From the Department of Life Science, Tokyo Institute
of Technology, Nagatsuda, Yokohama 226-0026, Japan, the
¶ Department of Biological Sciences, Graduate School of Science,
University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and the
Unit Process and Combined Circuit, Precursory Research for
Embryonic Science and Technology, Japan Science and Technology
Corporation, Graduate School of Science, University of Tokyo, Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P2), an important element in eukaryotic signal transduction, is synthesized either by phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P 5K) from phosphatidylinositol 4-phosphate (PtdIns(4)P) or by phosphatidylinositol-5-phosphate 4-kinase (PtdIns(5)P 4K) from phosphatidylinositol 5-phosphate (PtdIns(5)P). Two Saccharomyces cerevisiae genes, MSS4 and FAB1, are homologous to mammalian PtdIns(4)P 5Ks and PtdIns(5)P 4Ks. We show here that MSS4 is a functional homolog of mammalian PtdIns(4)P 5K but not of PtdIns(5)P 4K in vivo. We constructed a hemagglutinin epitope-tagged form of Mss4p and found that Mss4p has PtdIns(4)P 5K activity. Immunofluorescent and fractionation studies of the epitope-tagged Mss4p suggest that Mss4p is localized on the plasma membrane, whereas Fab1p is reportedly localized on the vacuolar membrane. A temperature-sensitive mss4-1 mutant was isolated, and its phenotypes at restrictive temperatures were found to include increased cell size, round shape, random distribution of actin patches, and delocalized staining of cell wall chitin. Thus, biochemical and genetic analyses on Mss4p indicated that yeast PtdIns(4)P 5K localized on the plasma membrane is required for actin organization.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Phosphatidylinositol 4,5-biphosphate
(PtdIns(4,5)P2)1
has been recognized as an important element in eukaryotic signal
transduction. Hydrolysis of PtdIns(4,5)P2 by phospholipase
C produces two second messengers, inositol 1,4,5-triphosphate
(IP3) and diacylglycerol. IP3 mobilizes
Ca2+ from intracellular stores, such as the endoplasmic
reticulum in animal cells (1) and vacuoles in plants (2) and yeast (3).
It is well known that the elevated intracellular Ca2+
stimulates a variety of calcium-modulating signaling enzymes, including
calmodulin-dependent protein kinases and calcineurin, a
type II B phosphoprotein phosphatase (4). Diacylglycerol, on the other
hand, activates the conventional isoforms of protein kinase C, which in
turn play a critical role in the regulation of a number of cellular
functions in mammalian cells (5). In the budding yeast
Saccharomyces cerevisiae, a protein kinase C-homologous gene
(PKC1) was isolated (6), whose product was shown to function in cell wall integrity and cell cycle progression (7, 8). In
vitro studies of Pkc1p, however, indicated that Pkc1p is strongly activated by phosphatidylserine in the presence of Rho1p, but not by
diacylglycerol (9). The stimulation by phosphatidylserine alone is
characteristic of the atypical isoform of protein kinase C, which
is stimulated by phosphatidylserine alone. Since the biochemical
property of Pkc1p is different from that of the conventional isoforms
of mammalian protein kinase C, it remains unclear whether and how
diacylglycerol acts as an important second messenger in S. cerevisiae.
PtdIns(4,5)P2 is also known to function as a regulator of
actin-binding proteins (10) such as profilin (11), gelsolin (12), and
-actinin of vertebrates (13). Recently, profilin was reported to be
localized both in the plasma membrane and cytosolic fractions in
S. cerevisiae, with the membrane association presumably facilitated by its interaction with phosphatidylinositol metabolites (14). Therefore, it is likely that through its regulation of actin-binding proteins, phosphatidylinositol metabolites affect the
cytoskeleton in yeast.
