From the Departments of Pharmacology and Cancer Biology and of Biochemistry, Duke Medical Center, Durham, North Carolina 27710
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sequence analysis of Saccharomyces cerevisiae chromosome IX identified a 946 amino acid open reading frame (YIL002C), designated here as INP51, that has carboxyl- and amino-terminal regions similar to mammalian inositol polyphosphate 5-phosphatases and to yeast SAC1. This two-domain primary structure resembles the mammalian 5-phosphatase, synaptojanin. We report that Inp51p is associated with a particulate fraction and that recombinant Inp51p exhibits intrinsic phosphatidylinositol 4,5-bisphosphate 5-phosphatase activity. Deletion of INP51 (inp51) results in a "cold-tolerant" phenotype, enabling significantly faster growth at temperatures below 15 °C as compared with a parental strain. Complementation analysis of an inp51 mutant strain demonstrates that the cold tolerance is strictly due to loss of 5-phosphatase catalytic activity. Furthermore, deletion of PLC1 in an inp51 mutant does not abrogate cold tolerance, indicating that Plc1p-mediated production of soluble inositol phosphates is not required. Cells lacking INP51 have a 2-4-fold increase in levels of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, whereas cells overexpressing Inp51p exhibit a 35% decrease in levels of phosphatidylinositol 4,5-bisphosphate. We conclude that INP51 function is critical for proper phosphatidylinositol 4,5-bisphosphate homeostasis. In addition, we define a novel role for a 5-phosphatase loss of function mutant that improves the growth of cells at colder temperatures without alteration of growth at normal temperatures, which may have useful commercial applications.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Of central importance to the inositol signaling pathway is
phosphatidylinositol 4,5-bisphosphate
(PI(4,5)P2),1
which serves as a precursor to second messengers and is a signaling molecule itself (1-3). PI(4,5)P2 has a direct functional
role in signaling via interactions with actin-binding proteins,
phospholipase D, and pleckstrin homology domains (3-7). Levels of
PI(4,5)P2 are tightly maintained by a balance of kinase,
lipase, transferase, and phosphatase activities. The importance of this
regulation is manifested by disease states that arise as a result of
mutation in these enzymes. For example, in Drosophila
deletion of the norpA phospholipase C gene results in
blindness (8); ablation of an eye-specific CDP-diacylglycerol
synthetase results in retinal degeneration that can be
pharmacologically rescued by re-addition of PI(4,5)P2 (9);
and recent studies by Milligan et al. (10) demonstrate that
the phosphatidylinositol transfer protein domain of the
rdgB gene is necessary for proper photoreceptor function in
response to prolonged light stimuli and serves to protect photoreceptor degeneration. In addition, the human disease oculocerebrorenal syndrome
(Lowe's syndrome) arises from mutations in the OCRL-1 gene
(11), which encodes an inositol polyphosphate 5-phosphatase (5-ptase)
(12).
5-Ptase enzymes comprise a family of critical proteins that remove the 5-position phosphate from either soluble or lipid inositol polyphosphates, or both (reviewed in Refs. 13 and 14). Initially, types I and II 5-ptases were postulated to terminate inositol 1,4,5-trisphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate-mediated Ca2+ mobilization (15, 16). However, recent characterization of several new 5-ptases provides evidence for their expanded role in cell function, including vesicular trafficking and Ras and cytokine signaling (13, 17). These members include OCRL-1, synaptojanin (18), GA-5pt (19), p150SHIP (20, 21), and PIP35pt (22). The mammalian 5-ptase family consists of at least 10 distinct members and is identified by two short peptide motifs, "(i/l)W(l/f/m)GD(l/f)D(y/f)R" and "P(a/s)W(c/t/s)DRIL" (23). Mutational analyses of these residues demonstrate their importance for substrate recognition and catalysis (23-25). It is unclear why so many 5-ptases exist and why in the case of Lowe's syndrome other 5-ptases are unable to compensate for defects in OCRL-1.
