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INTRODUCTION |
The yeast Saccharomyces cerevisiae is an important
model system for determining the molecular mechanisms of eukaryotic
intracellular protein transport. Of particular interest is the yeast
secretory pathway in which SEC genes essential for protein
secretion by S. cerevisiae have been identified (1). These
genes were discovered as conditional lethal mutations that interrupt
secretory flow at specific points along the secretory pathway.
Biochemical and functional identification of the SEC gene
products has increased the understanding of mechanisms involved in the
yeast secretory pathway.
The yeast SEC14 gene is required for the formation of
transport vesicles from the Golgi (2). However, the mechanism by which
its encoded protein, Sec14p, facilitates this process is not precisely
known. When conditional lethal mutant sec14-1ts
strains are shifted to nonpermissive temperatures, protein secretion halts (1). Sec14p is a phosphatidylinositol transfer protein with the
ability to transfer both phosphatidylinositol and phosphatidylcholine between membranes (2). A unique protein in S. cerevisiae,
Sec14p, is set apart from other phospholipid transfer proteins by its ability to interact specifically with the Golgi (3), an association that is required for secretory competence in yeast (4). Sec14p is
proposed to have a sensor function that maintains a critical phosphatidylinositol to phosphatidylcholine ratio required for vesicle
formation at the Golgi (3).
To further understand the mechanism of SEC14 action in the
secretory pathway, suppressor mutants of
sec14-1ts were identified (3). Characterization
of the genes defective in these mutants revealed that genes of three of
six gene complementation groups identified were involved in the
CDP-choline pathway for phosphatidylcholine biosynthesis. Apparently,
inhibition of phosphatidylcholine synthesis through the CDP-choline
pathway leads to efficient bypass of sec14-1ts,
probably by altering phospholipid composition of the Golgi membrane.
Other suppressors of sec14-1ts are mutations of
the SAC1 gene (5). Originally identified as a suppressor of
act1 alleles when mutated, SAC1 encodes an
integral membrane protein that is located in the endoplasmic reticulum
and Golgi (6). Generally, SAC1 mutations lead to a
cold-sensitive phenotype and inositol auxotrophy. However, mutants with
the sac1-22 allele do not display inositol auxotrophy,
despite an inositol requirement for its bypass of
sec14-1ts. Sac1p has significant homology with
the noncatalytic domains of the yeast and mammalian
polyphosphoinositide 5-phosphatases (7), but its function is not known.
Phospholipase D activity was recently shown to be essential for bypass
of the sec14-1ts phenotypes in several bypass
mutants, but not for normal secretion (8, 9). The formation of
phosphatidic acid from phospholipase D-mediated hydrolysis of
phosphatidylcholine has been implicated in this process. This is
supported by results showing that phosphatidic acid promotes Golgi
vesicle formation in a mammalian cell system (10).
Kearns et al. (11) suggested that
sec14-1ts bypass by sac1-22 occurs by
increasing the amount of diacylglycerol
(DAG)1 in the Golgi through
stimulated sphingolipid biosynthesis. This model was based on an
apparent 6-fold increase in the cellular levels of
mannosyl-diinositolphosphoryl-ceramide (M(IP)2C).
M(IP)2C synthesis occurs in the Golgi with the transfer of
phosphorylinositol from phosphatidylinositol to
mannosyl-monoinositolphosphoryl-ceramide (MIPC) to yield a molar
equivalent of DAG (12). The sac1-22 bypass was perturbed by
heterologous expression of Escherichia coli DAG kinase,
presumably due to the conversion of DAG to phosphatidic acid.
The recent identification of IPT1 as M(IP)2C
synthase (13) allowed us to test the role of M(IP)2C
production in the bypass of sec14-1ts by
sac1-22. IPT1 was also recently found to be
identical to SYR4, a gene necessary for yeast growth
inhibition by the bacterial metabolite syringomycin
E.2 In this work, we report
that disruption of IPT1 has no effect on the bypass,
indicating that M(IP)2C production is not important in the
sac1-22 suppression of sec14-1ts.
Instead, phosphatidylinositol 4-phosphate (PtdIns(4)P) is shown to
increase in the sec14-1ts sac1-22
bypass mutants, suggesting a role for this lipid in Golgi-localized secretory pathway events.
