From the Departments of Pharmacology and Cancer Biology and of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
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ABSTRACT |
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The SAC1 gene product has been
implicated in the regulation of actin cytoskeleton, secretion from the
Golgi, and microsomal ATP transport; yet its function is unknown.
Within SAC1 is an evolutionarily conserved 300-amino acid
region, designated a SAC1-like domain, that is also present
at the amino termini of the inositol polyphosphate 5-phosphatases,
mammalian synaptojanin, and certain yeast INP5 gene
products. Here we report that SAC1-like domains have
intrinsic enzymatic activity that defines a new class of polyphosphoinositide phosphatase (PPIPase). Purified recombinant SAC1-like domains convert yeast lipids phosphatidylinositol
(PI) 3-phosphate, PI 4-phosphate, and PI 3,5-bisphosphate to PI,
whereas PI 4,5-bisphosphate is not a substrate. Yeast lacking Sac1p
exhibit 10-, 2.5-, and 2-fold increases in the cellular levels of PI
4-phosphate, PI 3,5-bisphosphate, and PI 3-phosphate, respectively. The
5-phosphatase domains of synaptojanin, Inp52p, and Inp53p are also
catalytic, thus representing the first examples of an inositol
signaling protein with two distinct lipid phosphatase active sites
within a single polypeptide chain. Together, our data provide a long sought mechanism as to how defects in Sac1p overcome certain actin mutants and bypass the requirement for yeast
phosphatidylinositol/phosphatidylcholine transfer protein, Sec14p. We
demonstrate that PPIPase activity is a key regulator of membrane
trafficking and actin cytoskeleton organization and suggest signaling
roles for phosphoinositides other than PI 4,5-bisphosphate in these
processes. Additionally, the tethering of PPIPase and 5-phosphatase
activities indicate a novel mechanism by which concerted
phosphoinositide hydrolysis participates in membrane trafficking.
Phosphoinositides are essential components of eukaryotic membranes
and are key regulators of membrane trafficking and actin cytoskeleton
(1-3). Phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2)1
serves as a precursor to second messengers and as a signaling molecule
itself by regulating protein activities and through interactions with
protein modules (3-5). Additionally, roles for PI(3)P,
PI(3,5)P2 and PI(3,4,5)P3 in membrane movement
have been defined (1, 6-10). Homeostasis of phosphoinositides occurs
both spatially and temporally via a plethora of lipase, kinase, and
phosphatase activities, thereby providing several unique points of
regulation (for reviews see Refs. 11-17).
A role for inositol lipid phosphatases in membrane trafficking has come
from the characterization and cloning of synaptojanin, a mammalian
neuronal inositol polyphosphate 5-phosphatase (5-ptase) involved in
synaptic vesicle recycling (18). Additionally, studies of three yeast
INP5 gene products (also known as SJLs)
demonstrate that although they are collectively essential, the
individual proteins also have nonredundant roles in regulating membrane
trafficking, cell wall synthesis, osmo-sensitivity, and actin
cytoskeleton structure (19-22). Synaptojanin and the Inp5ps are
members of a large 5-ptase gene family, conserved from yeast to humans,
that specifically remove the D-5 position phosphate from certain
phosphoinositides and/or inositol polyphosphates (15-21). Of interest,
the domain structures of Inp52p and Inp53p, and to a lesser extent
Inp51p, are analogous to synaptojanin, having a 300-amino acid 5-ptase domain that is flanked by amino- and carboxyl-terminal
SAC1-like and proline-rich domains. Although the function of
SAC1-like domains in synaptojanin, the Inp5ps, and Sac1p is
unknown, genetic studies have shown that mutations in Sac1p overcome
certain actin defects and bypass the requirement for Sec14p, a
phosphatidylinositol/phosphatidylcholine transfer protein required for
generation of secretory vesicles from Golgi (23-26). In
addition, Sac1p function is required for proper transport of
ATP into the endoplasmic reticulum (27).