To understand better the roles of 5-ptases in cell growth and in inositol metabolism in vivo, we initiated studies in a genetically tractable yeast Saccharomyces cerevisiae. Several genes involved in yeast inositol lipid biosynthesis and metabolism have been identified (see reviews in Refs. 26 and 27). In contrast, genes encoding inositol lipid phosphatases have not yet been defined. Importantly, the yeast genome sequencing project has enabled identification of open reading frames sharing similarity to mammalian inositol signaling genes. One such open reading frame, YIL002c, designated here as INP51, is highly similar to mammalian 5-ptases. We demonstrate that INP51 encodes a 108-kDa protein which has intrinsic PI(4,5)P2 5-ptase activity. Characterization of both loss and gain of function mutants of inp51 demonstrates that 5-ptase activity is required for proper in vivo PI(4,5)P2 maintenance. Our studies also suggest a role for Inp51p-regulated inositol metabolites in the growth of yeast at cold temperatures.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strains, Media, and Genetic Methods-- Yeast strains used in this study are listed in Table I. The cells were propagated in standard rich (YPD) medium or in complete minimal medium (CM) containing either 2% glucose or galactose and, when appropriate, lacking the nutrient(s) to maintain selection for plasmids or markers. Standard procedures for yeast genetic manipulations were used (28, 29). Strains were sporulated on 0.3% potassium acetate plates, and tetrads were dissected using a Zeiss Micromanipulator. The ability of a given yeast strain to propagate at temperatures below 15 °C was assessed by dispersing cells onto an appropriate solid media plate and, after incubation for 2 weeks, observing the resulting growth either visually or under a dissecting microscope, as appropriate.
|
Targeted Gene Disruption and Complementation of INP51--
The
INP51 gene was obtained from cosmid 9687 (the gift of Mark
Johnston, Washington University, St. Louis) which contained a 21,431-bp
segment of chromosome IX. The cosmid DNA was digested with
SacI and PstI endonucleases, and the 3.8-kb
fragment containing the INP51 locus was purified and
subcloned into pBluescript II SK + (Stratagene, La Jolla, CA) to yield
pBluINP51. The majority of the INP51 gene was
removed and replaced with the selectable marker LEU2 (Fig.
1B). The LEU2 gene, obtained from an
XbaI/PstI digestion of plasmid pJJ282 (obtained
from Susan Wente, Washington University), was inserted at these sites
into pBluINP51, to yield pinp51::LEU2.
This plasmid DNA was linearized and transformed into W303
(MATa/MAT) cells (obtained from
Susan Wente) by the standard Li+ acetate protocol (28).
Stable Leu+ transformants were selected on
Leu
plates and verified by Southern blot analysis (28) of
genomic DNA digested with KpnI and PvuII
endonucleases and probed with a radiolabeled 1.2-kb INP51
SacI/XbaI fragment (see Fig. 1B).
Inp51 Antibody Generation and Western Analysis-- A carboxyl-terminal peptide of Inp51p having the sequence "CVENEDEPLFIER" was chemically synthesized (Protein and Nucleic Acid Chemistry Lab, Washington University, St. Louis) and injected into rabbits using standard protocols (Pocono Rabbit Farm, Canadensis, PA). Immunoreactive antisera was affinity purified using peptide sulfolink-agarose gel (prepared according to Pierce)
Soluble and particulate protein fractions were made from appropriate haploid yeast strains as follows. Approximately 3 × 108 cells were harvested at logarithmic phase by centrifugation (2,000 × g), washed twice in 1 ml of ice-cold water, and resuspended in 250 µl of 50 mM HEPES, pH 7.5, supplemented with 10 mM MgCl2, 50 mM KCl, and 1 mM phenylmethylsulfonyl fluoride (Lysis buffer). Cells were lysed by addition of 100 µl of acid-washed glass beads (425-600 µm diameter, Sigma) followed by vigorous reciprocal shaking (Mini Bead-BeaterTM, BioSpec, Inc., Bartlesville, OK) for 2 min. Lysates were centrifuged at 20,800 × g for 5 min, and the upper soluble fraction was transferred to a new tube. The particulate fraction was resuspended in 250 µl Lysis buffer and homogenized by vigorous reciprocal shaking for 2 min. Proteins were separated by SDS-PAGE, immobilized onto nitrocellulose filters by electroblotting, and immunoblotted using affinity purified anti-Inp51p antibodies according to standard protocols (28).Bacterial Expression of INP51 and Enzymatic Analysis-- pBluINP51 was digested with XbaI and PstI, releasing a 2610-bp partial INP51 fragment encoding residues 163-946 that was gel-purified. INP51 residues 1-162 were made by PCR amplification of pBluINP51 template using sense and antisense primers, 5'-AGCTTGAGGATCCAAAATGAGACTCTTCATCGGTAGAAGA-3' and 5'-AGTGCGTATCTAGAATAGTAGAATGTTCCATCACT-3', under conditions recommended by the manufacturer. The 516-bp product was digested with BamHI and XbaI, gel-purified, and co-inserted with the 2610-bp XbaI/PstI fragment into the BamHI and PstI sites of pCMV (31) to make pCMVINP51. The region generated by PCR was sequenced to confirm that no unwanted mutations were introduced. pCMVINP51 was digested with BamHI and PstI, and the 3.2-kb fragment containing the entire INP51 coding sequence was inserted into pTrcHisA (Invitrogen, Carlsbad, CA) at these sites to yield pTrcINP51.