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EXPERIMENTAL PROCEDURES |
Strains and Media--
S. cerevisiae strains used in
this work were CTY182 (MATa ura3-52
his3-200 lys2-801) (2), CTY1-1A (MATa
ura3-52
his3-200 lys2-801 sec
14-1ts) (2), CTY165 (MATa
ura3-52
his3-200 ade2-101
sec14-1ts sac1-22) (11), and CTY-SR (CTY165
ipt1::URA3, this work). Cells were typically
grown in synthetic complete (SC) media as described by Kaiser et
al. (15). For lipid analyses, cells were grown in synthetic
minimal medium (16).
Disruption of IPT1 in CTY165--
The IPT1 disruptant
strain CTY-SR was constructed by the one-step disruption method (17).
The IPT1 disruption construct described
elsewhere2 was made by replacing a 1-kilobase
PvuII-PvuII fragment, which included the 5'
portion of IPT1, with a 1.1-kilobase URA3
fragment. This construct was linearized and used to transform CTY165.
Disruptants were selected on SC-ura, and IPT1 disruption was
confirmed by Southern blot analysis.
Liquid Secretion Assay--
Ten-ml cultures grown in SC medium
at 28 °C were harvested at an optical density between 0.5 and 1 at
600 nm, washed once in SC low-glucose medium (1 mM
glucose), resuspended into 10 ml of low-glucose medium, and split into
two 5-ml cultures. Low-glucose cultures were incubated at either
28 °C or 37 °C for 1 h. Secretory indices were determined by
assaying the total and secreted invertase activity as described by
Westphal et al. (18).
Quantification of Sphingolipids--
Sphingolipid extractions
were modified from the methods described by Smith and Lester (19).
Steady-state measurements of sphingolipid levels were conducted by
growing 20-ml cultures for 18-24 h at 28 °C in the presence of 200 µCi of H332PO4. Growth was
terminated by the addition of trichloroacetic acid to a final
concentration of 5%. Cells were washed twice with 1 ml of
H2O and freeze-dried overnight. Dried cells were
resuspended in 1 ml of H2O and extracted by the addition of
1.4 ml of ethanol:diethyl ether:pyridine (15:5:1) for 30 min at
57 °C. Debris was pelleted by centrifugation, and the supernatant
was removed to a fresh tube. After drying under N2, lipids
were resuspended in 1 ml of solvent A (chloroform:methanol:water
(16:16:5 v/v/v)), and glycerophospholipids were deacylated by the
addition of 1 ml of 0.2 N NaOH in methanol and incubation
at 30 °C for 45 min. One ml of 5% EDTA was added, and samples were
neutralized by the addition of 0.2 ml of 1 N acetic acid.
Lipids were then extracted with 1 ml of chloroform and dried under
N2. Lipids were resuspended in 0.1 ml of solvent A and
analyzed by thin layer chromatography on 1-mm Silica Gel G plates
(Analtech, Newark, DE) treated with 2.5% EDTA (pH 7.2) and developed
in chloroform:methanol:4.2 N ammonium hydroxide (9:7:2
v/v/v). Radiolabeled lipids were identified by autoradiography and
quantified by scraping relevant spots followed by liquid scintillation counting. The identities of inositolphosphoryl-ceramide, MIPC, and
M(IP)2C were confirmed by electrospray ionization mass
spectroscopy (Utah State University Biotechnology Center).
Quantification of Diacylglycerol--
Steady-state and pulse
analyses of DAG levels were measured by labeling cells with
[14C]acetate (ICN Pharmaceuticals, Costa Mesa, CA).