Moreover, PtdIns(4,5)P2 stimulates GDP to GTP exchange of ADP-ribosylation factor 1 (ARF1) (15). As the GTP-bound form of ARF1 triggers the attachment of the coat proteins (16-18), PtdIns(4,5)P2 may play a critical role in coat assembly. Interestingly, PtdIns(4,5)P2 was found to work as a cofactor for brain membrane phospholipase D (PLD) (19). These findings led to the proposal that PLD and phosphatidylinositol 4-phosphate 5-kinase (PtdIns(4)P 5-kinase) with their respective products, PtdIns(4,5)P2 and phosphatidic acid, form a positive feedback loop that causes a vesicle fusion with the acceptor membrane (19). Since PtdIns(4,5)P2 as well as phosphatidic acid activates an ARF GTPase-activating protein (20), they further postulated that the positive feedback loop is halted by the conversion of active ARF-GTP to ARF-GDP. Thus, PtdIns(4,5)P2 may work as a crucial factor in membrane trafficking.
Ins(4,5)P2 is synthesized either from PtdIns(4)P by the phosphorylation on the fifth hydroxyl group of the myo-inositol ring or from PtdIns(5)P by the phosphorylation on the fourth hydroxyl group (21). Phosphatidylinositol-4-phosphate 5-kinase (PtdIns(4)P 5K) and phosphatidylinositol-5-phosphate 4-kinase (PtdIns(5)P 4K), both of which catalyze PtdIns(4,5)P2 synthesis, are functionally different (22) but structurally similar to each other (23-26). Although mammalian PtdIns(5)P 4K was previously known as type II PtdIns(4)P 5K (23-26), it was reidentified as PtdIns(5)P 4K by careful examination (21). Physiological functions of mammalian PtdIns(5)P 4K and PtdIns(4)P 5K, however, remain to be elucidated. The sequences of mammalian PtdIns(4)P 5K and PtdIns(5)P 4K isoforms have homology to those of two yeast gene products, Fab1p and Mss4p (23-26). Though the FAB1 gene is not essential, the product, localized on the vacuolar membrane, is required for the vacuolar function and morphology (27). MSS4 was originally identified as a multicopy suppressor of the temperature-sensitive mutation in the STT4 gene (28), which encodes an PtdIns 4-kinase, suggesting involvement of Mss4p in PtdIns(4)P metabolism (29). Since a deletion of the MSS4 gene is lethal, characterization of conditional-lethal mutants of mss4 is useful for understanding the function of MSS4.
We report here that Mss4p has PtdIns(4)P 5K activity in
vitro and that expression of murine type I PtdIns(4)P 5K
functionally replaces MSS4 in vivo. Unlike Fab1p, Mss4p is
located primarily on the plasma membrane. Analyses of a
temperature-sensitive mss4 mutant revealed that Mss4p is
involved in the establishment of cell morphology.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Yeast Strains and Genetic Manipulations--
The yeast strains
used are listed in Table I. The complete
and minimal yeast media as well as the sporulation medium and procedures of tetrad analysis were as described (30). YPGS medium contains 2% galactose, 0.1% sucrose, 1% Bacto-yeast extract, and 2%
polypepton, whereas YPA medium for pre-sporulation consists of 1%
Bacto-yeast extract, 2% polypepton, and 1% potassium acetate (Wako
Pure Chemical Industries, Osaka, Japan). Yeast transformation was
carried out with lithium acetate (31). Plates containing 0.2%
5-fluoroorotic acid (FOA, Sigma) were used to select yeast cells
capable of losing a URA3 marked plasmid. E. coli
strains, DH5 (Life Technologies, Inc.) and SCS1 (Stratagene), were
used for gene manipulation. DNA sequencing was carried out with an automated DNA sequencer (model 373A, Applied Biosystems, Foster City,
CA).
