Báo cáo khoa học: Regulation of phospholipid biosynthesis by phosphatidylinositol transfer protein Sec14p and its homologues pot

The INO1 gene is deregulated derepressed when inositol is present under the conditions of increased phosphatidylcholine PtdCho turnover, as occurs in the sec14D cki1D strain SEC14 encode

Regulation of phospholipid biosynthesis by phosphatidylinositol

transfer protein Sec14p and its homologues

A critical role for phosphatidic acid

Roman Holicˇ, Milosˇ Za´gorsˇek and Peter Griacˇ

Institute of Animal Biochemistry and Genetics, Slovak Academy of Sciences, Slovakia

Transcription of yeast phospholipid biosynthesis structural

genes, which contain an inositol-sensitive upstream

activa-ting sequence in their promoters, responds to the availability

of the soluble precursors inositol and choline and to changes

in phospholipid metabolism The INO1 gene is deregulated

(derepressed when inositol is present) under the conditions of

increased phosphatidylcholine (PtdCho) turnover, as occurs

in the sec14D cki1D strain (SEC14 encodes the major yeast

phosphatidylinositol transfer protein; CKI1 encodes choline

kinase of the cytidine diphosphate choline pathway of

PtdCho biosynthesis) Five proteins (Sfhp) share sequence

homology with phosphatidylinositol transfer protein

Sec14p Two (Sfh2p and Sfh4p), when overexpressed largely

complement the otherwise essential Sec14p requirement

concerning growth and secretion In this study, we analysed

the ability of Sec14 homologues to correct the defect in

regulation of phospholipid biosynthesis resulting from

defective or missing Sec14p We also analysed how PtdCho turnover relates to the transcriptional regulation of phos-pholipid biosynthesis The results show that (a) none of the Sec14 homologues was able to substitute for Sec14p in its regulatory aspects of phospholipid biosynthesis, (b) removal

of phospholipase D activity corrected the aberrant INO1 gene regulation in yeast strains with otherwise high PtdCho turnover, and (c) increased steady-state phosphatidic acid levels correlated with derepressed levels of the INO1 gene Overall, the results support the model in which high phos-phatidic acid levels lead to derepression of the genes of phospholipid biosynthesis [Henry, S.A & Patton-Vogt, J.L (1998) Prog Nucleic Acid Res Mol Biol 61, 133–179] Keywords: SEC14 homologues; INO1 regulation; phos-pholipid turnover; transcription; Saccharomyces cerevisiae

The majority of yeast phospholipid structural genes contain

in their promoters inositol-sensitive upstream activating

sequence (UASINO) and are regulated in response to the

availability of the soluble precursors inositol and choline

Yeast phospholipid structural genes are derepressed in the

absence of inositol and repressed in the presence of inositol

[1] UASINO-containing genes are activated by two

tran-scription factors – Ino2p and Ino4p [2] – and repressed by a

negative regulator – Opi1p [3] The INO1 gene (encoding

inositol 1-phosphate synthase) [4] is the most regulated gene

of the entire regulon and thus frequently serves as a reporter

gene for the whole set of coordinately regulated genes of

phospholipid biosynthesis [1,5] In addition to the presence

or absence of inositol, transcription of the INO1 gene responds to changes in phospholipid metabolism [6], correct INO1 regulation being dependent on ongoing phosphati-dylcholine (PtdCho) biosynthesis [7] The INO1 gene is deregulated (derepressed when inositol is present) also under conditions of increased PtdCho degradation [8,9] Critical analysis of conditions with defective regulation of the INO1 gene led to the development of a model in which the metabolic signal for INO1 derepression is generated by phosphatidic acid (PA) or a closely related metabolite [6] This model proposes that the relative increase of PA, as a result of increased production, versus its utilization provides the signal for derepression of coordinately regulated UASINO-containing genes of phospholipid biosynthesis A recent study by Loewen et al [10] has demonstrated the mechanism by which Saccharomyces cerevisiae can regulate phospholipid biosynthesis After the rapid consumption of

PA following the addition of inositol (the negative regulator

of phospholipid biosynthesis), Opi1 protein was released from the endoplasmic reticulum, entered the nucleus and repressed its target genes

Sec14p was originally identified as a phosphatidylinositol (PtdIns) transfer protein which catalyses the in vitro trans-port of PtdIns and PtdCho between artificial and biological membranes [11] In vivo, Sec14p performs an essential role in protein transport from the Golgi apparatus [12] Connec-tion between the funcConnec-tion of Sec14p and metabolism of the major membrane phospholipid, PtdCho, was established

Correspondence to P Griacˇ, Institute of Animal Biochemistry and

genetics, Slovak Academy of Sciences, Moyzesova 61, 900 28 Ivanka

pri Dunaji, Slovakia Fax: +421 245943932, Tel.: +421 245943151,

E-mail: Peter.Griac@savba.sk

Abbreviations: CDP, cytidine diphosphate; INO1, gene encoding

inositol 1-phosphate synthase; PA, phosphatidic acid; Pld1p,

phos-pholipase D1; PtdCho, phosphatidylcholine

phosphatidyl-inositol

3,4 ; PtdSer, phosphatidylserine

transfer protein; Sfh, Sec14 homologues; UAS INO , inositol-sensitive

upstream activating sequence.

