(Received for publication, November 1, 1995; and in revised form, December 7, 1995)
From the
We have identified an open reading frame on chromosome XI of the
yeast, Saccharomyces cerevisiae, as encoding a protein with
phospholipase D (PLD) activity. We have named this open reading frame, PLD1, and show that yeast bearing a disruption in this gene
are unable to catalyze the hydrolysis of phosphatidylcholine. PLD1 encodes a hypothetical protein of 1683 amino acids and has a
predicted molecular mass of 195 kDa. Yeast bearing disruptions at the PLD1 locus are morphologically normal and grow vegetatively
like wild-type cells. In contrast, homozygous pld1 diploid cells are unable to sporulate and do not produce asci
under conditions that induce meiosis and sporulation in wild-type
cells. Thus, PLD1 is likely to be essential for the meiotic
cycle in yeast cells.
This is the first identification of a
eukaryotic, non-plant, phosphatidylcholine-hydrolyzing phospholipase D
gene. Because the biological role of PLD is not well understood, we
expect that pld1 yeast will become a useful tool for the
characterization of PLD functions as well as for the identification of
mammalian PLD homologs.
Phospholipase D activity has been identified in a wide variety
of cell types and organisms from bacteria(1) ,
yeast(2) , plants(3) , and mammals (reviewed in (4) and (5) ). PLD acts to catalyze the hydrolysis of
certain phospholipids, i.e. catalyzing the hydrolysis of
phosphatidylcholine (PC) ()to yield phosphatidic acid (PA)
and choline. PA, itself, is thought to act as an intracellular second
messenger in PLD-induced signaling processes (reviewed in (5) )
and may also be involved in regulating the synthesis of
phosphatidylinositol 4,5-bisphosphate (PIP
) (6) .
Activation of PLD in mammals occurs via known receptor-mediated
mechanisms (reviewed in (5) ), and evidence exists for the
involvement of protein kinase C(5, 7) , heterotrimeric
GTP-binding (G) proteins (reviewed in (5) and (8) ),
and small G proteins of the ARF and Rho families in its
activation(9, 10, 11, 12) . Two
distinct activation requirements for mammalian PLD have been described:
one type of enzymatic activity is GTP- and ARF-dependent, while another
is oleate-dependent(13) . However, both types of enzymatic
activities can be stimulated by
PIP(13, 14) . Interestingly, recent
studies have shown that PIP
biosynthesis, itself, is a
direct prerequisite for PLD activation(15) , which suggests
that PIP
is a co-factor of PLD.
Recent work has
implicated the activation of both phosphoinositide (PI) metabolism and
ARF as necessary steps for the intracellular trafficking of
membrane-bound compartments (reviewed in (16, 17, 18) ). Since PLD activity in mammals
may be activated by either ARF or PIP, it is intriguing to
think that PLD could play an important role in mediating membrane
trafficking. Yet, despite the wealth of information concerning the
biochemical properties of PLD, little is known about the structural and
molecular properties of the enzyme(s), while even less is known about
its biological role. To date, the only published sequence for a protein
from eukaryotes having PC-specific PLD activity is from
plants(3) , and its role in either cell signaling or membrane
trafficking remains virtually unknown.
Here we describe the identification and characterization of a gene encoding a PLD from yeast. We show that the yeast enzyme is homologous to plant PLD and shares some homology with a bacterial phosphatidyltransferase. Disruption of the PLD1 gene in yeast has no effects upon cell growth and morphology during vegetative growth but is likely to be required for meiosis and sporulation.
Figure 1: Homologous regions of ORF YKR031c and plant PLD. The diagram in A illustrates ORF YKR031c. Numbers above the diagram indicate those regions (marked boxes) ranging between 11 and 42 amino acids that are highly homologous to regions of plant PLD. Beneath each box is the percentage identity shared between residues of YKR031c and plant PLD within that designated region. The corresponding number of amino acids for each given region is: 1-25, 2-20, 3-19, 4-42, 5-28, 6-11, 7-37, and 8-24, respectively. B shows sequence comparisons between plant PLD and YKR031c contained within boxes 4, 5, and 7. An additional comparison between YKR031c and phosphatidylserine synthase from E. coli (PSS) is shown. Numbers correspond to amino acid sequence of the proteins; identities are marked with a vertical bar, and conserved residues are marked with a colon.
To create
disruptions of PLD1 in yeast, plasmids pPLD::HIS3 and
p
PLDFS::HIS3 were digested with EcoRI and SphI
to release fragments containing the disrupted PLD1 gene. These
were transformed into yeast cells using standard procedures to create
the strains described above. All disruptions were verified by Southern
analysis.
The hydrolysis of C-NBD-PC
in lysates was monitored by the production of PA, essentially as
described by Danin et al. (23) . Briefly, the reaction
mixture contained 0.3 mg/ml protein, 35 mM HEPES pH 7.2, 150
mM NaCl, 0.4 mM C
-NBD-PC, and 4 mol %
PIP
. Reaction volume was 120 µl, and the reactions were
incubated at 30 °C for 30 min. Transphosphatidylation assays
contained between 0 and 500 mM primary alcohol as substrate.
