From the Department of Biochemistry and Molecular
Biology, Monash University, Clayton, Victoria 3800, Australia, the
PeterMacCallum Cancer Institute, East Melbourne, Victoria 3002, Australia, and the ** Department of Biochemistry and Molecular Biology,
Melbourne University, Parkville, Victoria 3052, Australia
Received for publication, November 20, 2000, and in revised form, December 13, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The budding yeast Saccharomyces
cerevisiae has four inositol polyphosphate 5-phosphatase
(5-phosphatase) genes, INP51, INP52, INP53, and
INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which
consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich
region found in the other three yeast 5-phosphatases. We report that
Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The
hydrophobic tail comprises the last 13 amino acids of the protein and
is sufficient to target green fluorescent protein to the endoplasmic
reticulum. Protease protection assays demonstrated that the N terminus
of Inp54p is oriented toward the cytoplasm of the cell, with the C
terminus of the protein also exposed to the cytosol. Null mutation of
INP54 resulted in a 2-fold increase in secretion of a
reporter protein, compared with wild-type yeast or cells deleted for
any of the SacI domain-containing 5-phosphatases. We
propose that Inp54p plays a role in regulating secretion, possibly by
modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the
cytoplasmic surface of the endoplasmic reticulum membrane.
Phosphoinositides are ubiquitous membrane components of various
intracellular compartments, which regulate many diverse cellular functions including membrane trafficking events, secretion, actin cytoskeletal organization, cellular proliferation, and inhibition of
apoptosis (reviewed in Refs. 1-4). Many of these functions are
mediated by binding and recruiting signaling proteins which contain
specific phosphoinositide-binding domains such as SH2 domains,
pleckstrin homology domains, FYVE domains, C2 domains or
polybasic domains, thereby localizing these effector proteins to
specific membranes (reviewed in Refs. 3 and 4).
Phosphatidylinositol (4,5)-bisphosphate
(PtdIns(4,5)P2)1
serves as a precursor to second messenger molecules such as inositol (1,4,5)-trisphosphate and phosphatidylinositol (3,4,5)-trisphosphate, but also independent of further modification regulates the actin cytoskeleton and membrane trafficking (1, 5). PtdIns(4,5)P2 binds to actin-binding proteins such as profilin and gelsolin (6) and
displaces capping proteins from actin filaments, allowing polymerization and formation of actin stress fibers (7-9).
PtdIns(4,5)P2 also plays a role in regulating vesicle
budding and in the recruitment and activation of proteins involved in
the coating of vesicles (2).
Cellular levels of PtdIns(4,5)P2 are regulated by a series
of lipid phosphorylation and dephosphorylation reactions mediated by
specific lipid kinases and phosphatases. Inositol polyphosphate 5-phosphatases (5-phosphatases) regulate cellular
PtdIns(4,5)P2 levels by hydrolyzing the 5-position
phosphate from the inositol ring forming phosphatidylinositol
4-phosphate (PtdIns(4)P) (10, 11). The budding yeast
Saccharomyces cerevisiae has four 5-phosphatase genes,
INP51, INP52, INP53, and INP54. Inp51p,
Inp52p, and Inp53p each comprise an N-terminal SacI domain,
a central 5-phosphatase domain, and a C-terminal proline-rich region
(12, 13). These enzymes share significant sequence homology with the
mammalian homologue synaptojanin, which regulates the recycling of
synaptic vesicles in nerve terminals (14). Synaptojanin, Inp52p, and Inp53p contain two catalytic domains, a central 5-phosphatase domain
and an N-terminal SacI domain which hydrolyzes
PtdIns(3,5)P2, PtdIns(4)P, and PtdIns(3)P forming PtdIns
(15). Null mutation of any two SacI domain-containing
5-phosphatases results in plasma membrane invaginations and thickened
cell walls, defects in polarization of the actin cytoskeleton, and
impaired endocytosis (12, 13). However, double SacI
domain-containing 5-phosphatase null mutants display normal secretion
of invertase suggesting that Inp51p, Inp52p, and Inp53p do not play a
role in regulating secretion (16). A triple SacI
domain-containing 5-phosphatase null mutant is nonviable suggesting
Inp54p cannot function to rescue the loss of these three
5-phosphatases.