Here we report studies aimed at elucidating the unique and overlapping
roles of the INP5 gene products. We demonstrate that Inp52p,
Inp53p, and synaptojanin have two lipid phosphatase activities, one as
a novel polyphosphoinositide phosphatase (PPIPase) and the other as a
PI(4,5)P2 5-ptase. Remarkably, we show that these activities are encoded by two autonomous active sites provided by the
SAC1-like and 5-ptase domains, respectively. PPIPase
activity is biologically relevant because loss of this activity in
cells results in significant changes in the levels of certain
phosphoinositides. The unique substrate selectivity and regulatory
properties of each activity suggests novel hypotheses regarding how
individual and/or combinatorial control of PI(4)P, PI(3)P,
PI(3,5)P2, and PI(4,5)P2 are intimately
involved in the regulation of vesicle trafficking and actin
cytoskeleton organization.
Strains, Media, and Genetic Methods--
Yeast strains used in
this study are listed in Table I.
Standard procedures for growth and yeast genetic manipulations were used as described (28). SacI mutant strain was kindly
provided by Dr. Vytas A. Bankaitis (University of Alabama at
Birmingham). In all cases, DNA produced by PCR amplification was
sequenced to confirm that unwanted mutations were not present.
Production of Inp5 Proteins--
Overexpression of Inp51p,
Inp52p, and Inp53p in yeast was achieved on a triple knockout
background. Construction of triple mutant strains required deletion of
INP54 in previously reported double mutants (20). The
INP54 coding region was deleted and replaced in a W303
diploid strain with the LEU2 gene by a PCR-based method
(20). Haploid inp54 and appropriate haploid double mutants were mated, sporulated, and dissected to yield inp51 inp52
inp54, inp51 inp53 inp54, and inp52 inp53
inp54 triple mutants.
Overexpression of Inp5ps was accomplished in triple mutant yeast
harboring plasmids containing the coding sequence of each Inp5 under
the control of a galactose-inducible promoter. Plasmids were
constructed using a PCR-based strategy in which the coding sequences
were inserted into pBJ246 (ATCC 777452) to yield pGAL51, pGAL52, and
pGAL53. Cells harboring plasmids were grown in CM/ura Preparation of Radiolabeled Substrates and Enzymatic
Assays--
Radiolabeled substrate was either purchased
(3H-PI(4,5)P2; NEN Life Science Products) or
prepared from steady-state inositol-labeled cells (3H-PI,
3H-PI(3)P, 3H-PI(4)P,
3H-PI(3,5)P2, and
3H-PI(4,5)P2). Inp51 mutant strains
were grown in CM containing 200 µCi/ml of
myo-[2-3H(N)]inositol over 10 doublings,
harvested at log-phase (1 × 108 cells),
"stressed" by treatment with 5 mg of zymolyase 20T (Seikagaku Corp., Tokyo, Japan) for 30 min, and lipids were chloroform/methanol extracted (21). Purified 3H-PIP (PI(3)P and PI(4)P) was
obtained by separating crude lipids using base-oxalate TLC (29) and
extraction of appropriate regions of silica. Purified
3H-PIP2 (PI(4,5)P2 and
PI(3,5)P2) was obtained by separating crude lipids on
acid-oxalate TLC (21) and recovered as described above.
Mixed vesicle substrates were prepared by drying total cell or purified
lipids under a N2 stream and briefly sonicating in buffer
containing RB buffer (50 mM HEPES, pH 7.4, 50 mM KCl, 3 mM EGTA). Detergent micelle substrate
was prepared using RBT buffer (RB buffer supplemented with 0.2% Triton
X-100). Enzymatic assays were performed in either RB or RBT buffer
supplemented with 0.2 mg/ml bovine serum albumin, 2 mM
MgCl2, and 10 mM dithiothreitol for 1 h at
37 °C. Where noted, MgCl2 was omitted to optimize
PPIPase activity. Reactions were stopped, extracted with
chloroform/methanol, deacylated with methylamine (21), and analyzed by
HPLC (see below).
Expression of Recombinant Protein in Bacteria--
The coding
region of INP53 was inserted in-frame into pGEX-3X vector
(Amersham Pharmacia Biotech) to yield pGEX53. The 5-ptase domain of
INP53 (residues 490-1107) was amplified by PCR and inserted in-frame into pGEX-3X to yield pGEX53-5pt. The SAC1-like
domain of INP53 (residues 1-548) was constructed by
digestion of pGEX53 with SnaBI and SmaI and
religated to produce pGEX53-SAC. The SAC1-like domain of
synaptojanin (residues 1-523) was amplified by PCR from a human brain
pCMV sport library DNA (Life Technologies, Inc.) and inserted into
pGEX-3X yielding pGEXSyn-SAC.