Steady-state labeling was conducted by adding 5 µCi of
[14C]acetate to 20-ml cultures and growing the cells for
18-24 h at 28 °C. Pulse labeling was done by adding 40 µCi of
[14C]acetate to 20-ml cultures, with an optical density
at 600 nm between 0.7 and 0.8, for 20 min at 28 °C. DAG extraction
was done as described by Buttke and Pyle (20). The total lipid
fractions from 14C pulse-labeled and steady-state-labeled
cells were extracted with 2 ml of methanol under reflux for 1 h,
followed by the addition of 4 ml of chloroform and incubation at room
temperature for 18 h. Phases were separated by the addition of 1.2 ml of 0.1 M potassium chloride. The chloroform phase was
removed to a fresh tube and dried under N2. Labeled lipids
were suspended in 100 µl of chloroform and separated by thin layer
chromatography with the solvent petroleum ether:diethyl ether:acetic
acid (85:15:1 v/v/v). 14C-labeled lipids were detected by
autoradiography, scraped, and quantified by liquid scintillation
counting. Nonlabeled DAG and other lipid standards were detected by
charring after spraying with 50% sulfuric acid.
Phospholipid Analysis--
Cells were grown at 26 °C for
18 h in synthetic defined minimal media supplemented with 1 mM myo-inositol in the presence of 40 µCi/ml
H332PO4 (ICN Pharmaceuticals) as
performed in the work of Kearns et al. (11). Total lipids
were extracted from 20 A590 units of each cell
culture as described for sphingolipid analysis without deacylation.
Dried lipids were suspended in 400 µl of solvent A and separated by
two-dimensional thin layer chromatography as described above and
visualized by autoradiography. The relevant lipid spot was scraped from
plates and extracted three times with 500 µl of solvent A, dried
under N2, and processed for high performance liquid
chromatography (HPLC) analysis as described below.
Phosphoinositide Analysis--
Yeast cells were grown at
26 °C in synthetic minimal media supplemented with 100 µM myo-inositol and 5 µCi/ml
myo-[3H]inositol (23 Ci/mmol; NEN Life Science
Products, Boston, MA) to a mid-log phase. Lipids were extracted as
described under "Quantification of Sphingolipids." Deacylation was
carried out by the method of Serunian et al. (21). Dried
glycerophosphoinositols were subjected to anion exchange chromatography
using a Whatman Partisil 5 SAX column (25 cm × 4.6 mm; Whatman
Inc., Clifton, NJ) on a Beckman System Gold HPLC system. The column was
pre-equilibrated with 10 mM ammonium phosphate, pH 3.8. A
portion of each sample (2.5 × 106 cpm) was applied to
the column, washed with 5 ml of 10 mM ammonium phosphate
(pH 3.8), and eluted with a 40-ml linear gradient from 10 mM to 0.7 M ammonium phosphate (pH 3.8) at a
flow rate of 1 ml/min. [32P]Glycerophosphoinositol
3-phosphate and [32P]glycerophosphoinositol 4-phosphate
standards were generated by in vitro phosphorylation of
phosphatidylinositol with
-[32P]ATP followed by
deacylation. For the production of PtdIns(3)P, an extract enriched in
PtdIns 3-kinase was prepared from a 25-30% ammonium sulfate
precipitate of a yeast cytosolic fraction of strain TVY614 (22)
carrying a multicopy plasmid of the VPS15 gene (pJSY324.15)
(23) and the VPS34 gene (pPHY52) (24). The PtdIns 4-kinase
source was a crude cell extract from a yeast strain (PHY102) that lacks
the VPS34 gene (24). The glycerophosphoinositol 3,5-bisphosphate peak was identified as a species that increased 16-fold by osmotic stress (1 M NaCl for 20 min; data not
shown) (25). [3H]Glycerophosphoinositol 4,5-bisphosphate
was generated by deacylation of
[3H]PtdIns(4,5)P2 (NEN Life Science Products).
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RESULTS |
IPT1 Disruption Does Not Affect sac1-22 Bypass of
sec14-1ts--
The proposed involvement of
M(IP)2C synthesis in the sac1-22 bypass of
sec14-1ts prompted us to disrupt
IPT1, the structural gene for M(IP)2C synthase,
in strain CTY165 (sec14-1ts sac1-22).
If increased M(IP)2C synthesis in this strain promotes bypass, then disruption of IPT1 should alleviate the bypass.