Inp54p is a PtdIns(4,5)P2 5-phosphatase (17), as are all
the yeast 5-phosphatases. Therefore it is not surprising that single null mutation of INP54 is not lethal (18). However, further characterization of the phenotype of this mutant has not been reported.
In this study we demonstrate Inp54p is a C-terminal tail-anchored
protein that localizes to the cytosolic face of the endoplasmic
reticulum. This localization is mediated by a short 13-amino acid
leucine-rich region at the extreme C terminus of the protein. Null
mutation of INP54, but not any of the SacI domain-containing 5-phosphatases, results in increased levels of
secretion from the endoplasmic reticulum. We propose the enzyme regulates PtdIns(4,5)P2 levels on the cytoplasmic surface
of the endoplasmic reticulum and thereby regulates secretion.
Materials--
Restriction and DNA modifying enzymes were
supplied by New England Biolabs, Fermentas, or Promega. Biomol
GreenTM Reagent for phosphate detection was obtained from
Biomol. Oligonucleotides were obtained from Bresatec, Australia, and
the Department of Microbiology, Monash University, Australia. The Big
Dye Terminator Cycle Sequencing kit was from PE Applied Systems (Foster
City, CA). All other reagents were from Sigma unless otherwise stated. The plasmid pPS1303 for GFP expression was a kind gift from Professor Pamela Silver, Dana Farber Cancer Institute, Harvard University, the
pRS416 vector from Dr. Mark Prescott, Monash University, Australia, the
plasmid pJJ250 was a gift from Dr. Doris Germain, Peter MacCallum Cancer Institute, Australia. Bovine pancreatic trypsin inhibitor (BPTI)
expression plasmid pEB316U was a kind gift from Professor Dane Wittrup,
MIT. Yeast strains used in the study are listed in Table
I. Yeast strains were cultured at
30 °C in standard YPD media or complete minimal media lacking
specific amino acids to maintain selection of markers where
appropriate.
Disruption of INP54 in S. cerevisiae--
The genomic sequence
containing the INP54 ORF (open reading frame), 1600 bp
upstream of the start codon and 700 bp downstream of the stop codon was
amplified by PCR (incorporating a XhoI site at the 5' end
and a NotI site at the 3' end) and cloned into the XhoI/NotI site of Bluescript. The construct was
digested with PstI/SpeI to release a 1.7-kilobase
pair fragment containing the full sequence of INP54, which
was replaced with a LEU2 expression cassette obtained by
digesting the plasmid pJJ250 with XbaI/PstI (19).
The LEU2 gene flanked by the sequence upstream and
downstream of INP54 ORF was recovered from the Bluescript
vector by XhoI/NotI digestion. The DNA fragment
was then transformed into W303 diploid cells by electroporation as
described previously (20). Transformants were selected by their ability
to grow on complete minimal media lacking leucine, and were
subsequently screened for homologous integration of the LEU2
marker by PCR. Null mutations of the SacI domain-containing
5-phosphatases has been described previously (21).
Cloning of INP54--
INP54was amplified from SEY6210
genomic DNA by PCR using synthetic oligonucleotide primers as described
in Table II (GenBank accession number
Z74807). The primers amplified the complete coding region of the
5-phosphatase from nucleotides 1 to 1156 and incorporated an
EcoRI site at the 5' end and a BamHI site at the
3' end. The PCR product was ligated into the pCRBlunt vector (Invitrogen), released by EcoRI restriction digest, and
subcloned into the EcoRI site of the pTrcHisB vector to give
the construct pTrcHisB-INP54. The identity of the PCR
product was confirmed as INP54 by dideoxy sequence analysis.
INP54331 was amplified and cloned as above with
the 3' oligonucleotide incorporating a stop codon plus an
EcoRI site after nucleotide 1093 (Table II).