|
Construction of Plasmids--
The plasmids used in this study
are described in Table II. Plasmid
pYO1953 was cloned from the YEp13 genomic library (32). The insertion
of the 3.9-kb BamHI-XhoI fragment of
MSS4 into the vector pBluescript SK+ resulted in
pYO1956, which was used for the construction of other MSS4-containing plasmids and as a template for error-prone
polymerase chain reaction. pYO1958, which was designed to aid
MSS4 gene disruption, mss4::HIS3, was
constructed by ligation of the 5.0-kb EcoRI-EcoRI fragment of pYO1956 and the 1.3-kb BamHI-XhoI
fragment of pJJ215 containing the HIS3 gene. pYO1959,
pYO1960, and pYO1962 were made by inserting the 3.9-kb
BamHI-XhoI fragment of pYO1956 containing MSS4 into the BamHI-XhoI gap of
pRS315, pRS314, and pRS316, respectively. pYO1964 was formed by
replacing the 1.2-kb NdeI-KpnI fragment of
pYO1960 with the NdeI-KpnI linker, which was made
by annealing the oligomers TATGTGAGATCTGGTAC and CAGATCTCACA. The
murine PtdIns(4)P 5K type I and human PtdIns(5)P 4K genes were
obtained by polymerase chain reaction using the published sequences
(25, 23) with the BamHI and BclI restriction
sites, respectively, attached at both ends.
|
Immunoprecipitation and PtdIns(4)P 5K Assay--
Cell lysates
were made in RIPA buffer (50 mM Tris-HCl, pH 8.0, 1%
Nonidet P-40, 0.15 M NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mM sodium orthovanadate) by
vortexing six times for 30 s each with acid-washed glass beads
(425-600 µm in diameter, Sigma). After preadsorption with protein A
cellulofine (Seikagaku-kogyo, Tokyo), the samples (300 µg of protein,
assayed by Bio-Rad protein assay kit) were subjected to
immunoprecipitation with saturating amounts of 16B12 anti-HA monoclonal
antibody (Berkeley Antibody, Richmond, CA) and then adsorbed to protein
A cellulofine. The adsorbed immunoprecipitates were then washed four
times with RIPA buffer and four times further with buffer T (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 50 µM ATP, 0.25 M sucrose, and 0.15 M NaCl). To determine the PtdIns(4)P 5K activity in the
immunoprecipitates, we incubated 10 µl of sample in 50 mM
Tris-HCl, pH 7.5, 1 mM EGTA, 10 mM
MgCl2, 50 µM ATP, 80 µM PtdIns(4)P (Sigma), and 5.0 or 0.5 µCi of [-32P]ATP
(Amersham Pharmacia Biotech) in the presence or absence of 50 µM phosphatidic acid in a total volume of 50 µl. After
60 min, the reaction was terminated by the addition of 0.4 ml of chloroform/methanol/12 N HCl (100:200:1 by volume). The
lipids were extracted by the method of Bligh and Dyer (33), dried, and,
together with PtdIns(4,5)P2, which was used as standard, were spotted on Merck Silica gel 60 TLC plates impregnated with 1.2%
potassium oxalate, with the exception of the experiment whose result is
shown in lanes 4 and 5 of Fig. 1C, in
which a similarly treated Whatman 60A plate was utilized. The samples
were separated with the solvent system of
chloroform/methanol/acetone/acetic acid/water (42:30:12:12:12 by
volume), and [32P]Ins(4,5)P2 was visualized
by autoradiography except for the product on the Whatman plate, which
was processed by BAS2000 Fuji bioImaging analyzer.
Isolation of Temperature-sensitive mss4 Mutants-- We first made an mss4 strain carrying the mutant gene on a centromer plasmid; the 3.1-kb BamHI-XhoI fragment of pYO1958 carrying the mss4::HIS3 gene was used to transform the diploid strain, YPH501. His+ transformants were selected, and the disruption of one of the chromosomal MSS4 gene copies was confirmed by Southern hybridization. The MSS4/mss4::HIS3 diploid strain, named YOC801, was transformed with pYO1962 carrying MSS4 and URA3, and the transformants were subjected to tetrad dissection. His+ Ura+ asci were selected and designated YOC802 (mss4::HIS3 (pYO1962)).