(Received 17 August 2004, revised 23 September 2004,

accepted 24 September 2004)

by the following lines of evidence (Fig 1), namely that

(a) mutations in genes encoding structural enzymes of the

cytidine diphosphate choline (CDP-choline) pathway for

PtdCho biosynthesis bypass the essential requirement for

Sec14p in secretion and cell viability [13], (b) PtdCho-bound

Sec14p down-regulates a key enzyme of the CDP-choline

pathway, namely choline phosphate cytidylyltransferase

(Pct1p) [14], and (c) Sec14p regulates phospholipase D1

(Pld1p)-mediated PtdCho turnover, Sec14p being a negative

regulator of this pathway in vegetatively growing cells

[8,9,15] and having a positive effect on the production of PA

originating from PtdCho in sporulation [16] A connection

between Sec14p and phospholipid biosynthesis exists also at

the transcriptional level The INO1 gene is not regulated

properly in the absence of functional Sec14p, as

demon-strated in the sec14tscki1D (the CKI1 gene encodes choline

kinase, the structural gene of the CDP-choline pathway)

strain when cultured at a non-permissive temperature of

sec14ts[8,9]

In yeast, five proteins (Sec14 homologues, Sfh) share

primary sequence homology with the major PtdIns transfer

protein encoded by the SEC14 gene [17,18] Sec14

homo-logues Sfh2p and Sfh4p, when overexpressed,

complemen-ted very well the sec14 growth and secretion defects Sfh1p

and Sfh5p were also able to complement these defects to

some degree [17,18] In this study, we analysed the ability of

the Sec14 homologues to correct the regulatory defect in

INO1 transcription resulting from defective or missing

Sec14p We show that none of the Sec14 homologues is able

to substitute for Sec14p in reverting the derepressed levels of

the INO1 transcript in the presence of inositol when the

CDP-choline pathway is blocked The reason why even the

best growth phenotype-complementing homologue, Sfh2p,

cannot substitute for Sec14p in its regulatory role toward

phospholipid biosynthesis, lies in its inability to regulate

phospholipase D-mediated PtdCho turnover Using the

sec14D ckiD pld1D triple mutant with overexpressed Sfh2p,

we were able to perform an in vivo test of the model that

INO1 derepression can occur in response to metabolic

signal generated via Pld1p-mediated PtdCho turnover Moreover, we show that higher steady-state cellular levels

of PA correlate with deregulated levels of INO1 transcript

in cells grown in the presence of inositol, providing support for the model that PA acts as a metabolic signal for INO1derepression [6]

Experimental procedures Strains and culture conditions The yeast strains used in this study are listed in Table 1 Recipient yeast strain PGY170, used to study INO1–lacZ expression, was a spore from a genetic cross of PGY145 [18] and BRS1069 [19]

Cells were grown aerobically at 30C with shaking in chemically defined synthetic media lacking inositol and choline (I–C–), supplemented with 75 lMinozitol (I+C–) or supplemented with 75 lM inositol and 1 mM choline (I+C+) [20]

Plasmid and strain construction Standard genetic methods were used throughout this work [21] Yeast transformation was performed by using the lithium acetate method [22], with minor modifications Plasmids Episomal plasmids containing SEC14 and its homologues under their own promoters based on a yeast

2 lm plasmid, YEplac181 (yeast LEU2 marker) [23], were

as described previously [18] Episomal plasmid YEplac112-SFH2(TRP1 marker) was constructed by subcloning SFH2 from YEplac181-SFH2 [18] into YEplac112, by using SalI and SphI restriction enzymes [23] Centromeric plasmid YCplac22-SEC14 (TRP1 marker) was constructed by subcloning SEC14 from YCp(SEC14) (URA3 marker) [24] (kindly provided by V Bankaitis, University of North Carolina, Chapel Hill, NC, USA) into YCplac22, by using EcoRI and HindIII restriction enzymess [23]

Gene disruption Disruption of the Pld1p gene in strain PGY209 was performed by integrative transformation using the NotI/XhoI disruption cassette from plasmid B913 (kindly provided by J Engebrecht, University of California San Diego, La Jolla, CA, USA)

Assay for the Opi–(overproduction of inositol) phenotype

To test for the excretion of inositol, as described in detail previously [25], yeast strain S3 was transformed with YEplac181 [23] based episomal plasmids containing SEC14or SFH genes Strains were patched on synthetic

I–C– plates and allowed to grow at 30C for 2 days The plates were then sprayed with a suspension of a diploid tester strain (AID), homozygous for ino1 and ade1 (Table 1), and incubated for another 2 days

b-Galactosidase assay

b-Galactosidase production was derived from the integrated version of the fully regulated INO1–lacZ construct [19] Yeast strains were grown to mid-logarithmic phase in I+

Fig 1 Phospholipid metabolic pathways in yeast The pathways shown

include the relevant steps discussed in the text The dashed line

rep-resents degradation of phosphatidylcholine via a phospholipase

D-mediated route.

and I– media at 30C The b-galactosidase assays were

performed as described by Lopes et al [19], except that

aliquots were removed from the reaction mix at 5, 10 and

15 or 20 min b-Galactosidase units are defined as

(A420Æ

5 min)1Æmg)1of protein)· 10 000 Protein was

quanti-fied by using the method of Lowry et al [26]