Fifty-milliliter cultures of DH5Inositol Radiolabeling of Yeast Strains--
Approximately
1 × 105 cells were inoculated into 1 ml of
appropriately modified CM containing 10-20 µCi of
[3H]myoinositol (American Radiolabel Corp., St. Louis,
MO) or YPD containing 50 µCi/ml [3H]myoinositol, grown
to a density of 3 × 107 cells/ml, harvested by
centrifugation, washed twice with 1 ml of ice-cold water, and
resuspended in 100 µl of 0.5 N HCl. Extraction of soluble
and lipid inositol phosphates was performed by adding 372 µl of
chloroform/methanol (1:2 v/v) and 100 µl of glass beads, bead beating
for 2 min, adding 125 µl each of chloroform and 2 M KCl,
bead beating again for 2 min, and centrifuging at 20,800 × g for 5 min. The upper and lower phases containing
water-soluble and lipid inositol phosphates, respectively, were
separated and stored at 80 °C no longer than 3 days prior to
use.
Separation of Radiolabeled Inositol Phosphates-- Silica-60 TLC plates (20 × 20 cm, Merck) were prepared by submersion in 54 mM potassium oxalate containing 47.5% ethanol, 2 mM EDTA and baked a minimum of 60 min at 100 °C prior to use. TLC plates were developed in a solvent mixture of 160 ml of chloroform, 60 ml of acetone, 52 ml of methanol, 48 ml of glacial acetic acid, and 28 ml of water. Radiolabeled lipids were detected by autoradiography after spraying plates with En[3H]ance (DuPont) and quantified by scraping appropriate regions of silica into 10 ml of scintillation fluid and counting after equilibration for 10 h.
Alternatively, crude or TLC-separated radiolabeled lipids were subjected to deacylation (32), and resulting glycerophosphoinositols (groPI) were analyzed by HPLC. Crude radiolabeled lipids were dried by vacuum centrifugation and incubated with 300 µl of methylamine reagent (prepared by bubbling gaseous methylamine (CH3NH2, Fluka, Buchs, Switzerland) into ice-cold 6.2 ml of methanol, 4.6 ml of H2O, and 1.5 ml of 1-butanol until the total volume reached 20 ml) at 53 °C for 30 min and vortexing every 10 min. Cold 1-propanol (150 µl) was added, and the mixture was vacuum centrifuged to dryness. The residue was resuspended in 200 µl of H2O, extracted twice with 300 µl of 1-butanol/petroleum ether/ethyl formate (20:4:1 v/v/v), each time discarding the upper layer, and stored atOCRL-1 Digestion of Radiolabeled PI(4,5)P2 Obtained from inp51 Mutant Cells-- Approximately 10,000 cpm of TLC-purified [3H]PI(4,5)P2 derived from radiolabeled inp51 mutant cells was combined with 100 µg of PI, [32P]PI(3)P, and [32P]PI(3,4)P2 standards and dried under a steady N2 stream, and the residue was sonicated briefly in 100 µl of 50 mM MES/Tris, pH 6.5, containing 5 mM MgCl2. Lipid substrate was incubated with 100 ng of purified recombinant OCRL-1 5-ptase (obtained from Xiaoling Zhang, Washington University) for 30 min at 37 °C. Reactions were stopped by the addition of 300 µl of methylamine reagent. The products were then deacylated and characterized by Partisil SAX HPLC as detailed above.