Random mutations were introduced by error-prone polymerase chain reaction mutagenesis (34) in the PI(4)P 5-kinase-conserved region of MSS4 using the two synthetic oligonucleotides, CCTTCTCAAAAGTCAAAGCA and TCGTACTACCGTTCCGGTA, corresponding to bases 841-860 and 2055-2025, respectively. The amplified 1.2-kb fragment was purified, digested with NdeI and KpnI, and then inserted to the NdeI-KpnI gap of pYO1964. Approximately 4,000 independent clones were made, and DNA of the plasmid pool was employed for transformation of YOC802 strain. The transformants that grew on SD-Trp medium at 23 °C were streaked on FOA (Immunofluorescence Microscopy-- Immunofluorescent staining of yeast cells was carried out according to Pringle et al. (35). Cells were grown to early exponential phase at 30 °C in YPD medium. HA-tagged Mss4p was visualized by indirect immunofluorescence using 16B12 anti-HA mouse monoclonal antibody as the first antibody and an fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Wako Pure Chemical Industries, Osaka, Japan) as the second antibody. DNA, actin, and chitin were stained with 4',6'-diamidino-2-phenylindole dihydrochloride, rhodamine-phalloidin (Molecular Probes), and calcofluor white M2R new (Sigma), respectively. Cell morphology and fluorescent staining were observed and photographed using a BX60 microscope (Olympus, Tokyo). Cell fractionation experiments were performed using the previously described techniques (36).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mss4p Is a Functional Homolog of Mammalian PtdIns(4)P
5-Kinase--
A BLAST search of protein sequence data bases revealed
that yeast Mss4p has 36, 33, and 31% identity with murine type I, type I
PtdIns(4)P 5K, and human PtdIns(5)P 4K, respectively, in
agreement with previous reports. To examine whether Mss4p is a functional homolog of any of the mammalian phosphatidylinositol phosphokinases (PIPKs) in yeast, we constructed plasmids carrying the
genes encoding murine type I
PtdIns(4)P 5K and human PtdIns(5)P 4K
hooked up to either the constitutive GAP promoter or the
galactose-inducible GAL1 promoter. After these expression
plasmids were introduced to YOC802 strain carrying
mss4::HIS3 and a URA3-MSS4 plasmid, the
growth on FOA plates was examined. We found that all the transformants expressing the type I
PtdIns(4)P 5K gene were capable of growing on
FOA plates (Fig. 1A,
panels b and c). On the other hand, expression of
the PtdIns(5)P 4K gene failed to complement the MSS4 gene
disruption, irrespective of copy number or the promoters used (Fig.
1A, panels b and c).
|
Expression of an Epitope-tagged MSS4 in Yeast Cells-- To analyze the Mss4p functions, we inserted the 3HA-epitope tag at the N-terminal of Mss4p. Introduction of the 3HA-epitope tag preserves its essential function because the tagged MSS4 with a single copy plasmid can fully complement mss4::HIS3 at all the temperatures examined (23, 30, and 37 °C). These results indicate that the 3HA-tagged Mss4p is functional in vivo. Western blotting analysis of the cells expressing the tagged Mss4p has shown that the anti-HA monoclonal antibody recognized a single band with a molecular mass of 86 kDa, which matched the predicted molecular weight of Mss4p (data not shown).