PtdCho turnover

PtdCho turnover in strains with a cki1 genetic background

was analysed as reported previously [8,27] Yeast strains

were grown overnight at 30C in I+C–media containing

1 lCiÆmL)1of [methyl-14C]choline chloride Cultures were

harvested during mid-logarithmic growth phase, washed

twice with sterile distilled water and resuspended in 5 mL of

fresh unlabelled I+C–media At the time-points indicated,

1.4 mL of the culture was removed and the cells were

collected by centrifugation

supernatant was saved as the medium fraction The cell

pellet was suspended in 0.5 mL of 5% (v/v) trichloroacetic

acid and incubated on ice for 20 min After centrifugation

7

(6000 g, 2C, 3 min), the supernatant was saved as the

intracellular water-soluble fraction The pellet was

resus-pended in 0.5 mL of 1MTris/HCl buffer (pH 8), centifuged

(6000 g, 2C, 3 min), and the resulting supernatant was

combined with the intracellular water-soluble fraction,

effectively neutralizing the acidic extract The final pellet

was saved as the total membrane fraction To solubilize the

cell pellet, it was suspended in 100 lL of 1% (v/v)

Triton-X-100, frozen at)70 C and incubated in the presence of 10%

(w/v) deoxycholate, overnight at 37C Radioactivity of all

fractions was determined by liquid scintillation counting

Radioactivity in glycerophosphocholine and choline in the

medium fraction was determined after separation on a

cation-exchange column, Dowex 50 WX 8 (200–400 mesh;

Serva), as described by Cook & Wakelam [28]

Phospholipid composition

Steady-state labelling with 32P-labelled orthophosphate

(2 mCiÆmL)1; ICN) was performed by using the method

of Atkinson et al [29] Cells were labelled overnight, for

at least six generations, with 5 lCiÆmL)1 of 32P-labelled

orthophosphate in 5 mL of vitamin-defined synthetic I+C–

media, and harvested during the late logarithmic phase of

growth Labelled lipids were extracted as described by Atkinson et al [29] 2D paper chromatography on silica-impregnated paper was performed according to the method

of Steiner & Lester [30] Labelled spots, corresponding to specific lipids, were cut out after autoradiography and quantified by liquid scintillation

Results Two of the Sec14 homologues, namely Sfh2p and Sfh4p, when overexpressed under their own promoters from multicopy episomal plasmids, efficiently complemented the growth and secretion defects of the sec14tsstrain To a low (but significant) degree, Sfh1 protein also complemented the sec14ts-associated growth defect [18] The Opi– (production of inositol) phenotype is indicative of over-expression of inositol-1-phosphate synthase owing to misregulation of the INO1 gene [1,25] The S3 strain (sec14D pct1D), containing an empty cloning vector, dis-plays a strong Opi–phenotype (Fig 2), in agreement with a previous report [8] showing that sec14 mutants containing

a block in the CDP-choline pathway exhibit overexpession

of the INO1 gene The Opi–phenotype disappeared when the S3 strain was transformed with an episomal plasmid containing the SEC14 gene (Fig 2) However, none of the Sec14 homologues was able to fully suppress the Opi– phenotype in the sec14D pct1D strain

To measure in a quantitative manner the levels of INO1 gene expression, we used the fully regulated INO1–lacZ reporter construct integrated into the genome of the sec14D cki1D strain As expected, SEC14 was able to restore wild-type regulation of the INO1 gene, derepression

of the gene in medium lacking inositol, and repression in medium containing inositol None of the Sec14 homo-logues, even those that complemented the sec14-associated growth defect fairly well, restored the correct regulation of the INO1 gene in response to inositol (Fig 3)

Next, we investigated the inability of Sfh2p, which complemented very well the growth defect associated with the sec14 mutation, to complement the INO1 gene regula-tory defect PtdCho turnover, consistent with previously published data [8], was enhanced in the sec14D cki1D strain under the conditions in which the regulatory defect occurred (inositol-containing media) (Fig 4A) The sec14D cki1D strain, containing SEC14 on a centromeric plasmid

Table 1 Yeast strains.

BRS1069 Mat a ade2–1, his3–11,15, leu2–3,112, can1–100, trp1–1, ura3–1, ura3–1::INO1-lacZ(URA3) (pJH334) S Henry [19]

PGY170 Mat a ade2, his3, ura3, trp1, leu2, cki1::HIS3, sec14::kanMX, ura3::INO1-lacZ (URA3) This work PGY209 Mat a ade2, his3, ura3, trp1, leu2, cki1::HIS3, sec14::kanMX, ura3::INO1-lacZ (URA3),

YEplac112 – SFH2 (TRP1)

This work PGY210 Mat a ade2, his3, ura3, trp1, leu2, cki1::HIS3, sec14::kanMX, pld1::LEU2, ura3::INO1-lacZ (URA3),

YEplac112 – SFH2 (TRP1)

This work PGY216 Mat a ade2, his3, ura3, trp1, leu2, cki1::HIS3, sec14::kanMX, ura3::INO1-lacZ (URA3), YCplac22 (TRP1) This work PGY218 Mat a ade2, his3, ura3, trp1, leu2, cki1::HIS3, sec14::kanMX, ura3::INO1-lacZ (URA3),