Expression of GST fusion proteins was performed in Escherichia
coli strain BL21(DE3) (Novagen) as described previously (21) with
the exception that 1 liter of LB medium was used for a 24-h induction
at 23 °C, and cells were disrupted by French press. Fusion proteins
were purified by glutathione-Sepharose 4B chromatography according to
manufacturer's directions (Amersham Pharmacia Biotech).
Yeast Mutant Radiolabeling Studies and HPLC Analysis--
Yeast
strains were labeled over 10 cell doublings to steady-state in
appropriate minimal medium containing
[3H]myo-inositol. Lipids were deacylated with
methylamine reagent essentially as described (21) except that 1 mg of
carrier lipid (brain extract, Sigma) was added to improve yield.
Glycerophosphoinositols (groPI) were resolved by Partisphere SAX HPLC
(Whatman 4.6 × 125 mm). Samples were equilibrated to 10 mM ammonium phosphate, pH 3.5 (AP), applied to column, and
eluted using the following gradient: 10 mM AP to 340 mM AP over 15 min, 340 mM AP to 1.02 M AP over 7.5 min, and isocratic 1.02 M AP for
additional 5 min. Radioactivity was measured using the
BetaRAMTM in-line detector (INUS, Tampa, FL).
Sequencing of the Saccharomyces cerevisiae genome
reveals four gene products, designated INP5s, that have the
canonical catalytic motifs present in all inositol polyphosphate
5-phosphatases. Previously, we reported that recombinant Inp51p
functions as a lipid-selective 5-ptase that converts
PI(4,5)P2 to PI(4)P but does not hydrolyze Ins(1,4,5)P3 (21). We have recently found that Inp54p is
also a PI(4,5)P2 lipid selective
5-ptase.2 These data coupled
with published reports that yeast extracts do not have detectable
Ins(1,4,5)P3 5-ptase activity (21, 30) suggest that the
Inp5ps utilize lipid substrates. Candidate D-5 phosphorylated lipid
substrates present in yeast include PI(4,5)P2 and the newly
discovered osmotic stress-induced PI(3,5)P2 (31, 32). To
help explain the individual and overlapping cellular functions of the
Inp5ps, we characterized their substrate selectivities.
Polyphosphoinositide Phosphatase and 5-ptase Activities of the
Inp5ps--
In our initial biochemical assays, we used native Inp5
proteins and presented lipid substrate in two distinct contexts: mixed vesicles and detergent micelles. Individual native Inp5ps were overproduced in a yeast strain that lacked the other three 5-ptases (GAL51 in inp52 inp53 inp54, GAL52 in inp51 inp53
inp54, and GAL53 in inp51 inp52 inp54). Total cellular
lipids harvested from 3H-inositol-labeled yeast were used
to prepare the substrates as a means to approximate the phospholipid
stoichiometry and composition encountered in vivo. The
abundance of PI(4,5)P2 and PI(3,5)P2 candidate
substrates was increased 2- and 10-fold by preparing lipids from
stressed inp51 mutant strains, resulting in readily detectable 3H-PI, 3H-PI(3)P,
3H-PI(4)P, 3H-PI(3,5)P2, and
3H-PI(4,5)P2 (Fig.
1).
Individual Inp5ps show distinct substrate selectivities and sensitivity
to detergent (Fig. 1). Treatment of mixed vesicle substrate (Fig.