The Tagged MSS4 Gene Product Has PtdIns(4)P 5-Kinase Activity-- To examine PtdIns(4)P 5K activity of the MSS4 gene product, we immunoprecipitated the 3HA-tagged MSS4 protein expressed in yeast with the anti-HA monoclonal antibody and determined the kinase activity in the immunoprecipitate (Fig. 1B). The immunoprecipitate from the YOC804 cells carrying the tagged MSS4 gene on a multicopy plasmid had the highest PtdIns(4)P 5K activity, followed by that from the YOC803 cells, which harbored the same gene on a single copy plasmid, whereas that from the cells with untagged Mss4p (YOC806) exhibited little activity. Furthermore, the PtdIns(4)P 5K activity in the immunoprecipitates was found to be stimulated by the addition of 50 µM phosphatidic acid (Fig. 1B), a characteristic property of PtdIns(4)P 5K but not of PtdIns(5)P 4K (37). These results demonstrate that the tagged Mss4p possesses PtdIns(4)P 5K activity and that the amount of the kinase activity is copy number-dependent.
The mss4-1 Protein Has Less PtdIns(4)P 5K Activity When Cultured at the Restrictive Temperature-- We examined whether the PtdIns(4)P 5K activity of the mss4-1 mutant changes at the restrictive temperature. We first made a strain with the MSS4 gene disrupted but harboring a 3HA-tagged mss4-1 gene on a multicopy plasmid and designated it YOC823. The strain was cultured at 23 °C, was transferred to 38 °C at early exponential growth phase, and was further cultivated for 0, 2, 4, 6, or 8 h before being harvested. The lysates were made, immunoprecipitated with the anti-HA antibody, and the PtdIns(4)P 5K activities in the immunoprecipitates were assayed. As can be seen in Fig. 1C, the kinase activity starts decreasing immediately upon the temperature shift, and the reduction is complete by 4 h at the restrictive temperature. Western blotting of cell lysates showed that the strain had less amount of the mutant protein when cultured at the restrictive temperature than at the permissive temperature (data not shown). These results indicate a temperature-sensitive defect in the synthesis and/or heat lability of the mutant protein.
The MSS4 Gene Product Is Localized on the Plasma Membrane-- To examine intracellular localization of the tagged Mss4p, we first investigated the partitioning of 3HA-tagged Mss4p by cell fractionation experiments. Mss4p expressed either on the multicopy plasmid or on the single copy plasmid was mainly detected in the membrane fraction (Fig. 2A). Comparison with diluted samples as a standard showed that approximately 80% of the Mss4p was contained in the membrane fraction (data not shown). Next, immunofluorescence microscopy with the anti-HA monoclonal antibody revealed that the tagged Mss4p expressed on a single copy plasmid was almost exclusively localized on the cell surface (Fig. 2B). No polarized localization of the staining was observed during the cell cycle. The tagged Mss4p expressed on a multicopy plasmid gave stronger signals on the cell surface than on a single copy plasmid and occasionally gave a few additional internal punctuated signals (Fig. 2B, panel A), which were distinct from vacuoles. Cells expressing untagged Mss4p on a single copy plasmid did not give a detectable signal (Fig. 2B, panel C), indicating that the staining is not an artifact. Combined with the observation that the tagged Mss4p expressed on a single copy plasmid can fully complement the mss4 deletion, these results suggest that the MSS4 gene product is nearly exclusively localized on the plasma membrane.
|
Phenotypes of the Temperature-sensitive mss4-1 Mutant-- Growth of the temperature-sensitive mss4-1 mutant (YOC808) was compared with those of the wild-type strain (YOC807) and the mss4::HIS3 strain expressing untagged Mss4p on a single copy plasmid (YOC806) cultured on YPD plates (Fig. 3) and in YPD liquid media (Fig. 4). Judging from the colony size, the mss4-1 strain grew as well as the wild-type strain at 23 °C, grew slowly at 37 °C, and completely failed to grow at 37.5 °C (Fig. 3). The growth defect of the mss4 mutant was not suppressed by addition of 100 mM CaCl2. The doubling time of mss4-1 was 4.3 h at 23 °C, whereas that of the wild-type cells was 4.0 h, indicating that the mss4-1 mutant grows almost as fast as the wild type at the permissive temperature. At 38 °C, however, the growth of the mutant cells stopped within 4 h after the temperature shift (Fig. 4).
