addition of phosphatidylinositol 4-phosphate to the PSD2 assays leads to inhibition of enzyme activity. Little inhibition of PSD2 occurs at 1 µM phosphatidylinositol 4-phosphate, but up to 50% inhibition occurs at 10 µM (results are from two experiments). The data clearly indicate that the pstB1 mutant exhibits a defect in steps between PtdSer synthesis and decarboxylation. Our preferred interpretation of this result is that the pstB1 mutation affects transport.

Another possible role for the STT4/PSTB1 gene product might be in the regulation of protein traffic within the cell, thereby affecting the proper localization of PSD2 after its synthesis within the ER. This function seems improbable for two reasons. First, if PSD2 were not properly transported to the Golgi/vacuole compartment, it would likely remain in the ER, be degraded within the cell, or be secreted. If the enzyme were retained in the ER, the necessity for PtdSer transport from the ER to the Golgi/vacuole would be lost, and one would not expect such a mutant to require exogenous ethanolamine. If the enzyme were intracellularly degraded or bypassed the Golgi/vacuole and secreted one might expect the mutant to have a PSD2 activity that is significantly different from the parental strain, but this was not observed (Fig. 3B). Second, the STT4/PSTB1 gene has not been identified as important in the many screens for mutations in protein transport. Thus, based on the data presented, the STT4/PSTB1 gene product appears to be required for the efficient transport of PtdSer from its site of synthesis in the endoplasmic reticulum to its site of decarboxylation by PSD2 in the Golgi/vacuole compartment.

The mechanism by which STT4/PSTB1 gene product could regulate PtdSer transport to the location of PSD2 remains unresolved. Since this PI-4-K has been implicated in intracellular signaling, it is probable that it also functions as a regulator of other cellular processes. It will be of great interest in the future to identify additional proteins with which the STT4/PSTB1 gene product interacts and their effects upon lipid transport.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants GM 32453 (to D. R. V.) and F32-GM 16701 (to P. J. T.).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.

To whom correspondence should be addressed: Dept. of Medicine, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1300; Fax: 303-398-1806; E-mail: voelkerd{at}njc.org.

1 The abbreviations used are: PtdSer, phosphatidylserine; PI-4-K, phosphatidylinositol 4-kinase; PtdIns-4-P, phosphatidylinositol 4-phosphate; PSD, phosphatidylserine decarboxylase; PSS, phosphatidylserine synthase; PtdCho, phosphatidylcholine; PtdEtn, phosphatidylethanolamine; PtdIns, phosphatidylinositol; ER, endoplasmic reticulum; kb, kilobase pair(s); PCR, polymerase chain reaction.

2 J. M. Cherry, C. Adler, C. Ball, S. Dwight, S. Chervitz, Y. Hia, G. Juvik, T. Roe, S. Weng, and D. Botstein (1996) Saccharomyces genome database available on-line at the following address: http://genomewww.stanford.edu/Saccharomyces/.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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