1A) with buffer control or pGAL extract (not shown) yielded
identical profiles of groPIs, confirming the absence of background
phosphatase activity. Inp51p (pGAL51) treatment of mixed vesicle
substrate resulted in a selective reduction in PI(4,5)P2 (12,200 versus 9,400 cpm) and a corresponding increase in
PI(4)P (6,600 versus 9,800 cpm). The activity was time- and
dose-dependent under conditions of 10-40% conversion, and
a maximal hydrolysis of 80% was attainable using the mixed vesicle
substrate (not shown). Remarkably, treatment of mixed vesicles with
Inp52p (pGAL52) or Inp53p (pGAL53) resulted in 80% decreases in all
polyphosphoinositides. Strikingly, treatment of substrate prepared as a
detergent micelle (Fig. 1B) yielded significantly different
activities. Under these conditions, Inp51p did not hydrolyze any of the
phosphoinositides. Inp52p and Inp53p converted 100% of the
PI(4,5)P2 to PI(4)P; however, they were no longer able to
hydrolyze PI(3,5)P2, PI(3)P and PI(4)P. We also tested the
three proteins against soluble substrates including inositol
1,4-bisphosphate, Ins(1,4,5)P3, and inositol 1,3,4,5, tetrakisphosphate and found that they did not serve as substrates (not
shown). These data suggest that Inp51p is a detergent-sensitive PI(4,5)P2 5-ptase that does not utilize
PI(3,5)P2 or PIPs, consistent with our previously published
data (21). In contrast, Inp52p and Inp53p possess two distinct
activities: one is a detergent-insensitive PI(4,5)P2
5-ptase, and the other is a detergent-sensitive polyphosphoinositide 3-/4-/5-phosphatase activity (designated PPIPase). The 5-ptase and
PPIPase activities were found to be
Mg2+-dependent and -independent, respectively
(not shown).
To demonstrate that the PPIPase activity of Inp52p and Inp53p was
intrinsic and not associated, recombinant Inp53p was produced in
bacteria as a GST fusion protein (GST-53) and analyzed (Fig. 2A). Treatment of mixed
vesicle substrate with either control (not shown) or GST showed no
detectable phosphatase activity. Purified recombinant GST-53 harbored
activity that resulted in >60% decreases in all
polyphosphoinositides. Treatment of detergent micelle substrate
with GST-53 resulted in a 100% decrease in PI(4,5)P2 and a
quantitative increase in PI(4)P (not shown). These data are similar to
those obtained with native Inp53p and demonstrate that both the PPIPase
and 5-ptase activities are intrinsic properties of Inp53p and
presumably Inp52p.
The PPIPase Activity Is Attributable to SAC1-like Domains--
The
unique biochemical properties and substrate selectivity of PPIPase and
5-phosphatase activities suggest that they are separable. Therefore, we
separated partially proteolyzed recombinant Inp53p by size exclusion
chromatography and assayed individual fractions for PPIPase and 5-ptase
activities. This analysis confirmed that these activities are separable
(not shown). Furthermore, the PPIPase and 5-ptase activities were
detected in fractions eluting as low as 30 and 25 kDa, respectively
(not shown). This was intriguing because multiple sequence alignments
predict that the SAC1-like and 5-ptase domains are
approximately this size. With this in mind, the 5-ptase and
SAC1-like domains of INP53 were independently
expressed as GST fusion proteins and analyzed (Fig. 2A).
Treatment of mixed vesicle substrate with the 5-ptase domain resulted
in a 40% decrease in PI(4,5)P2 and a quantitative increase
in PI(4)P (GST53-5pt), demonstrating that it encodes a 5-ptase but not
PPIPase activity. Remarkably, treatment of mixed vesicles with the
SAC1-like domain resulted in >70% reduction in the levels
of PI(3,5)P2, PI(4)P, and PI(3)P, whereas
PI(4,5)P2 levels did not change (GST53-Sac). Using
detergent micellar substrate we found that the PPIPase activity of
GST53-Sac was ablated and that the 5-ptase activity of GST53-5pt was
stimulated (not shown), recapitulating data observed for the
full-length GST53 protein. The PPIPase activity was found to be
Mg2+-independent and inhibited by 2 mM
Cu2+ and Zn2+ (80%) and by Mg2+
(20%) (not shown). Addition of DTT markedly increased activity. Together these data demonstrate that both the SAC1-like and
5-ptase domains of Inp53p autonomously fold and are sufficient to
encode the intrinsic PPIPase and 5-ptase activities found in Inp53p.
The product(s) formed by the PPIPase activity were characterized using
purified PIP2 and PIP mixed vesicle substrates (Fig. 2,
B and C). Treatment of either lipid substrate
with GST protein alone did not produce detectable hydrolytic activity.