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The first piece of evidence that MSS4 encodes PtdIns(4)P 5K comes from the kinase assay; immunoprecipitates from cells expressing the epitope-tagged form of Mss4p had PtdIns(4)P 5K activity, and the tagged Mss4p is functional as demonstrated by the full complementation of the mss4 deletion by single-copy expression of 3HA-Mss4p. The substrate used, PtdIns(4)P, was purified from bovine brain, is approximately 98% pure on TLC according to the manufacturer, and may contain a small amount of PtdIns(5)P. It is formally possible that MSS4 actually encodes PtdIns(5)P 4K, and the PtdIns(5)P 4K activity produces PtdIns(4,5)P2 from the trace amount of PtdIns(5)P contained in the substrate. However, the following lines of evidence make this alternative explanation unlikely. The kinase activity of the gene product is enhanced in the presence of phosphatidic acid, a characteristic of mammalian PtdIns(4)P 5K isoforms, but not of PtdIns(5)P 4K (37). In addition, the murine PtdIns(4)P 5K gene, but not the human PtdIns(5)P 4K gene, complemented the mss4 gene disruption and, when the expression level was reasonably high, suppressed the temperature sensitivity of the mss4-1 strain. This identification is consistent with the higher homology Mss4p has with mammalian PtdIns(4)P 5K isoforms than with PtdIns(5) 4K. We therefore conclude that MSS4 encodes PtdIns(4)P 5K.
Our finding that MSS4 encodes PtdIns(4)P 5K explains the
previous genetic studies on MSS4 well. Since overexpression
of MSS4 suppresses the cell lysis phenotype of
stt4 at 23 °C and the temperature-sensitive
stt4-1 mutation, MSS4 was suggested to function downstream of Stt4p, a PtdIns 4-kinase (29, 53). The present demonstration is in agreement with the idea that Stt4p phosphorylates PtdIns to produce PtdIns(4)P, which in turn is further phosphorylated by the action of Mss4p to yield PtdIns(4,5)P2. In the same
paper, it was reported that overproduction of MSS4 did not
affect PtdIns 4-kinase activity of wild-type yeast cells and that
PtdIns 4-kinase activity of the
stt4 cells carrying a
multicopy MSS4 plasmid was as low as that in the
stt4 cells. These results are consistent with the notion
that Mss4p is a PtdIns(4)P 5K and has little if any PtdIns 4-kinase
activity.
Another S. cerevisiae gene, FAB1, whose product has a significant homology to mammalian PIPKs, is not essential (27). Fab1p was suggested to have PtdIns(4)P 5K activity on the vacuolar membrane, and the product of the kinase reaction, PtdIns(4,5)P2, was proposed to function as a regulator of vacuole homeostasis (27). However, the possibility that FAB1 encodes PtdIns(5)P 4K cannot be excluded, especially because no kinase assay has been reported. On the other hand, MSS4 is an essential gene (29) whose product is mainly localized to the plasma membrane (Fig. 2). Thus, it seems that Mss4p functions as PtdIns(4)P 5K on the plasma membrane and the product of the reaction, PtdIns(4,5)P2, plays an essential function at or near the plasma membrane. The idea that the two PIPKs that produce PtdIns(4,5)P2 play different roles at different compartments is supported by our recent observation that MSS4 on a multicopy plasmid suppresses the temperature sensitivity of cmd1-228, a calmodulin mutant with defect in calmodulin localization (43), whereas FAB1 does not (data not shown).
One explanation of the phenotypes of the temperature-sensitive mss4-1 mutant cells is that the phenotypes are caused by a reduced level of PtdIns(4,5)P2, whereas another interpretation is that they are brought about by defects in hitherto unidentified function(s) of Mss4p. We favor the former possibility because it is consistent with our finding that, when the temperature-sensitive mutant cells are shifted to the restrictive temperature, PtdIns(4)P 5K activity decreases before the mutant phenotypes become evident (Figs. 1C and 6).