Overexpression of Inp51 in Yeast--
pCMVINP51 was
digested with BamHI and SacII, releasing a 3.2-kb
fragment containing the complete coding sequence which was inserted
into pBJ246 (ATCC 777452) at these sites to yield
pBJ246INP51. pBJ246INP51 or pBJ246
plasmids were transformed into the haploid W303 by the standard
Li+ acetate protocol (28). Ura+ strains were
grown in 1 ml of CM lacking uracil (CM/ura) supplemented
with 2% glucose to a density of 5 × 107 cells/ml,
washed 3 times in 1 ml of CM/ura
without sugar,
resuspended in 1 ml of CM/ura
supplemented with either
2% glucose (uninduced) or 2% galactose (induced), and grown for
9 h at 30 °C. Cellular extracts were separated by SDS-PAGE and
analyzed by immunoblotting as described above. For inositol labeling
studies the growth media were supplemented with 10 µCi/ml
[3H]myoinositol, and radiolabeled lipids were analyzed by
TLC as described above.
Disruption of PLC1-- The complete coding region of PLC1 was replaced with the G418 gene using a PCR-based approach. G418 was amplified from plasmid pFA6-kanMX2 (34) by PCR using the sense primer 5'-AAACGTACAACGGTAAGGTCATTCACGCAGTGTATATGCAGCTGAAGCTTCGTACGC-3' and the antisense primer 5'-CGTATTTATGAATATGTGTATTTGGCCGGAAAAAGATCGCCGCATAGGCCACTAGTGGATCTG-3' (obtained from Joseph Heitman, Duke University) (where underlined bases correspond to PLC1 sequence and the remainder of the primer is from the pFA6-kanMX2 plasmid) according to conditions recommended by the manufacturer. The resulting plc1::G418 PCR product was introduced into diploid cells by the standard Li+ acetate protocol (28). Stable G418R cells were selected for on YPD plates supplemented with 200 µg/ml G418 (Calbiochem) and verified by PCR of genomic DNA template using the above sense primer and an antisense primer 5'-ATATACTCGAGAACTTTCTGCTGAAGAGAAGGC-3' (obtained from Joseph Heitman) derived from the 3'-untranslated region of the PLC1 locus.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of an Inositol Polyphosphate 5-Phosphatase in
Yeast--
The TBLASTN search algorithm (35) was used to compare the
amino acid sequences of the mammalian 5-ptases, type I, type II, and
OCRL-1 (GenBankTM accession numbers Z31695, M74161, and
M88162, respectively) to the predicted open reading frames in S. cerevisiae genomic sequence (available January, 1995). We
identified a highly related open reading frame corresponding to locus
YIL002c, located on chromosome IX, encoding a 946-residue gene product,
designated here as INP51 for INositol
polyphosphate 5-Phosphatase number 1. The carboxyl-terminal region of Inp51p is approximately
30% identical (>50% similar) over 300 amino acids to the catalytic domains of type II and OCRL-1 5-ptases. Importantly, Inp51p contains the two short sequence motifs common to all 5-ptases characterized to
date (Fig. 1A). Two
substitutions of the "PAWCDRILW" sequence of type II and OCRL-1 are
found in Inp51p, Cys787 Thr and Trp792
Ser. In addition, the amino-terminal half of Inp51p has significant similarity to the S. cerevisiae 623-residue SAC1
and the predicted 879-residue YNL325c gene products (Fig.