Full-length GST53 quantitatively hydrolyzed PIP2 to form
PI, and no PIP intermediates were detected. Treatment of
PIP2 with GST53-5pt protein converted PI(4,5)P2 but not PI(3,5)P2 to PI(4)P. In
contrast, GST53-Sac protein hydrolyzed PI(3,5)P2 to PI
without producing detectable intermediates and did not utilize
PI(4,5)P2. Treatment of PIP with GST alone results in no
hydrolytic activity. Addition of GST53-Sac results in conversion of
both PI(3)P and PI(4)P to PI. In contrast, PIP is not hydrolyzed by
GST53-5pt protein (not shown).
To examine whether the SAC1-like domain of human
synaptojanin harbors similar PPIPase activity, we expressed this domain
as a GST fusion protein. Of interest, although the majority of reports of the biochemical activity of synaptojanin show that it is restricted to a type II 5-ptase, Chung et al. (33) reported that
purified rat brain synaptojanin exhibits 4-/5-phosphatase activity,
which converts PI(4,5)P2 to PI, although it was not
determined whether this activity was intrinsic or a co-purifying
contaminant such as the polyphosphoinositidase activity reported by
Hope and Pike (34). Treatment of total cell substrate with purified
GSTSyn-Sac resulted in >50% decreases in levels of PI(3)P, PI(4)P,
and PI(3,5)P2 (not shown). Again, PI(4,5)P2 was
not a substrate, consistent with the results obtained using GST53-Sac
protein. Treatment of purified 3H-PIP with GSTSyn-Sac
results in 50% conversion of both isomers to PI (Fig. 2C).
The activity is time- and dose-dependent and has similar
biochemical properties to GST53-Sac PPIPase in terms of metal
inhibition and DTT stimulation (not shown).
The inability of any SAC1-like domains tested to hydrolyze
PI(4,5)P2 is surprising because they are able to function
as 3-, 4-, and 5-phosphatases. Attempts to detect PI(4,5)P2
hydrolytic activity under a variety of conditions, including using
commercially available PI(4,5)P2, have reproducibly failed
(not shown). Thus, the mechanism by which SAC1-like domains
discern their substrates remains uncertain and will be of significant
future interest.
SAC1-like Domain PPIPase Activity Is Functional in the
Cell--
To ascertain if PPIPase activity of SAC1-like
domains was functional in vivo, we examined the
phosphoinositide levels in inp52 inp53 mutant strains.
Previously, we reported that steady-state levels of
PI(4,5)P2 did not change in inp52 inp53 mutants
(20), whereas two-fold increases were detected in inp51 null
cells (21). However, these studies were performed using thin layer
chromatography system not capable of resolving PI(4,5)P2
and PI(3,5)P2. We therefore analyzed
3H-inositol lipid levels in wild type and inp52
inp53 mutant strains using a deacylation and HPLC strategy. The
levels of PI(4,5)P2, PI(3)P, and PI(4)P were similar in
both strains (not shown). However, at early log phase (3 × 106) the levels of PI(3,5)P2 increased
5-7-fold in inp52 inp53 mutant cells (Fig.
3A). It is noteworthy that
even at the increased levels, the amount of PI(3,5)P2 was
only 30% of the PI(4,5)P2, thereby explaining why this may
have been missed in our previous study. Additionally, this difference
was not observed at late log and stationary phase. These data are
consistent with a role for Inp52p and Inp53p in regulating levels of
PI(3,5)P2.