YCplac22 – SEC14 (TRP1)

This work

(Fig 4B), displayed greatly reduced PtdCho turnover In

contrast to Sec14p, Sfh2p was unable to attenuate high

PtdCho turnover (Fig 4C) Deletion of the Pld1p gene,

pld1D, in the sec14D cki1D strain containing Sfh2p

expressed from the episomal plasmid, diminished the majority of PtdCho turnover (Fig 4D)

To test the hypothesis that the inability of Sfh2p to decrease PtdCho turnover to wild-type levels is responsible for the inability of Sfh2p to complement the INO1 regulatory defect caused by the absence of Sec14p, we measured INO1 expression in the sec14D cki1D pld1D strain containing SFH2 on an episomal plasmid It was shown previously that the phospholipase D1 (PLD1) gene product plays an essential role in the suppression of the otherwise lethal sec14D defect by CDP-choline pathway muta-tions [9,17] As a consequence, the triple mutant sec14D cki1D pld1D is not viable Introduction of the SFH2gene on a multicopy episomal plasmid renders the triple mutant sec14D cki1D pld1D viable [18] Note that introduction of Sfh2p from the episomal plasmid does not decrease the high PtdCho turnover of the sec14D cki1D strain (Fig 4A,C) Next, we compared INO1 gene expres-sion in the triple mutant sec14D cki1D pld1D (SFH2) to the same strain with an intact PLD1 gene, sec14D cki1D (SFH2) (Fig 5) The results show clearly that INO1 expression in the triple mutant, sec14D cki1D pld1D (SFH2), corresponds

to the
normal INO1 expression, as represented by the sec14D cki1D strain containing the SEC14 gene on a centromeric plasmid, and not to the deregulated situation found in the sec14D cki1D or sec14D cki1D (SFH2) strains Two products – PA and choline – are generated from every molecule of PtdCho catabolized via a phospholipase D-mediated route In previous experiments [20], no corre-lation was found between the relative content of PtdCho

Fig 2 Overproduction of the inositol (Opi–) phenotype The sec14 pct1 mutant strain (S3, Table 1) was transformed with plasmids con-taining SEC14, SEC14 homologues SFH1– SFH5, and an empty cloning vector, YEplac181 Excretion of inositol (an indicator

of aberrant INO1 gene regulation) results in halo-type growth of the tester strain around the strains being tested The sec14 pct1 strain, containing SEC14 plasmid, represents wild-type INO1 regulation, and the empty cloning vector serves as a negative control.

Fig 3 Sec14 homologues are not able to correct aberrant regulation of

the INO1 gene The sec14 cki1 strain (PGY170, Table 1) containing

SEC14, SEC14 homologues SFH1–SFH5, and an empty cloning

vector (YEplac181) was grown at 30 C in derepressing (I – C – ) or

repressing (I + C + ) conditions to the mid-logarithmic phase of growth.

The b-galactosidase assay was performed as described in the

Experi-mental procedures Data are expressed as the mean ± SD of three

independent experiments The sec14 cki1 strain containing the SEC14

plasmid represents wild-type INO1 regulation, and the empty cloning

vector serves as a negative control.

and/or free choline availability, and INO1 gene regulation.

Therefore, we focused on the correlation between the other

PtdCho degradation product, PA, and INO1 gene

regula-tion Steady-state phospholipid compositions of the

corres-ponding strains were directly compared Figure 6A

represents comparison of the phospholipid composition in

the sec14D cki1D (SEC14) strain (
normal PtdCho turnover

and
normal INO1 expression) and the sec14D cki1D strain

(enhanced PtdCho turnover and deregulated INO1

expres-sion) Figure 6B represents comparison of the phospholipid

composition in the sec14D cki1D pld1D (SFH2) strain

(genetically blocked Pld1p-mediated PtdCho turnover and

normal INO1 expression) and the sec14D cki1D (SFH2)

strain (enhanced PtdCho turnover and deregulated

INO1 expression) In both pairs of strains, enhanced

Fig 4 The Sec14 homologue, Sfh2, is not able to control phospholipase D-mediated phosphatidylcholine turnover The sec14 cki1 strain (PGY170, Table 1), containing empty cloning vector YCplac22 (A), SEC14 (B), the SEC14 homologue, SFH2 (C), and the sec14 cki1 pld1 strain containing the SEC14 homologue, SFH2 (PGY210) (D), were cultured at 30 C to the mid-logarithmic phase of growth in I +

C–medium containing

1 lCiÆlL)1of14C-labelled choline chloride At time zero, the cells were centrifuged, washed and reinoculated into nonradioactive I+C–medium Data are expressed as a percentage of total label at each time-point: (m), radioactivity in the culture medium; (j), radioactivity in the cellular, water-soluble fraction; (r), radioactivity in the membrane fraction The data represent the average of at least two independent experiments.