1A). Although SAC1 is not an essential gene,
sac1
mutants are inositol auxotrophs and are
cold-sensitive; moreover, particular sac1 alleles suppress certain act1ts alleles (36) and a
sac1
mutation bypasses the effects of the sec14-1ts mutation (37, 38). The segment of
Inp51p with the greatest similarity to SacI
(RTNCMDCLDRTN motif in Fig. 1A) corresponds to
the portion of SAC1 that when altered enables suppression of act1 and sec14 mutations (37, 39). For example,
sac1-8 and sac1-22 mutant alleles were found to
have a Asp337 to Asn mutation (39). The YNL325c gene
product acts as a dosage suppressor of sac1 mutations,
although a ynl325c null mutation does not bypass
sec14 mutations (39). The two-domain structure of Inp51p
most resembles the mammalian 5-ptase synaptojanin, a protein associated
with synaptic vesicles (Fig. 1A).
|
|
Inp51 Has Intrinsic 5-Phosphatase Activity-- Based on the similarity of INP51 to synaptojanin, OCRL-1, and type II mammalian 5-ptases, we predicted that the Inp51p may function as a dual specificity phosphatase that hydrolyzes both lipid and soluble inositol polyphosphates. In the yeast S. cerevisiae, both putative substrates IP3 and PI(4,5)P2 have been identified (40-42). Initially, we measured 5-ptase activity in extracts prepared from either haploid wild type derived from parental strain or inp51 mutants using IP3 or PI(4,5)P2 as substrates. The only detectable 5-ptase activity we observed was against the lipid substrate (data not shown). The inability to find 5-ptase activity using IP3 was puzzling in light of metabolism in mammalian cells but not unexpected as others have previously reported that yeast do not contain IP3 5-phosphomonoesterase activity (43).
In order to verify that Inp51p harbored intrinsic 5-ptase activity, we heterologously expressed recombinant protein in the bacteria. Bacteria are especially useful because they lack measurable 5-ptase activity. The coding sequence of the INP51 gene was inserted into an isopropyl-1-thio-Loss of INP51 5-Ptase Function Results in a Cold-resistant Phenotype-- The cold-sensitive phenotype of sac1 mutants and the possibility that INP51 may regulate levels of a lipid substrate prompted us to analyze growth of an inp51 mutant at temperatures ranging from 12 to 37 °C. Haploid cells derived from the wild-type parental strain and inp51 mutant strains were dispersed onto solid media and incubated at temperatures of 12, 14, 23, 30, and 37 °C for appropriate times. No differences in the size or numbers of colonies were observed between these strains at 23 °C and above (Fig. 3A). In contrast, after 14 days at 12 or 14 °C, we observed that the parental wild-type cells are unable to grow, whereas the inp51 mutants are able to grow as single colonies (Fig. 3A, W303a and JYY3).
|
Inp51 Regulates Levels of PIP2 and IP3 in Vivo-- We next determined the effects of INP51 loss of function on metabolism of lipid and soluble inositol phosphates in vivo. Appropriate yeast strains were labeled to steady state with [3H]inositol such that radioactivity reflected the relative mass levels of various inositol phosphates (labeling throughout eight cell doublings was sufficient to reach equilibrium). Radiolabeled lipids were isolated from haploid inp51 mutants, parental-derived wild-type, inp51 pRSINP51, and inp51 pRSINP51D788A strains, separated by oxalate TLC, and visualized by autoradiography. Five major and several minor lipid species are present in all four strains (Fig. 4A). Studies of inositol incorporation into phospholipids (PL) by Smith and Lester (44) demonstrated that S. cerevisiae possesses PI, three inositol phosphorylceramides (IPC-I, -II, and -III), a mannosyl-inositol phosphorylceramide (MIPC), a mannosyldi-inositol diphosphorylceramide (M[IP]2C), PIP, and PIP2. Based on migration of appropriate standards, we have assigned the five major species to include PI (labeled as such), IPC-I, and IPC-II (spot a), IPC-III and MIPC (spot b), M[IP]2C and PIP (spot c), and PIP2 (labeled as such). Visual inspection of the five major inositol PL in these strains shows that deletion of INP51 results in an accumulation of PIP2 (lane 1), whereas levels of other lipids do not appear to change. Inspection of PL in the complemented strains demonstrates that introduction of Inp51p (lane 3) but not Inp51pD788A (lane 4) into the inp51 mutant restores PIP2 levels to those observed in wild type (lane 2). Radioactivity in regions of silica corresponding to PI and PIP2 were quantified from several independent experiments (Fig. 4B). Amounts of PI recovered were not significantly different in any of the strains. In contrast, levels of PIP2 increased 1.9-fold in an inp51 mutant (sample 1) as compared with parental wild type (sample 2); moreover, this increase was reverted by introduction of INP51 (sample 3) but not INP51D788A (sample 4). These data confirm that accumulation of PIP2 requires loss of Inp51p 5-ptase activity. Additionally, similar patterns of PIP2 lipid accumulation were observed in these strains grown at 12 °C.