Previous work of Kearns et al. (25) suggested that
sac1 mutant strains had large increases in mannosyl
di-inositol diphosphorylceramide (M[IP]2C). Our
biochemical studies and the observation that TLC systems, such as those
used in the Kearns study, may not effectively resolve PIPs from
M[IP]2C suggested that the M[IP]2C spot may have been misidentified. Therefore, we analyzed the levels of phosphoinositides in sac1 mutant strains using a
deacylation/HPLC system. Analysis of groPIs from steady-state labeled
CTY182 (wild type) and CTY244 (sac1 Conservation of a Mg2+-independent Catalytic Motif
within Inositol and Protein Phosphatases--
SAC1-like
domains contain a highly conserved sequence motif, RTNCLDCLDRTN
(Fig. 4A). Within this
sequence is the CX5R(T/S) motif found in other
metal-independent protein and inositol polyphosphate phosphatases
including: the tyrosine and dual function protein phosphatases PTP1B
and VHR; the inositol polyphosphate 4-phosphatase family; and the
PI(3,4,5)P3 3-phosphatase encoded by the tumor suppressor
PTEN (36-38). The observed effects of metal and DTT on PPIPase
activity are consistent with the role of this motif in catalysis. These
biochemical and sequence similarities indicate a possible evolutionary
relationship among these proteins. Additionally, the lack of PPIPase
activity in both native and recombinant Inp5p is likely explained by
the presence of extensive mutations within this motif to RisafDsiekpN.
Most notable are the mutations in the consensus residues C Concerted and Independent Action of PPIPase Activity--
The
assignment of PPIPase activity to SAC1-like domains
represents a significant step forward in understanding how its
substrates or products may regulate vesicle trafficking, actin
organization, and other cellular events as summarized in Fig.
4B. Loss of PPIPase activity in sac1 mutants
results in profound changes in the cellular levels of
phosphoinositides, especially PI(4)P. In the case of all the "sec14
bypass" mutants, it is thought that the common downstream component
is expansion of the diacylglycerol pool (25). The accumulation of
phosphoinositides in sac1 mutants may expand the pool of
diacylglycerol directly through Plc1p-mediated turnover of PI(4)P or
indirectly through alterations in novel phosphoinositide-regulated lipid metabolic pathways that lead to increased diacylglycerol production. Although the largest metabolic changes are observed in
PI(4)P, significant changes are also found in PI(3)P and
PI(3,5)P2. Thus, it is possible that these lipids regulate
events related to diacylglycerol production. The ability of defects in
Sac1p and presumably PPIPase activity to overcome defects in
act1-1 mutant is intriguing. Much focus has been placed on
PI(4,5)P2 in regulating actin cytoskeleton; however, our
data indicate levels of this lipid do not significantly change in
sac1 mutants. Thus we suggest a novel role for PI(4)P,
PI(3)P, and/or PI(3,5)P2 in regulating actin organization.
Furthermore, our work provides insight into possible roles for PPIPase
activity and phosphoinositides in the regulation of ATP transport into
the lumen of the endoplasmic reticulum (27).
The fact that Inp52p, Inp53p, and synaptojanin possess both PPIPase and
5-ptase activities is novel and necessitates re-evaluation of the
relative roles of PI(4,5)P2 versus PI(4)P,
PI(3)P, and PI(3,5)P2 in synaptic and general vesicle
trafficking. It is intriguing to speculate as to why the two catalytic
domains are found in nature to be tethered or alone (Fig.
4B). One possible reason may be that tethering enables
localized substrate hydrolysis. In a concerted model, the 5-ptase
domain may hydrolyze PI(4,5)P2 to produce PI(4)P, signal
event one, which is then sequentially converted to PI by the
SAC1-like domain, signal event two. These events may occur
rapidly or slowly awaiting the appropriate regulatory queues.
Alternatively, the lipid substrates for 5-ptase and PPIPase activities
may be independent and/or localized at different membranes. To this
end, each activity may be involved at different steps in vesicle
trafficking, for example one during vesicle formation and the other
during vesicle docking or fusion. Perhaps PPIPase activity functions to
reduce polyanion charge facilitating changes in protein recruitment or
by directly reducing anionic lipid-lipid repulsion forces encountered
during membrane fusion. Additionally, PPIPase activity may be required
to replenish PI stores that are critical for re-initiation of signaling
events and general membrane homeostasis. Importantly, the distinct
biochemical properties and substrate selectivity of each domain
indicate multiple points of potential regulation, thereby enabling
combinatorial signaling modes for proteins such as Inp52p, Inp53p, and
synaptojanin. Clearly, both 5-ptase and PPIPase activities are
important in regulating membrane trafficking. It is interesting that
Inp51p and certain alternative splice variants of synaptojanin have
lost the PPIPase functional domain.