Through what pathways does PtdIns(4,5)P2 give rise to the
mutant phenotypes? The mss4-1 mutant phenotypes are
strikingly similar to those of mutants of two actin-binding proteins,
i.e. profilin null mutants (38) and the capping protein
deletion mutants, cap1 and
cap2 (39).
Profilin is a ubiquitous actin- and PtdIns(4,5)P2-binding protein in eukaryotic cells (44) and is required for the proper organization of actin cytoskeleton into actin cables, which occur at
regions of active growth and for proper maintenance of cell polarity
(38). It was also shown that depletion of PtdIns(4,5)P2 in
the plasma membrane leads to profilin translocation to the cytosol
(14). The binding of capping protein to the growing end of actin
filaments was demonstrated to be prevented by micromolar concentrations
of PtdIns(4,5)P2 (39). We show here a synthetic lethal
interaction between PtdIns(4)P 5K and profilin. Thus, it is plausible
that a lower level of PtdIns(4,5)P2 in mss4-1
cells hinders proper functioning of profilin and capping protein,
leading to disorganization of actin cables.
Ins(4,5)P2 is known to be hydrolyzed by phospholipase C, encoded by the PLC1 gene in S. cerevisiae, to produce IP3 and diacylglycerol. Temperature-sensitive plc1 mutant cells were reported to be swollen with large buds and two nuclei at the restrictive temperature, and the growth defect of the mutant was suppressed by addition of 100 mM CaCl2 (45). An increased incidence of aberrant chromosomal segregation was also observed with another temperature-sensitive mutant, plc1-1 (46). Since mss4-1 cells at the restrictive temperature do not show these phenotypes, we consider it improbable that the primary effect of Mss4p is through PtdIns(4,5)P2 hydrolysis. Alternatively, PtdIns(4,5)P2 may be required to stimulate GDP to GTP exchange of yeast ARF, which is important for secretion (47). The localization and the mutant phenotypes, however, do not support the idea that the major pathway related to Mss4p involves ARF. PtdIns(4,5)P2 is also known to work as a cofactor for PLD. Yeast PLD encoded by SPO14 (48) is essential for meiosis but not for vegetative growth. The necessity of this gene only for meiosis again makes PLD an unlikely candidate for the major target of PtdIns(4,5)P2.
In summary, we propose that the MSS4 gene product functions in regulation of actin-binding proteins through generation of PtdIns(4,5)P2 from PtdIns(4)P in or near the plasma membrane. We believe that novel factors involved in the PtdIns(4,5)P2 signaling pathway can be identified by genetic approach with the conditional mutant of mss4. Further investigations of the MSS4 gene will greatly elucidate phosphatidylinositol signal transduction cascade.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Mike Hall for communicating results before publication and Fumiko Naito for preparing the manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan (to K. H. and Y. O.) and by funds from the Takeda Science Foundation and the Kowa Life Science Foundation (to Y. O.)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.
§ Present address: Division of Pediatric Pharmacology, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-ku, Tokyo 154-8509, Japan.
** To whom correspondence should be addressed. Fax: 81-3-5802-3366; E-mail: ohya{at}biol.s.u-tokyo.ac.jp.
1 The abbreviations used are: PtdIns(4,5)P2, phosphatidylinositol 4,5-biphosphate; PtdIns(4)P 5K, phosphatidylinositol-4-phosphate 5-kinase; IP3, inositol 1,4,5-triphosphate; PLD, phospholipase D; PIPK, phosphatidylinositol phosphokinase; FOA, 5-fluoroorotic acid; kb, kilobase(s); anti-HA, anti-hemagglutinin.
2 Y. Takita, M. Nakaya, Y. Anraku, and Y. Ohya, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|