|
|
|
Cold Tolerance Does Not Require IP3 Produced via a
Plc1p Pathway--
Since loss of INP51 function leads to
increases in both PI(4,5)P2 and IP3, we were
interested in determining whether phospholipase C activity is necessary
for cold resistance. In yeast PLC1 encodes a
PI-phospholipase C that hydrolyzes PI(4,5)P2 to form
diacylglycerol and IP3 (45-47). TBLASTN comparison of the
PLC1 sequence with the complete S. cerevisiae
genome fails to identify another significantly related gene product
indicating that a single gene is present. Therefore, we constructed
plc1::G418 single and
plc1
::G418 inp51
::LEU2 double
mutants as described under "Materials and Methods." Cells from
these strains as well as from parental derived wild-type and
inp51 mutant strains were dispersed onto solid media and
grown at 23 and 12 °C for 3 and 12 days. Consistent with previously published data at 23 °C (45), on rich medium plc1
deletion results in a partially growth-compromised phenotype; moreover,
the plc1 inp51 double mutant did not exhibit a significantly
improved growth rate (data not shown). To quantify better the
cold-resistant phenotype, we photographed regions of single colonies of
strains grown at 12 °C using a dissecting microscope (Fig.
7). We observed very slow growth in both
wild-type (A) and plc1 mutant (B)
cells, which have an average colony diameter (obtained from at least
five individual colonies) of 19 ± 0.3 and 13 ± 0.4 µm
(p < 0.01 determined by two-tailed Student's
t test). In marked contrast, the average colony diameter of
inp51 mutant cells is 197.5 ± 45 µm, whereas that of
inp51 plc1 double mutant cells is 102.5 ± 13.9 µm
(p < 0.001 for each as compared with wild type). These
data demonstrate that production of IP3 via a Plc1p pathway
is not required for cold resistance induced by Inp51 loss of
function.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Many PI biosynthetic and metabolic enzymes are conserved from yeast to man. In the yeast S. cerevisiae, inositol is utilized to form PI, PI(3)P, PI(4)P, and PI(4,5)P2 (reviewed in Ref. 26) as well as IPC-I, IPC-II, IPC-III, MIPC, and M(IP)2C (see review in Ref. 48). Genes encoding synthase, kinase, and lipase enzymes have been cloned from S. cerevisiae (reviewed in Refs. 26 and 27) including inositol 1-phosphate synthase, INO1 (49); PI synthase, PIS1 (50, 51); PI 4-kinases, PIK1 (52) and STT4 (53); PI(4)P 5-kinases, FAB1 and MSS4 based on strong homology to mammalian proteins (54-56); PI 3-kinase, VPS34 (57); and phospholipase C, PLC1 (45-47). Sequencing the genomes of a multitude of organisms has resulted in the powerful ability to identify gene families conserved throughout evolution. We used this information to identify a 5-ptase, INP51, in the yeast S. cerevisiae.
Our studies indicate that INP51 encodes a lipid-selective 5-ptase whose activity is critically required to maintain proper cellular levels of PI(4,5)P2. In mammalian cells, several distinct 5-ptases have been classified into four groups based mainly on substrate selectivity (58) as follows: group I 5-ptases utilize only soluble inositol polyphosphates; group II enzymes utilize both lipid and soluble substrates; group III enzymes associate with PI 3-kinase and only utilize PI(3,4,5)P3; and group IV enzymes associate with tyrosine-phosphorylated growth factor receptors and appear to utilize lipid and soluble substrates containing a D3 position phosphate. The domain structure of Inp51p indicates it is most like synaptojanin, a group II 5-ptase that hydrolyzes both PI(4,5)P2 and IP3 in vitro (18). However, since Inp51p does not utilize IP3 in vitro, we suggest it should not be strictly classified as a group II enzyme and may represent a novel PI(4,5)P2-selective class. In vivo studies of inositol polyphosphate levels in cells lacking Inp51p show measurable increases in the levels of PI(4,5)P2 and IP3. The increase in IP3 in inp51 mutant cells is most likely a mass action effect that results from an accumulation of PIP2. The lack of measurable IP3 5-ptase activity from either recombinant Inp51p or parental yeast cell extracts supports this hypothesis. Furthermore, overexpression of Inp51p decreases levels of PI(4,5)P2. Together these data clearly show that changes in cellular 5-ptase activity directly contribute to PI(4,5)P2 homeostasis.