The assays were terminated by chloroform/methanol/HCl extraction
(100:100;0.6), and the phospholipid products were separated by thin
layer chromatography (TLC), as described(23) . Fluorescent
phospholipids were visualized by UV illumination, scraped from TLC
plates, methanol-extracted, and counted in a fluorimeter (excitation at
468 nm; emission at 520 nm). Results are expressed as the average of
two duplicate samples measured in arbitrary fluorescence units.
Sequence comparison between the proteins showed that they had low overall homology. However, at least eight specific regions of 11-42 amino acids in length were found to be highly homologous, ranging from 50 to 76% (Fig. 1). The percentage of shared residues in these regions varied between 20 and 62% (Fig. 1). One region, in particular, was also identified as having homology to a phosphatidylserine synthase from bacteria (Fig. 1, homology box 7). Thus, ORF YKR031c seemed a likely candidate for a yeast protein having PLD activity and was tentatively renamed PLD1.
In order to verify that pld1 cells lack PLD
activity, we performed PLD assays in vitro on yeast cell
lysates, using the fluorescent PC analog, C
-NBD-PC, as
substrate. Wild-type cells of either mating type were found to have
significant levels of PLD activity, as judged by PA production (Fig. 2). In contrast,
pld1 cells were found to
have no detectable PLD activity, indicating that the disruption of PLD1 destroys the protein responsible for PLD function. Thus,
the PLD1 gene product (Pld1) is likely to confer PLD-like
activity.
Figure 2:
Measurement of PLD activity in wild-type
cells and pld1 disruption strains. The production of PA
from fluorescent-labeled phosphatidylcholine (C
-NBD-PC) in
lysates prepared from wild-type (WT) and
pld1 cells was performed (see ``Experimental Procedures'').
Included in the assay were EGTA (5 mM), EDTA (1 mM),
and octyl glucoside (0.2 mM). PA production is measured at an
emission wavelength of 520 nm and given in fluorescence units. A shows a photograph of the fluorescence-based emission taken of the
samples chromatographed on TLC plates. B represents a
histogram of the data shown in A. Lanes 1 and 20 indicate PA production in the absence of added protein. Lanes
2-4 and 11-13 indicate PA production in
membrane preparations from JC1 and JC2 wild-type cells, respectively. Lanes 5-7 indicate PA production in
pld1 strain PLD
-1, while lanes 8-10 indicate PA
production in
pld1 strain PLD
FS-1. Lanes
14-16 indicate PA production in pld1 strain
PLD
-2, while lanes 17-19 indicate PA production in
pld1 strain PLD
FS-2. Lane 21 contains a
standard for phosphatidic acid.
To
verify that the activity of the PLD1 gene product is similar
to that described previously, we have characterized PLD activity in
wild-type and pld1 cells (Fig. 3). We first
examined the effect of primary alcohol addition upon PA production and
alcohol transphosphatidylation in haploid cells. We found that an
increase in alcohol chain length resulted in a decrease in the overall
activity of the enzyme, which was characterized by both a decrease in
PA formation and an increase in phosphatidyl alcohol production (Fig. 3A). Next, we examined the effect of various
agents and carbon sources upon PLD activity. The addition of chelators
of divalent metal cations or oleate resulted in a significant decrease
in PLD activity (Fig. 3A). Likewise, the omission of
PIP
led to a substantial decrease (>65%) in activity (Fig. 3A). Thus, the PLD activity examined here is
highly similar to that described previously (2) and is similar
to the oleate-independent PLD from mammals(13) .
Figure 3:
Characterization of yeast PLD activity.
PLD activity (PA production) was assayed under different conditions. A, the ability of straight chain alcohols to serve as
substrates for the transphosphatidylation reaction is shown in lanes 1-4. PA production was assayed with the addition
of 0.5 M primary alcohol (e.g. ethanol (Eth), propanol (Prop), and butanol (But))
and is shown as a solid bar. In addition, the amount of
phosphatidyl alcohol produced was also assayed and is shown above as a hatched bar. The effect of divalent cation chelators
(EGTA/EDTA) upon PA production is shown in lane 5, while the
effects of oleate addition (60 mol%, lane 6) or the omission
of PIP from the assay (lane 7) is also given. The
amount of PA/phosphatidyl alcohol production is normalized (in percent)
to the amount of activity generated under standard assay conditions in
the presence of PIP
. Activity was assayed in lysates
prepared from JC1 wild-type yeast. B, the effect upon PLD
activity of growing cells on different carbon sources. Wild-type yeast
(JC2) were grown for 6 h on a variety of rich media containing
different carbon sources (lane 1, glucose (YPD); lane
2, acetate (YPA); lane 3, ethanol/glycerol (YPEG); and lane 4, galactose (YPG)). PA production in cell lysates was
assayed as described (see ``Experimental Procedures''). In a
different experiment, homozygous
pld1::HIS3/
pld1::HIS3 (PLD
FS-1
PLD
FS-2 cells) and heterozygous
pld1::HIS3/PLD1 (PLD
FS-1
JC2) cells
were assayed for PA production when shifted to acetate-containing
medium (YPA) (lanes 6 and 8) for 2 h or maintained
continually on glucose (YPD) (lanes 5 and 7). The
activities for these representative experiments are listed in arbitrary
fluorescence units.