A disruption strain was obtained by transforming CTY165 with a
linearized ipt1::URA3 construct. After
transformation, disruptants were selected on SC-ura plates. Three
Ura+ transformants were randomly selected and found to be
resistant to syringomycin E (1.0 µg/ml), which is characteristic of
mutants that lack M(IP)2C.2 Disruption was
confirmed by Southern blot analysis. One strain, CTY-SR, was selected
and used for the remaining studies. To examine the effect of
IPT1 disruption, strains CTY182 (wild type), CTY1-1A (sec14-1ts), CTY165
(sec14-1ts sac1-22), and CTY-SR
(sec14-1ts sac1-22
ipt1) were streaked onto minimal medium plates with or
without myo-inositol (0.1 mM) and incubated at
permissive (28 °C) and nonpermissive (37 °C) temperatures (Fig.
1). All strains grew with and without
inositol at 28 °C. CTY1-1A (sec14-1ts) did
not grow in either case at 37 °C, as expected, whereas CTY182 (wild
type) CTY165 (sec14-1ts sac1-22), and
CTY-SR (sec14-1ts sac1-22
ipt1) grew at 37 °C with inositol, but only CTY182 was able to grow at 37 °C on medium lacking inositol. These phenotypes are characteristic of the myo-inositol requirement for
sac1-22 bypass of sec14-1ts and
demonstrate that disruption of IPT1 does not abolish the bypass.

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Fig. 1.
Disruption of IPT1 does not
affect suppression by sac1-22. Strains CTY182 (wild
type), CTY165 (sec14-1ts sac1-22),
CTY-SR (sec14-1ts sac1-22
ipt1), and CTY1-1A (sec14-1ts)
were streaked onto synthetic minimal media with 0.1 mM
inositol or without inositol at permissive (28 °C) or nonpermissive
(37 °C) temperatures.
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Secretory indices were determined for each strain by assaying
whole cell and secreted invertase activities at 28 °C and 37 °C
(Fig. 2). All strains showed similar
levels of invertase secretion at 28 °C. At 37 °C, strains CTY182
(wild type) and CTY165 (sec14-1ts
sac1-22) had similar secretory indices, secretion was impeded in
strain CTY1-1A (sec14-1ts), and strain CTY-SR
(sec14-1ts sac1-22
ipt1) showed a slightly enhanced secretory index. The results confirm the growth phenotypes of these strains, showing that
IPT1-encoded M(IP)2C synthesis is not involved
in the sac1-22 bypass of the
sec14-1ts secretory defect.

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Fig. 2.
Secretory indices of CTY strains.
Secretory indices were determined for strains CTY182 (wild type),
CTY1-1A (sec14-1ts), CTY165
(sec14-1ts sac1-22), and CTY-SR
(sec14-1ts sac1-22
ipt1) at a permissive temperature (28 °C; top
panel) and a nonpermissive temperature (37 °C; bottom
panel). The secretory index is the percentage of total cellular
invertase activity that is secreted. Data presented are averages of
three experiments, with standard deviations shown as error
bars.
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Sphingolipid Levels--
The above observation that
IPT1 disruption did not influence sac1-22
suppression of sec14-1ts prompted a
re-examination of the sphingolipid levels in the relevant strains.
Strains CTY182 (wild type), CTY1-1A (sec14-1ts),
CTY165 (sec14-1ts sac1-22), and
CTY-SR (sec14-1ts sac1-22
ipt1) were steady-state labeled with
H332PO4 to quantify sphingolipids.
Quantification of M(IP)2C levels in strains CTY182 (wild
type), CTY1-1A (sec14-1ts), and CTY165
(sec14-1ts sac1-22) revealed similar
degrees of 32P incorporation into M(IP)2C (in
cpm per mg dry weight of cells: 836 ± 356 (n = 3), 1080 ± 516 (n = 3), and 853 ± 341 (n = 3), respectively), and, as expected, CTY-SR
(sec14-1ts sac1-22
ipt1) did not produce M(IP)2C (Fig.
3). Differences were observed in the
relative MIPC levels. CTY1-1A (sec14-1ts) had
higher cellular levels of MIPC (29% of total sphingolipids) compared
with the isogenic wild type strain CTY182 (15% of total sphingolipids)
and the sac1-22 bypass strain CTY165 (7.5% of total sphingolipids) (Fig. 3). These results show that the sac1-22
bypass of sec14-1ts does not involve increased
M(IP)2C production.

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Fig. 3.