Lastly, our report represents the first description at the molecular
level of a PI(3)P 3-phosphatase, a PI(4)P 4-phosphatase, a
PI(3,5)P2 3-/5-phosphatase, and collectively a PPIPase. Of
significant recent interest is the regulation of PI(3,5)P2.
PI(3,5)P2 is transiently present during osmotic stress
(32), and its synthesis by Fab1p, a PI(3)P 5-kinase, may be involved in
delivering vacuolar proteins via endosome pathway, especially the
formation of multi-vesicular bodies (MVB) (7-10). The osmo-sensitive
phenotype and abnormal vacuolar structure of certain inp5
mutants suggest their involvement in the regulation of
PI(3,5)P2 (19-21). Our data demonstrate that the PPIPase
activity of Inp52p, Inp53p, synaptojanin, and Sac1p are biologically
relevant regulators of PI(3,5)P2. Whether or not any of
these proteins and PPIPase activity plays a role in regulation of MVB
formation or MVB recycling remains to be established.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Yeast strains used in this study
containing 2% raffinose to an A600 of 0.2, induced by the addition of galactose (2% final) for 6 h and
harvested. Soluble extracts were prepared from 1 × 108 cells by bead beating in 200 µl of 250 mM
Tris-HCl, pH 7.5, supplemented with 25 mM EDTA, 5 mM sodium pyrophosphate, 750 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mM
NaVO4.
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Phosphoinositide substrate selectivity of
Inp51p, Inp52p, and Inp53p overproduced in yeast. Native Inp5ps
were analyzed for activity toward PI, PI(3)P, PI(4)P,
PI(3,5)P2, and PI(4,5)P2 (total cell substrate
assay). Radiolabeled substrates were presented as either mixed vesicle
(A) or detergent micelle (B). Approximately 1 ng
of individual Inp5ps was incubated with substrate in the presence of
Mg2+. Reactants were extracted, deacylated, and separated
by HPLC. Similar results were obtained in a minimum of three
independent experiments, and a representative radio trace is shown.
Elution position of various groPIs derived from PIs are appropriately
labeled.
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Fig. 2.
PPIPase and 5-ptase activity of recombinant
Inp53p are attributed to SAC1-like and 5-ptase
domains, respectively. Approximately 1 ng of purified Inp53p
full-length (GST53), 5-ptase domain only (GST53-5pt),
SAC1-like domain only (GST53-Sac), or control GST only (1 µg) were analyzed using total cell substrate assay (A),
purified PIP2 assay (B), or PIP assay
(C). A, enzymatic activity of Inp53p domains
toward yeast total cellular lipid substrate. No detectable activity was
observed in vector control in the presence (shown) or absence of
Mg2+. B, activity of GST53, GST53-5pt, and
GST53-Sac proteins on purified PIP2 substrate.
C, GST53-Sac and GSTSyn-Sac activity on purified PIP
substrate. Mg2+ was omitted from the SAC1-like
domain reactions in B and C.
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Fig. 3.
Cellular levels of phosphoinositides in
inp52inp53 and sac1 mutant
yeast. A, wild type (black bar) or
inp52 inp53 mutant (stippled bar) yeast were
steady-state labeled at 20 µCi/ml 3H-inositol, groPIs
prepared from lipids extracted were subjected to HPLC analysis, and
radioactivity was quantified. The levels of PI(3,5)P2 are
reported. B, CTY182 (wild type, black bars) and
CTY244 (sac1 , stippled bars) strains were
labeled at 40 µCi/ml 3H-inositol, and individual groPIs
were quantified as described above. Results were compiled from at least
three independent experiments. ** and * indicate p values
from two-tailed Student's t test of <0.001 and <0.01,
respectively.
) strains lipids
demonstrate that sac1 mutants exhibit ~10-, 2.5-, and
2-fold increases in PI(4)P, PI(3,5)P2, and PI(3)P,
respectively (Fig. 3B). In contrast, levels of
PI(4,5)P2 did not significantly change. We then labeled
cells with [3H]serine, which labels ceramide but not PI
lipids, and found no changes in any inositol ceramide lipids (not
shown). Because inositol ceramides are resistant to cleavage by
methylamine, we analyzed the nondeacylatable lipids from inositol
labeled cells and again found no differences (not shown). Collectively,
these data demonstrate that polyphosphoinositides and not
M[IP]2C are regulated by Sac1p PPIPase activity. The
dramatic accumulation of PI(4)P in sac1 mutants suggest that
Sac1p regulates a major pool of PI(4)P, possibly at the Golgi.