Fig 5 Phospholipase D-mediated high phosphatidylcholine turnover is

responsible for deregulation of the INO1 gene The sec14 cki1 strain

(PGY170, Table 1), containing empty cloning vector YCplac22,

SEC14, the SEC14 homologue (SFH2) and the sec14 cki1 pld1 strain

containing the SEC14 homologue, SFH2 (PGY210), were cultured at

30 C to the mid-logarithmic phase of growth at 30 C in derepressing

(I–C–) or repressing (I+C–) conditions The sec14 cki1 strain,

con-taining SEC14 plasmid, represents wild-type INO1 regulation; the

sec14 cki1 strain, containing the empty cloning vector, YCplac22,

serves as a deregulated INO1 control The b-galactosidase assay was

performed as described in the Materials and methods Data represent

the mean ± SD of three to five independent experiments.

Fig 6 Steady-state phospholipid composition The sec14 cki1 strain (PGY170, Table 1) containing empty cloning vector, YCplac22, SEC14 (A), the SEC14 homologue, SFH2, and the sec14 cki1 pld1 strain containing the SEC14 homologue, SFH2 (PGY210), (B) were cultured for five to six generations in I+C–medium at 30 C con-taining 5 lCiÆmL)1of 32P-labelled orthophosphate The cells were harvested and the phospholipids extracted and resolved as described in the Experimental procedures The term
others includes lipids remaining near the origin, and cytidine diphosphate diacylglycerol, cardiolipin

8,9 and other minor lipids Values represent the percentage of total lipid-associated32P incorporated into each phospholipid species Data are expressed as the mean value of two independent experiments.

PtdCho turnover resulted in a lower steady-state level of

PtdCho and a corresponding compensatory increase in

phosphatidylethanolamine (PtdEtn) levels compared with

their counterparts that have a
normal PtdCho turnover

Both strains sec14D cki1D and sec14D cki1D (SFH2), with a

derepressed expression of the INO1 gene in the medium

containing inositol, display significantly higher steady-state

levels of PA than their counterparts that have a
normal

(repressed) expression of the INO1 gene (and low levels of

PtdCho turnover) Thus, high steady-state levels of PA

correlate with the inability of the INO1 gene to repress in

response to inositol availability, and low steady-state levels

of PA correlate with the normal regulation of the INO1 gene

– its ability to repress in response to inositol availability

Discussion

Products of five yeast genes, named SFH1–5, exhibit

significant sequence homology to Sec14p [17,18] Sfh1p,

which shows greatest similarity to Sec14p, conserves all

recognized critical structural motifs of Sec14p [31,32] Sfh2,

Sfh3 and Sfh4 proteins share modest homology with Sec14p

throughout their primary sequences The recognized

signa-ture motifs LLRFLRARKF, DGRPVY,

YYPERMGK-FY and INAP of fungal Sec14 proteins [32] are clearly

recognizable in Sfh2p, Sfh3p and Sfh4p primary sequences

These motifs are conserved only to a limited degree in

Sfh5p Primary sequence homology shared by Sec14p and

Sfh proteins assumes functional significance because some

of the Sec14 homologues (namely Sfh2 and Sfh4), when

overexpressed, complemented the sec14-related growth and

secretory defects [17,18] Sfh3p and Sfh5p, under the

transcriptional control of their own promoters, failed to

do so Sfh1p, which displays the highest degree of similarity

to Sec14p, complemented the sec14-related growth defect

only to a limited degree [18] We hypothesize that the reason

for this weak growth complementation could be a result of

the different subcellular localization of Sec14p from Sfh1p

[18] All Sec14 homologues, except (interestingly) for Sfh1p,

display PtdIns transfer activity in vitro Sec14 homologues

do not, however, show PtdCho transfer activity typical for

Sec14p [17]

Our goal was to determine whether any of the Sec14

homologues could substitute for Sec14p in its role as a

regulator of phospholipid biosynthesis Under conditions

where Sec14p is nonfunctional and the CDP-choline

pathway for PtdCho biosynthesis is blocked, the INO1

gene, the most highly regulated of a set of genes encoding

enzymes of phospholipid biosynthesis, is deregulated

(dere-pressed in the presence of inositol) [8] Using two criteria –

Opi–phenotype and the b-galactosidase activity assay – we

tested the ability of all five Sec14 homologues to revert the

INO1regulatory defect caused by the absence of functional

Sec14p Our results (Fig 2) demonstrate that none of

the Sec14 homologues, when expressed from multicopy

plasmids in the sec14D pct1D strain, was able to fully

suppress the Opi– phenotype The overexpression of two

genes (SFH2 and SFH4) that rescue growth of sec14 cells

seems, to some extent, to decrease the inositol secretion The

Opi–phenotype is an excellent indicator of INO1 regulation,

reflecting INO1 expression in media without inositol

Nevertheless, it is a multifactorial phenotype (depending

also on the metabolic status of the cells, their growth rate and the ability of the cells to transport surplus inositol to the media) and therefore the Opi–phenotype is being considered

a qualitative, rather than a quantitative, indicator of INO1 gene regulation Therefore, we also assessed INO1 expres-sion using the INO–lacZ construct b-Galactosidase activity derived from the INO1 promoter in a yeast strain without Sec14p (sec14D), and containing a genetic block in the CDP-choline pathway (this time cki1D to demonstrate the independence on the nature of the CDP-choline pathway block) (Fig 3), confirmed the Opi– phenotype results, showing that none of the Sec14 homologues is able to suppress the INO1 gene regulatory defect imposed by the absence of Sec14p when the CDP-choline pathway is blocked What is the special function of Sec14p that neither the SFH gene product most similar to Sec14p (Sfh1p, showing 62.5% identity to Sec14p), nor the SFH gene product that almost completely complements the sec14-associated growth and secretion defects (Sfh2p) [17,18], can substitute for? Sec14p acts as a negative regulator of Pld1p-mediated PtdCho turnover pathway in vegetatively growing cells [8,9,15] Paradoxically, however, the simultaneous deletion of several SFH genes significantly reduced phos-pholipase D activity in vivo [17], and overexpression of Sfh2p and Sfh4p rather increased Pld1p-mediated PtdCho turnover [18] Many of these results were obtained by using yeast strains with a temperature-sensitive sec14ts allele grown at the restrictive temperature of 37C, and it has been demonstrated that PtdCho turnover in S cerevisiae varies as a function of temperature elevation, from 30 to