M(IP)2C levels of strains CTY1-1A
(sec14-1ts) and CTY165
(sec14-1ts sac1-22) are similar. Twenty-ml
cultures of strains CTY182 (wild type), CTY1-1A
(sec14-1ts), CTY165
(sec14-1ts sac1-22), and CTY-SR
(sec14-1ts sac1-22
ipt1) were radiolabeled with 200 µCi of
H332PO4. Sphingolipids were
extracted (see "Experimental Procedures"), and extracts equivalent
to 2-3 mg dry weight of cells were subjected to one-dimensional thin
layer chromatography and autoradiography (A). Individual
sphingolipids (M(IP)2C, ; MIPC, ;
inositolphosphoryl-ceramide, ) were quantified by scraping off the
radioactive spots and estimating the radioactivity by scintillation
counting (B). *, M(IP)2C was not detected in
lipid extracts of strain CTY-SR. The results shown are the averages of
three experiments, with standard deviations shown as error
bars.
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Phosphatidylinositol 4-Phosphate Levels Are Elevated in the sac1-22
Mutant--
The above finding that M(IP)2C levels are
unchanged in the CTY165 (sec14-1ts
sac1-22) bypass strain prompted us to revisit the effects of the
sac1-22 mutation on the cellular levels of
inositol-containing lipids. Strains CTY1-1A
(sec14-1ts) and CTY165
(sec14-1ts sac1-22) were grown at
permissive temperature in the presence of [32P]phosphate,
and the total lipids were extracted and analyzed by two-dimensional
thin layer chromatography (Fig. 4). A
preferential increase in the level of a phosphate-containing lipid that
resembled a diphosphoinositide (26) was observed in extracts of strain CTY165 (sec14-1ts sac1-22). To
identify this lipid, it was isolated from the silica matrix of the thin
layer chromatographic plates, extracted, deacylated, and subjected to
anionic exchange HPLC analyses (21) (Fig. 4). Comparisons to authentic
glycerophosphoinositol standards indicated that the lipid was
PtdIns(4)P. To confirm this identification, lipid extracts were
prepared from cultures of strains grown in the presence of
myo-[3H]inositol and deacylated, and the
glycerophosphoinositols were identified by anionic exchange HPLC
analyses (Fig. 5). A HPLC peak that
co-eluted with glycerophosphoinositol derived from PtdIns(4)P was
8-fold more abundant in the CTY165
(sec14-1ts sac1-22) samples as
compared with the CTY1-1A (sec14-1ts) samples.
The only other significant difference detected between the two strains
was a 60% reduction in the glycerophosphoinositol derived from
PtdIns(4,5)P2 in strain CTY165.

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Fig. 4.
PtdIns(4)P levels are elevated in the CTY165
(sec14-1ts sac1-22) bypass strain. The
autoradiographs of two-dimensional thin layer chromatograms of total
32P-labeled lipid extracts from (A) CTY1-1A
(sec14-1ts) and (B) CTY165
(sec14-1ts sac1-22) are shown. The
elevated spot in B is indicated by an arrow.
C, HPLC chromatogram of deacylated lipids isolated from the
thin layer chromatography media. Lipids were extracted from the spot
indicated by an arrow in B, deacylated, and
analyzed by HPLC as described under "Experimental Procedures"
(black line). Fractions (0.5 ml each) were collected from 0 to 40 ml. Counts in the fractions from 15 to 25 ml are shown. No
radioactivity above the background level was detected in the rest of
the fractions. The chromatogram with 32P-labeled deacylated
phosphatidylinositol 3-phosphate and PtdIns(4)P is superimposed
(gray line). gPI(3)P,
glycerophosphoinositol 3-phosphate; gPI(4)P,
glycerophosphoinositol 4-phosphate.
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Fig. 5.