The importance of PI(4,5)P2 in cell survival and as a regulator of several enzymes is well documented. Individual deletion of genes required for PI(4,5)P2 synthesis including PI synthase PIS1, PI 4-kinases PIK1 or STT4, or putative PI(4)P 5-kinase MSS4 results in lethality. Moreover, electroporation of PI(4,5)P2-specific antibodies into yeast potently inhibits cell growth (59). Additionally, PI(4,5)P2 modulates the activities of phospholipase D and certain actin-binding proteins such as profilin. Since only small changes in PI(4,5)P2 are observed in loss or gain of function inp51 mutant cells, mechanisms may exist that are capable of sensing PI(4,5)P2 levels enabling feedback compensation for changes in 5-ptase activity, for example by alterations in PI synthase, kinase, lipase, and/or other phosphatase activities. Alternatively, the small changes may reflect the possibility that Inp51p is restricted to hydrolyzing a distinct cellular pool of PI(4,5)P2 via compartmentalization or sequestration. Indeed, upon completion of the S. cerevisiae genome two additional INP5-predicted gene products were identified, designated INP52 (YNL106c) and INP53 (YOR109w). All three genes share highly similar sequences as well as the synaptojanin-like two-domain structure. We report elsewhere a thorough genetic analysis of this gene family, and we show that simultaneous deletion of all three genes is lethal (75). During preparation of this manuscript, it came to our attention that an independent genetic study has also concluded that the INP51, INP52, and INP53 genes (which were designated SJL1, SJL2, and SJL3) constitute an essential gene family (76).
The significance of the SacI-like domain present in Inp51p
is currently unclear. Mutant sac1 alleles suppress the
secretory defects caused by certain actin
(act1ts) alleles (36) and suppress the Golgi
transport defects and inviability of a sec14ts
mutation (37). Sec14 is a PL transfer protein exhibiting specificity toward PI and phosphatidylcholine. SacI is an integral
membrane protein, and null mutations result in inositol auxotrophy and cold sensitivity (36, 38). Recently, Kearns et al. (39) demonstrated that defects in SacI bypass the requirement for
Sec14 by increasing the pool of diacylglycerol in Golgi membranes. It was also reported that deletion of INP51 failed to
"bypass" the requirement of Sec14 (39). However, it was initially
reported that bypass suppression of act1ts or
sec14ts was achieved by sac1 mutation
and not sac1 deletion (36, 37). Thus, perhaps similar
mutations in the SacI domain of INP51 (see Fig.
1), for example Asp Asn (residue 426 in INP51)
substitution as found in sac1-8 or sac1-22
alleles (39), would be capable of bypass suppression. We also note that
deletion of residues 2-490 of Inp51p results in a 2-fold increase in
PI(4,5)P2 accumulation in vivo indicating loss
of 5-ptase function, whereas a point mutation of the Asp426
Ala in the SacI domain of Inp51p does not (data not
shown). It is uncertain at this time whether the loss of function of
the SacI domain-less Inp51p is due to improper localization
or loss of intrinsic activity.
The accumulation of PI(4,5)P2 upon loss of INP51 5-ptase function correlates with the cold-tolerant phenotype. Whereas mutations in numerous genes have been shown to result in cold-sensitive phenotypes, we were unable to find reports in which loss of function causes cold tolerance. It is clear that this phenotype is due to loss of 5-ptase catalytic activity and does not require the production IP3 via Plc1p. It is also of interest that deletion of INP51 in a cold-resistant strain (W303JT) further enhances growth at cold temperatures. Previous genetic analysis of mutations affecting growth of S. cerevisiae at low temperatures (60) demonstrates that mutations in the tryptophan biosynthesis machinery results in cold sensitivity even in rich media containing tryptophan. Presumably a tryptophan permease, such as Tat2 (61), is not functional at cold temperatures. Since our strain carries a trp1-1 allele we analyzed tryptophan permease function by measuring tryptophan uptake at 12 °C. We observed that inp51 mutants had significantly increased rates of uptake as compared with the very slow uptake of our parental strain (data not shown).