37C [27] Moreover, the thermosensitive sec14tsallele may contain some residual activity influencing Pld1p-mediated lipid turnover Thus, we compared PtdCho turnover in the sec14D cki1D strain (Fig 4A) and in the same strain transformed with a multicopy plasmid containing the Sec14 homologue gene, SFH2 (Fig 4C) PtdCho turnover was measured in I+C–medium at 30C, conditions under which the INO1 regulatory defect occurred The same sec14D cki1D strain, transformed with a centromeric, low-copy-number plasmid containing the SEC14 gene (Fig 4B), served as a control The fate of the label after shifting the cultures to unlabelled medium was compared There was a much higher PtdCho turnover rate in strains containing either empty vector or SFH2, as compared to the strain containing wild-type SEC14 Our results correspond very well with results obtained previously using the sec14tsallele and following PtdCho turnover at 37C in I–C– media [8,18] This experiment clearly demonstrates the inability of the Sec14 homologue, Sfh2, to substitute for Sec14p in regulating the turnover of PtdCho Next, we wanted to provide direct evidence that this elevated PtdCho turnover is responsible for deregulation of the INO1 gene To achieve this we disrupted the major route for PtdCho degrad-ation by Pld1p by using a triple mutant strain, sec14D cki1D pld1D, expressing Sfh2p The PLD1 gene product plays an essential role in suppression of the sec14 defect by CDP-choline pathway mutations [9], and, as a consequence, the triple mutant sec14D cki1D pld1D is not viable However, introduction of the SFH2 gene product expressed from the high-copy-number plasmid renders the triple mutant viable Introduction of the Sfh2p into the sec14D cki1D strain does not change the transcriptional

regulation of the INO1 gene (Figs 2 and 3) and also does

not suppress the high Pld1p-mediated PtdCho turnover in

the sec14D cki1D strain (Fig 4) Direct comparison of

INO1regulation in the sec14D cki1D (SFH2) strain, and in

the same strain in which the Pld1p-mediated PtdCho

turnover was abolished (Fig 4), shows the major role that

Pld1p-mediated turnover plays in the transcriptional

regu-lation of the INO1 gene under our experimental conditions

Henry & Patton-Vogt [6] proposed a model in which the

relatively high levels of PA, or another closely related

metabolite, leads to derepression of the UASINO-containing

genes Here, excess PA produced by phospholipase

D-mediated turnover in the sec14 cki1 strain leads to

dere-pression of the INO1 gene A still-controversial question is

whether the molecule to which the transcriptional regulation

responds is PA or some other closely related metabolite For

example, diacylglycerol and PA are related to each other by

a single phosphorylation/dephosphorylation step [1] PA is

a key intermediate in the formation of glycerophospholipids

and triacylglycerols There is always a fine balance between

the production and the utilization of PA In yeast, PA can

be formed de novo from glycerol 3-phosphate and

dihydro-acetone phosphate [33] Phospholipase D-mediated PtdCho

turnover produces one molecule of PA for every molecule of

PtdCho hydrolyzed On the demand site, PA is rapidly used

in the synthesis of phospholipids and triacylglycerols The

experiments in which we measured the steady-state levels of

phospholipids using32P-labelling, showed that deregulation

of the INO1 gene correlates with higher steady-state levels of

PA (Fig 6) The steady-state level of PA in the sec14D cki1D

strain (deregulated level of the INO1 transcript) is twice as

high as the PA level in the same strain with introduced

wild-type SEC14 (normal, regulated level of the INO1

tran-script) Similarly, the PA steady-state level is almost three

times higher in the sec14D cki1D strain with a

high-copy-number SFH2 gene (deregulated level of the INO1

transcript) as in the same strain in which Pld1p-mediated

turnover was abolished with pld1 disruption (normal,

regulated level of the INO1 transcript) Using steady-state

[32P]phospholipid labelling, we measured the overall cellular

levels of PA It is possible that in some membrane

com-partments local PA changes may far exceed those overall

cellular changes of PA, thus executing the change that is

critical for transmitting the signal(s) from alterations of

phospholipid metabolism to the transcriptional machinery

Recently, a mechanism was described, explaining how

changes in PA levels can be communicated to the

tran-scriptional apparatus [10] In an elegant series of

experi-ments it was shown that Opi1p, a negative transcriptional

regulator of phospholipid biosynthesis, is inhibited by

binding PA on the endoplasmic reticulum Following the

addition of inositol, the PA pool was rapidly consumed for

the biosynthesis of PtdIns, releasing Opi1p from the

endoplasmic reticulum and allowing its nuclear

transloca-tion and repression of target genes, including INO1 It is

possible that because of high phospholipase D-mediated

phospholipid turnover (e.g in the sec14D cki1D mutant)