Remarkably, despite the 10-fold increases in PI(4)P, the levels of
PI(4,5)P2 did not increase, if anything they appear to
decrease slightly (Fig. 3B). It is therefore possible that the pool of PI(4)P utilized by Sac1p is different or sequestered from a
pool used to make PI(4,5)P2. This may occur through spatial sequestration or by a protein masking as was recently proposed by
Stevenson et al. (35). Alternatively, the levels of
PI(4,5)P2 may be maintained by feedback mechanisms
controlling phosphatase, lipase, and/or kinase activities. It is also
noteworthy that upon deletion of Fig4p, another yeast
SAC1-like domain-containing protein, only small changes were
observed in steady-state phosphoinositide levels (not shown). One
explanation may be that the pool of lipid that Fig4p accesses is
relatively small or tightly regulated.
A, R
K, and (T/S)
P.
View larger version (38K):
[in a new window]
Fig. 4.
Conserved catalytic motif and regulation of
phosphoinositides by proteins harboring SAC1-like
domains. A, putative SAC1-like and
Mg2+-independent inositol and protein phosphatase catalytic
motifs. Sequences were aligned around the RTNCLDCLDRTN motif of dual
function 5-ptase/PPIPases, Inp52, Inp53, and synaptojanin, PPIPases,
Kiaa0274p, and a CX5R(T/S) motif found in an
protein or lipid phosphatases (PTP1B and VHR and
protein/PIP3 3-phosphatase (PTEN)) and a inositol
polyphosphate 4-phosphatase family (4ptase I and II, SopB and Ipg).
B, a model for concerted and independent mechanisms of a
PPIPase and 5-ptase activities in regulating phosphoinositides.
SAC1-like domains may exist alone as in Sac1p, Fig4p, and
Kiaa0274p enabling hydrolysis of PI(4)P, PI(3)P, and
PI(3,5)P2 to PI. 5-ptase domains are also found alone in
Inp51p, Inp54p, OCRL-l, and type I and II 5-ptases, which hydrolyze
PI(4,5)P2 to PI(4)P. Alternatively, SAC1-like
and 5-ptase domains are found tethered via an unconserved sequence
(indicated by the dashed line), as in Inp52p, Inp53p, and
synaptojanin. Some of the proposed roles in cell signaling and possible
routes of concerted or independent metabolism of relevant
phosphoinositides are designated.
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ACKNOWLEDGEMENTS |
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We thank members of the York and Shirish Shenolikar labs for helpful advice and discussions.
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FOOTNOTES |
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* This work was supported by a Burroughs Wellcome Fund Career Award in the Biomedical Sciences, a Whitehead Scholar Award, and National Institutes of Health R01-HL 55672.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology
and Cancer Biology, Duke University Medical Center, DUMC 3813, Durham,
NC 27710. Tel.: 919-681-6414; Fax: 919-684-8922; E-mail:
yorkj{at}acpub.duke.edu.
2 S. Guo and J. D. York, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: PI, phosphatidylinositol; PI(4, 5)P2, phosphatidylinositol (4,5)-bisphosphate; PI(3)P, phosphatidylinositol 3-phosphate; PI(4)P, phosphatidylinositol 4-phosphate; PI(3, 5)P2, phosphatidylinositol (3,5)-bisphosphate; PI(3, 4,5)P3, phosphatidylinositol 3,4,5-triphosphate; M[IP]2C, mannosyl di-inositol diphosphorylceramide; 5-ptase, inositol polyphosphate 5-phosphatase; PPIPase, polyphosphoinositide phosphatase; AP, ammonium phosphate; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; groPI, glycerophosphoinositols; PIP, phosphatidylinositol monophosphate; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; GST, glutathione S-transferase; DTT, dithiothreitol; MVB, multi-vesicular bodies.
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REFERENCES |
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