How increases in PI(4,5)P2 may function to regulate cold tolerance is unclear. PI(4,5)P2 may cause this effect directly, although this seems unlikely given it represents only a minor fraction (0.01%) of the total yeast phospholipid. It may be that increases in PI(4,5)P2 lead to increases in downstream metabolites which mediate the cold resistance; however, our studies demonstrate that PLC1 is not involved. Alternatively, it is attractive to suggest that PI(4,5)P2 mediates its effects through a signaling pathway which may regulate lipid biosynthesis, cytoskeletal organization, and/or vesicular trafficking.
The ability to induce cold tolerance by loss of a single enzyme
function may have useful applications in agriculture and fermentation industries. In plants, defining the molecular bases for chilling tolerance is of significant academic and commercial interest (reviewed in Ref. 62). The cold tolerance of inp51 mutants appears
quite similar to chilling tolerance in Arabidopsis. One
hypothesis for chilling tolerance in plants centers around membrane
fluidity and/or permeability. Several studies in Arabidopsis
provide a correlation between increases in fatty acid unsaturation and
resistance to cold (63-65). For example, FAD2 is believed
to encode a desaturase, and a fad2 mutant of
Arabidopsis is cold-sensitive (65, 66). Moreover, studies of
cold adaptation in carp demonstrate an up-regulation of
9-desaturase activity upon cooling fish from 30 to
10 °C (67). In S. cerevisiae the gene OLE1
encodes a
9-desaturase (68) which may be of particular
interest to test with our inp51 mutants. In addition, it has
been shown that cold shock induces an immediate rise in intracellular
free Ca2+ in Arabidopsis (69). In yeast,
IP3 does not appear to be involved in Ca2+
signaling and regulation of intracellular Ca2+ (70-74).
Thus, it would be worth testing whether increases in Ca2+
concentration in our solid media result in changes in growth at
12 °C. Alternatively, "cold-shock" exposure of organisms results in dramatic changes in protein synthesis. We therefore analyzed Inp51p
levels in cells grown at 30 and 12 °C; however, no changes were
observed (data not shown). Regardless of the mechanism, cloning of
INP51 homologs in Arabidopsis and studies of loss
of 5-ptase function may provide important clues in defining a molecular
basis for chilling tolerance.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Susan Wente, Joseph Heitman, Daniel Lew, and Jeremy Thorner for guidance, advice, and reagents without which this work would not have proceeded. We also thank Jeffery Datto for technical help and Bryan Spiegelberg for critical review of the manuscript. This work was initiated at Washington University, Division of Hematology/Oncology, and we are thankful to the members of this Division for support, especially Philip Majerus and Stuart Kornfeld.
![]() |
FOOTNOTES |
---|
* This work was supported by a Burroughs Wellcome Fund Career award in the Biomedical Sciences, a Whitehead Scholar award, funds from Merck Research Labs, and the National Institutes of Health Grant 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 the correspondence should be addressed: Depts. of
Pharmacology and Cancer Biology and of Biochemistry, Duke Medical Center, DUMC 3813, Durham, NC 27710. Tel.: 919-681-6414; Fax: 919-684-8922; E-mail: yorkj{at}acpub.duke.edu.
1 The abbreviations used are: PI(4,5)P2, phosphatidylinositol (4,5)-bisphosphate; IP3, inositol 1,4,5-trisphosphate; 5-ptase, 5-phosphatase; PIP, phosphatidylinositol bisphosphate; MIPC, mannosyl-inositol phosphorylceramide; M[IP]2C, mannosyldi-inositol diphosphorylceramide; IPC, inositol phosphorylceramides; bp, base pair; kb, kilobase pair; MES, 4-morpholineethanesulfonic acid; AF, ammonium formate; AP, ammonium phosphate; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; HPLC, high pressure liquid chromatography; groPI, glycerophosphoinositols; PL, phospholipids.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|