formation of PA (de novo formation combined with PA

from lipid turnover) exceeds its utilization, thus maintaining

the PA pool above the threshold level for effective release of

Opi1p to the nucleus Addition of inositol to the growth

medium under the condition of increased phospholipase

D-mediated turnover, nevertheless partially repressed INO1 transcription (Figs 3 and 5) when compared to media lacking inositol It is possible, that even under the condition

of high PtdCho turnover, the addition of inositol can consume part of the PA pool, permitting that partial repression of INO1 transcription Alternatively, the addi-tion of inositol can regulate INO1 transcripaddi-tion to a limited degree via another mechanism that is independent of the PA levels

In summary, our results show that (a) none of the Sec14 homologues is able to substitute for Sec14p in its regulatory aspects toward phospholipid biosynthesis, (b) removal of Pld1p activity in the strains with a high PtdCho turnover rate and resulting deregulation of the INO1 gene reverts this deregulation and simultaneously suppresses high PtdCho turnover, and (c) increased steady-state PA levels corres-pond with the deregulation of the INO1 gene, supporting a previous model [6] that excess PA (in this case produced by phospholipase D-mediated PtdCho turnover) leads to derepression of the INO1 gene

Acknowledgements

We thank Vytas Bankaitis (University of North Carolina at Chapel Hill, Chapel Hill, NC, USA), JoAnn Engebrecht (University of California, San Diego, La Jolla, CA, USA), and Susan Henry (Cornell University, Ithaca, NY, USA) for providing the strains and plasmids used in this study The expert technical assistance of Mariana Vitekova´

is acknowledged This study was supported by VEGA 2/4130/4 and Science and Technology Assistance Agency (Slovak Republic) APVT-51-016502 grants.

References

1 Carman, G.M & Henry, S.A (1989) Phospholipid biosynthesis in yeast Annu Rev Biochem 58, 635–669.

2 Ambroziak, J & Henry, S.A (1994) INO2 and INO4 gene pro-ducts, positive regulators of phospholipid biosynthesis in Sac-charomyces cerevisiae, form a complex that binds to the INO1 promoter J Biol Chem 269, 15344–15349.

3 White, M.J., Hirsch, J.P & Henry, S.A (1991) The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper J Biol Chem 266, 863–872.

4 Donahue, T.F & Henry, S.A (1981) myo-Inositol-1-phosphate synthase Characteristics of the enzyme and identification of its structural gene in yeast J Biol Chem 256, 7077–7085.

5 Greenberg, M.L & Lopes, J.M (1996) Genetic regulation of phospholipid biosynthesis in Saccharomyces cerevisiae Microbiol Rev 60, 1–20.

6 Henry, S.A & Patton-Vogt, J.L (1998) Genetic regulation of phospholipid metabolism: yeast as a model eukaryote Prog Nucleic Acid Res Mol Biol 61, 133–179.

7 McGraw, P & Henry, S.A (1989) Mutations in the Saccharo-myces cerevisiae opi3 gene: effects on phospholipid methylation, growth and cross-pathway regulation of inositol synthesis Genetics 122, 317–330.

8 Patton-Vogt, J.L., Griac, P., Sreenivas, A., Bruno, V., Dowd, S., Swede, M.J & Henry, S.A (1997) Role of the yeast phosphati-dylinositol/phosphatidylcholine transfer protein (Sec14p) in phosphatidylcholine turnover and INO1 regulation J Biol Chem.

272, 20873–20883.

9 Sreenivas, A., Patton-Vogt, J.L., Bruno, V., Griac, P & Henry, S.A (1998) A role for phospholipase D (Pld1p) in growth,

secretion, and regulation of membrane lipid synthesis in yeast.

J Biol Chem 273, 16635–16638.

10 Loewen, C.J., Gaspar, M.L., Jesch, S.A., Delon, C., Ktistakis,

N.T., Henry, S.A & Levine, T.P (2004) Phospholipid metabolism

regulated by a transcription factor sensing phosphatidic acid.

Science 304, 1644–1647.

11 Szolderits, G., Hermetter, A., Paltauf, F & Daum, G (1989)

Membrane properties modulate the activity of a

phosphatidyli-nositol transfer protein from the yeast, Saccharomyces cerevisiae.

Biochim Biophys Acta 986, 301–309.

12 Bankaitis, V.A., Aitken, J.R., Cleves, A.E & Dowhan, W (1990)

An essential role for a phospholipid transfer protein in yeast Golgi

function Nature 347, 561–562.

13 Cleves, A.E., McGee, T.P., Whitters, E.A., Champion, K.M.,

Aitken, J.R., Dowhan, W., Goebl, M & Bankaitis, V.A (1991)

Mutations in the CDP-choline pathway for phospholipid

bio-synthesis bypass the requirement for an essential phospholipid

transfer protein Cell 64, 789–800.

14 Skinner, H.B., McGee, T.P., McMaster, C.R., Fry, M.R., Bell,

R.M & Bankaitis, V.A (1995) The Saccharomyces cerevisiae

phosphatidylinositol-transfer protein effects a ligand-dependent

inhibition of choline-phosphate cytidylyltransferase activity Proc.