In contrast, growing cells in the presence of non-fermentable carbon sources, such as acetate and ethanol/glycerol, or in the presence of galactose resulted in an increase in PLD activity over cells maintained continually in glucose. Thus, the yeast enzyme may be under partial glucose-repressible control (Fig. 3B).
Since yeast
PLD is stimulated in the presence of acetate-containing medium (this
study and (2) ), we examined whether PLD activity is increased
in homozygous pld1::HIS3/
pld1::HIS3 and heterozygous
pld1::HIS3/PLD1 cells shifted from glucose-containing
medium to acetate-containing medium (sporulation conditions). Cells
bearing homozygous disruptions in PLD1 showed little basal
activity when grown in glucose-containing medium or when shifted to
acetate-containing medium. In contrast, heterozygous
pld1::HIS3/PLD1 diploid cells showed a strong
time-dependent induction of PLD activity that peaked within 2 h of
shifting the cells to acetate-containing medium (Fig. 3B and data not shown). Thus, PLD activity is induced under
sporulation conditions in a PLD1-dependent fashion.
The
stimulation of PLD activity upon transfer to acetate-containing medium
suggested to us that a functional PLD might be required for sporulation
in yeast. Indeed, diploid yeast bearing homozygous disruptions of PLD1 (pld1::HIS3/
pld1::HIS3) were found to
be unable to undergo sporulation to yield asci under conditions by
which wild-type cells are known to sporulate (data not shown). Thus,
there appears to be a requirement for PLD1 in the sporulation
of yeast cells.
We have identified a yeast protein that confers PLD activity
to yeast cell lysates. The gene encoding this activity, PLD1,
localizes to the GCN3-DAL80 intergenic region of the short arm
of chromosome XI and is the region that the spo14 sporulation
mutation maps to. Our results indicate that PLD1 encodes a
PC-hydrolyzing PLD that confers the significant, if not total
proportion, of PLD catalytic activity in yeast cells. Moreover, this
activity is stimulated during sporulation conditions and is similar in
requirements to that described previously by Ella et al. (2) . In addition, we have demonstrated that yeast Pld1 is
stimulated by PIP like mammalian PLD(14) .
Although the true role of PLD1 is not yet known, strong
evidence links PLD function to meiosis and sporulation in yeast cells.
First, PLD1 activity is stimulated during the shift to
sporulation-inducing medium (this study and (2) ). Second, pld1 cells fail to undergo sporulation. Third, while this
manuscript was in preparation, the sequence of SPO14 was
deposited into the NCBI data base (accession number L46807) and
although it was not identified as having PLD activity, we and others
have now identified it as being allelic to PLD1. (
)Thus, PLD activity appears necessary for yeast cells to
complete the meiotic cycle.
When diploid yeast are deprived of nitrogen and fermentable carbon sources they enter a meiotic cycle that leads to the development of four haploid nuclei. These nuclei are later enveloped by a membranous spore wall that arises from the coalescence of vesicles at the spindle pole (reviewed in (25) ). The spore wall consists of four layers, including at least two spore-specific layers that are induced only during the meiotic cycle. SPO14 was previously identified as a sporulation-specific gene that is required for the commitment to meiotic development as well as for meiotic segregation and spore formation(26) . Cells bearing the spo14 mutation are defective in sporulation but can return to mitotic division even from the late stages (i.e. tetraploid stage) of meiotic development(27) . In all likelihood, then, Spo14/Pld1 may be a critical component of the final stage of the meiotic cycle, spore wall assembly.
Converging lines of evidence
have implicated PLD function with PI metabolism and membrane
trafficking in mammals(17) . Activation of PLD by ARF, which
itself acts to recruit cytosolic coat proteins to newly forming
vesicles in the Golgi (reviewed in (18) ), may be an important
mechanism for generating PA and altering the lipid environment on
membranes destined to undergo fusion(17) . However, PLD
activation may also lead to PIP production(17) ,
which could have profound effects upon both PI metabolism and signaling
via PI-mediated signaling cascades, in addition to affecting the lipid
environment of membranes. Although this work is still in its infancy,
yeast may prove to be an ideal system in which to assay the role of PLD
upon PI metabolism, as well as in cell signaling and membrane
trafficking. Interestingly, our work already demonstrates that Pld1
activity is not necessary for vegetative cell growth, which implies
that membrane trafficking processes like secretion are not prominently
affected. Thus, we predict that Pld1 function is not likely to be
required for the anterograde trafficking of secreted essential
proteins, although it could play a role in other trafficking steps.
Note Added in Proof-After this work was accepted, a paper describing the identification of a human PLD gene was published (Hammond, S. M., Altshuller, Y. M., Sung, T-C., Rudge, S. A., Rose, K., Engebrecht, J., Morris, A. J., and Frohman, M. A.(1995) J. Biol. Chem.270, 29640-29643). This gene is highly homologous to [Abstract/Full Text] yeast PLD1.