Phosphoinositide levels of strains CTY1-1A
and CTY165. Ten-ml cultures of strains CTY1-1A ( ) and CTY165
(x) were labeled with 5 µCi/ml [3H]inositol at 26 °C
overnight. Lipids were extracted and deacylated, and extracts
containing 2.5 × 106 cpm were subjected to anion
exchange chromatography as described under "Experimental
Procedures." Lipid extracts from 10-ml cultures of strains CTY1-1A
and CTY165 contained 4 × 106 and 5.6 × 106 cpm, respectively. Fractions (0.3 ml each) were
collected from 15 to 45 ml. Counts of fractions 61-80 are shown in a
different scale in the inset. gPI(3)P,
glycerophosphoinositol 3-phosphate; gPI(4)P,
glycerophosphoinositol 4-phosphate; gPI(3,5)P2,
glycerophosphoinositol 3,5-bisphosphate;
gPI(4,5)P2, glycerophosphoinositol
4,5-bisphosphate. The chromatograms of deacylated lipids from CTY182
were indistinguishable from CTY1-1A (data not shown).
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DAG Production--
In the current model for sac1-22
suppression of sec14-1ts, bulk DAG levels are
hypothesized to increase as a result of elevated M(IP)2C
synthesis (11). However, the results presented above show that
M(IP)2C levels do not increase with sac1-22
suppression. To reconcile this discrepancy, the effects of
IPT1 disruption and sac1-22 suppression of
sec14-1ts on DAG production were determined.
Cultures of strains CTY182 (wild type), CTY1-1A
(sec14-1ts), CTY165
(sec14-1ts sac1-22), and CTY-SR
(sec14-1ts sac1-22
ipt1) were incubated with [14C]acetate
continually (steady-state labeling) or for a 20-min interval (pulse
labeling) before harvesting the cells. Total lipid extracts were
separated by thin layer chromatography, and DAG was identified by
comparison to an authentic standard. With steady-state labeling, no
significant differences in DAG levels were observed between the four
strains (data not shown). With pulse labeling, strains CTY182 (wild
type), CTY1-1A (sec14-1ts), and CTY165
(sec14-1ts sac1-22) showed similar
rates of net DAG production, but CTY-SR (sec14-1ts sac1-22
ipt1) produced approximately one-third less DAG (Fig. 6).

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Fig. 6.
Rates of diacylglycerol production in strains
CTY1-1A (sec14-1ts) and CTY165
(sec14-1ts sac1-22) are similar.
Exponentially growing cells (20-ml cultures) of strains CTY182 (wild
type), CTY1-1A (sec14-1ts), CTY165
(sec14-1ts sac1-22), and CTY-SR
(sec14-1ts sac1-22
ipt1) were pulse-labeled (20 min) with
[14C]acetate before lipid extraction, and the amounts of
14C recovered in diacylglycerol are shown as a percentage
of the total amount of [14C]acetate incorporated into the
cells. Five µl of each lipid extract (equal to one-twentieth of the
total lipids extracted from cells of a 20-ml culture) were subjected to
thin layer chromatography (see "Experimental Procedures"), and the
radioactivity in diacylglycerol was determined. The average
(n = 3) amounts of total radioactivity (in cpm) in the
extracted cells (from 20-ml cultures) were 177,757 (CTY182), 160,386 (CTY1-1A), 89,492 (CTY165), and 149,608 (CTY-SR). Results shown are the
averages of three separate experiments, with standard deviation shown
as error bars.
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DISCUSSION |
The main conclusion of our work is that M(IP)2C
synthesis, which is coupled to DAG production, is not involved in the
bypass of sec14-1ts by sac1-22. This
was determined by assessing the consequences of disrupting
IPT1, the gene that encodes the M(IP)2C
synthase, and also by examining the cellular levels of
M(IP)2C and DAG as a function of the bypass. These
conclusions differ from those of Kearns et al. (11), who
previously reported increases in both M(IP)2C and DAG in
the sec14-1ts sac1-22 bypass mutant.
It is clear that in the previous report, M(IP)2C was
misidentified. A re-evaluation in the present work of alterations in
lipid composition caused by the sac1-22 bypass mutation
revealed that the amount of a single phosphoinositol-containing lipid
species, PtdIns(4)P, was preferentially elevated rather than
M(IP)2C.
Our analyses, however, did reveal an increase in the relative level of
MIPC in CTY1-1A (sec14-1ts) at permissive
temperature (Fig. 3). This could be due to product inhibition of Ipt1p
by accumulated M(IP)2C in the Golgi compartment caused by a
defect in the secretory pathway that is nonetheless partially
functional at permissive temperature. Alleviation of this defect by
sac1-22 would account for the lowered MIPC levels observed
in strain CTY165 (sec14-1ts sac1-22).