Natl Acad Sci USA 92, 112–116.

15 Xie, Z., Fang, M., Rivas, M.P., Faulkner, A.J., Sternweis, P.C.,

Engebrecht, J.A & Bankaitis, V.A (1998) Phospholipase D

activity is required for suppression of yeast phosphatidylinositol

transfer protein defects Proc Natl Acad Sci USA 95, 12346–

12351.

16 Rudge, S.A., Sciorra, V.A., Iwamoto, M., Zhou, C., Strahl, T.,

Morris, A.J., Thorner, J & Engebrecht, J (2004) Roles of

phosphoinositides and of Spo14p (phospholipase D)-generated

phosphatidic acid during yeast sporulation Mol Biol Cell 15,

207–218.

17 Li, X., Routt, S.M., Xie, Z., Cui, X., Fang, M., Kearns, M.A.,

Bard, M., Kirsch, D.R & Bankaitis, V.A (2000) Identification of

a novel family of nonclassic yeast phosphatidylinositol transfer

proteins whose function modulates phospholipase D activity and

Sec14p-independent cell growth Mol Biol Cell 11, 1989–2005.

18 Schnabl, M., Oskolkova, O.V., Holic, R., Brezna, B., Pichler, H.,

Zagorsek, M., Kohlwein, S.D., Paltauf, F., Daum, G & Griac, P.

(2003) Subcellular localization of yeast Sec14 homologues and

their involvement in regulation of phospholipid turnover Eur J.

Biochem 270, 3133–3145.

19 Lopes, J.M., Hirsch, J.P., Chorgo, P.A., Schulze, K.L & Henry,

S.A (1991) Analysis of sequences in the INO1 promoter that are

involved in its regulation by phospholipid precursors Nucleic

Acids Res 19, 1687–1693.

20 Griac, P., Swede, M.J & Henry, S.A (1996) The role of

phos-phatidylcholine biosynthesis in the regulation of the INO1 gene of

yeast J Biol Chem 271, 25692–25698.

21 Burke, D., Dawson, D & Stearns, T (2000) Methods in Yeast Genetics, a Cold Spring Harbor Laboratory Course Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

22 Gietz, D., St Jean, A., Woods, R.A & Schiestl, R.H (1992) Improved method for high efficiency transformation of intact yeast cells Nucleic Acids Res 20, 1425.

23 Gietz, R.D & Sugino, A (1988) New yeast–Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites Gene 74, 527–534.

24 Phillips, S.E., Sha, B., Topalof, L., Xie, Z., Alb, J.G., Klenchin, V.A., Swigart, P., Cockcroft, S., Martin, T.F., Luo, M & Bank-aitis, V.A (1999) Yeast Sec14p deficient in phosphatidylinositol transfer activity is functional in vivo Mol Cell 4, 187–197.

25 Greenberg, M.L., Reiner, B & Henry, S.A (1982) Regulatory mutations of inositol biosynthesis in yeast: isolation of inositol-excreting mutants Genetics 100, 19–33.

26 Lowry, O.H., Rosebrough, N.J., Farr, A.L & Randall, R.J (1951) Protein measurement with the Folin phenol reagent J Biol Chem 193, 265–275.

27 Dowd, S.R., Bier, M.E & Patton-Vogt, J.L (2001) Turnover of phosphatidylcholine in Saccharomyces cerevisiae The role of the CDP-choline pathway J Biol Chem 276, 3756–3763.

28 Cook, S.J & Wakelam, M.J (1989) Analysis of the water-soluble products of phosphatidylcholine breakdown by ion-exchange chromatography Bombesin and TPA (12-O-tetradecanoyl-phorbol 13-acetate) stimulate choline generation in Swiss 3T3 cells

by a common mechanism Biochem J 263, 581–587.

29 Atkinson, K.D., Jensen, B., Kolat, A.I., Storm, E.M., Henry, S.A.

& Fogel, S (1980) Yeast mutants auxotrophic for choline or ethanolamine J Bacteriol 141, 558–564.

30 Steiner, M.R & Lester, R.L (1972) In vitro studies of phospho-lipid biosynthesis in Saccharomyces cerevisiae Biochim Biophys Acta 260, 222–243.

31 Sha, B., Phillips, S.E., Bankaitis, V.A & Luo, M (1998) Crystal structure of the Saccharomyces cerevisiae phosphatidylinositol-transfer protein Nature 391, 506–510.

32 Kearns, M.A., Monks, D.E., Fang, M., Rivas, M.P., Courtney, P.D., Chen, J., Prestwich, G.D., Theibert, A.B., Dewey, R.E & Bankaitis, V.A (1998) Novel developmentally regulated phos-phoinositide binding proteins from soybean whose expression bypasses the requirement for an essential phosphatidylinositol transfer protein in yeast EMBO J 17, 4004–4017.

33 Athenstaedt, K., Weys, S., Paltauf, F & Daum, G (1999) Redundant systems of phosphatidic acid biosynthesis via acylation of glycerol-3-phosphate or dihydroxyacetone phos-phate in the yeast Saccharomyces cerevisiae J Bacteriol 181, 1458–1463.