However, why the MIPC levels of strain CTY165 were lower than those of
strain CTY182 (wild type) (Fig. 3) remains unexplained.
It is premature to assign a specific role to PtdIns(4)P in the
SEC14-dependent protein secretory pathway. The
observed increased amounts of this lipid (Figs. 4 and 5) are only
correlated with the sec14-1ts sac1-22
bypass. Nevertheless, the potential importance of PtdIns(4)P in the
bypass is consistent with several observations that link phosphatidylinositol transfer protein function and phosphoinositide production. For example, it has been demonstrated that mammalian phosphatidylinositol transfer proteins are co-factors for
phosphoinositide production by PtdIns kinases that participate in
signaling and membrane traffic (27). In assays for certain signaling or
trafficking processes, Sec14p can substitute for mammalian
phosphatidylinositol transfer proteins (28-31). It is worth
noting that Sac1p has significant similarity to a noncatalytic domain
of mammalian polyphosphoinositide 5-phosphatases (7), and Sac1p may
conceivably be a novel phosphatase that regulates PtdIns(4)P levels. If
so, Sac1p inactivation would lead to elevated levels of PtdIns(4)P,
which, in turn, may compensate for insufficient PtdIns(4)P production
due to a defective Sec14p. In numerous studies of phosphoinositide
roles in membrane traffic, PtdIns(4)P is typically regarded as a
precursor to PtdIns(4,5)P2 (32). However, the present
finding that PtdIns(4,5)P2 levels are lowered in the
sec14-1ts sac1-22 bypass emphasizes
the potential importance of PtdIns(4)P, rather than
PtdIns(4,5)P2, in SEC14-dependent
secretion. In support of the significance of PtdIns(4)P in trafficking,
Matsuoka et al. (14) reported that either PtdIns(4)P or
PtdIns(4,5)P2 promoted the binding of coat proteins to
liposomes in an in vitro assay for COPII-coated vesicle formation.
Roles for phospholipase D and phosphatidic acid in the bypass have been
suggested recently (8, 9). How the presently observed increases in
PtdIns(4)P relate to phospholipase D function and phosphatidic acid
production in the Golgi secretory machinery is not clear. However, it
is conceivable that all of these elements are coordinately regulated in
yet unknown ways to allow efficient operation of the
SEC14-dependent secretory pathway.
We also examined the effect of IPT1 disruption on DAG
production after both steady-state and pulse labeling of cells with [14C]acetate. The pulse-labeling experiments revealed
that rates of DAG production were reduced by one-third in the strain
CTY-SR (sec 14-1ts sac1-22
ipt1)
compared with the CTY182 (wild type), CTY1-1A (sec14-1ts), or CTY165
(sec14-1ts sac1-22) strains. The
decrease in DAG production with IPT1 disruption is plausible
because elimination of M(IP)2C synthase, which is coupled
to phosphorylinositol transfer from PtdIns, would eliminate a source of
cellular DAG. Steady-state and pulse labeling of DAG did not reveal an
increase in DAG levels in strain CTY165 as reported previously (11).
This discrepancy between the previous work of Kearns et al.
(11) and the present work remains unresolved. However, the present
findings are consistent with results reported by Sreenivas et
al. (9), who found no difference in the DAG levels of other bypass
mutants of sec14-1ts. Furthermore, because
IPT1 disruption leads to lower DAG production levels despite the
retention of the sec14-1ts bypass phenotype, our
results suggest that an increase in DAG levels is not essential for
suppression by sac1-22.
Taken together, the present findings raise concerns about certain
features of the proposed mechanisms of sac1-22-mediated bypass of sec14-1ts (11). DAG production through
sphingolipid metabolism and M(IP)2C biosynthesis does not
appear to play a role in the bypass. Instead, another lipid,
PtdIns(4)P, is revealed to be of potential importance to
SEC14-dependent secretion. However, insight into
the mechanisms of the role of PtdIns(4)P in the secretion pathway will
require further investigation.