Expression and Purification of Recombinant
(His)6-Inp54p--
Two × 100-ml cultures of
pTrcHisB-INP54 were grown at 37 °C to an
A600 of 0.5-0.6 prior to induction with 0.1 mM isopropylthio- PtdIns(4,5)P2 5-Phosphatase Enzyme
Assays--
PtdIns(4,5)P2 substrate was a mixture of 33.3 µM PtdIns(4,5)P2 and 3 µg of
phosphatidylserine, dried under nitrogen, resuspended in 50 µl
of lipid resuspension buffer (20 mM Hepes, pH 7.5, 1 mM MgCl2, 1 mM EGTA), and sonicated
for 5 min. The recombinant Inp54p was incubated with the substrate in
the presence of kinase buffer (20 mM Hepes, pH 7.4, 1 mM EGTA, 5 mM MgCl2) in a 100-µl total reaction volume. Assays were performed at 37 °C for 30 min using two linear protein concentrations in duplicate. The reaction was
terminated by incubating with 1 ml of Biomol
GreenTM reagent at room temperature for 30 min. The
absorbance at 620 nm was measured and the amount of phosphate released
calculated by comparison with known standards supplied in the Biomol kit.
Expression of GFP-tagged Inp54p in S. cerevisiae--
All
INP54 constructs were amplified by PCR using appropriate
primers listed in Table II, from SEY6210 genomic DNA. The primers incorporated a BamHI site for cloning INP54 into
the pPS1303 vector, in-frame with the C-terminal GFP. The PCR product
was ligated into pCRBlunt, released by BamHI digestion, and
ligated to the compatible BglII site in pPS1303. GFP-tagged
LRD13, 12, 11, and 10 were amplified from pPS1303-INP54
using oligonucleotides listed in Table II and a 3' primer specific for
the 3' end of the GFP ORF. The resulting LRD-GFP products were cloned
into pCRBlunt, excised using BamHI and cloned into the
BglII site of pPS1303H. pPS1303H lacked the GFP ORF and was
constructed by excising GFP from pPS1303 via HindIII digest
followed by religation of the vector. The INP54-pPS1303
constructs were transformed into an inp54 null mutant strain
using the S. cerevisiae EasyComp Transformation kit
(Invitrogen) and the transformants were selected on complete minimal
media agar plates lacking uracil. The identity of all constructs were
confirmed using dideoxy sequencing analysis.
Expression of Inp54-GFP under Its Native Promoter in S. cerevisiae--
Full-length INP54 was amplified together
with 968 bp upstream of the open reading frame using oligonucleotides
listed in Table II. The PCR product was cloned into the
BamHI site of pRS416-GFP (21) in-frame with the C-terminal
GFP. The construct was then transformed into an inp54 null
mutant strain using the S. cerevisiae EasyComp
Transformation kit (Invitrogen) and the resulting transformants selected on complete minimal media lacking uracil. Single colonies were
grown to mid-log phase in minimal media lacking uracil at 30 °C,
fixed, and stained with an anti-GFP antibody
(CLONTECH) to amplify the signal, and analyzed
using confocal microscopy as described above.
Expression of HA-tagged Inp54p in S. cerevisiae--
The
HA-tagged Inp54p was cloned into the pPS1303H expression vector lacking
the GFP coding region. The primers used to amplify INP54
incorporated a HA tag at either the 5' or 3' end, with a BamHI site (see Table II), this facilitated cloning into the
BglII site of pPS1303H vector. The resulting constructs were
transformed into inp54 null mutants as described above.
Analysis of GFP and HA-tagged Inp54p in S. cerevisiae by Confocal
Microscopy--
Yeast cells were grown overnight in complete minimal
media + 2% glucose, diluted 1/200 in 2% raffinose, and induced the
next day in 2% galactose for 4 h. Cells were fixed according to
Franzusoff et al. (24) and stained with either an anti-HA
antibody (diluted 1/1000) to visualize the HA-tagged proteins or an
anti-BiP antibody (diluted 1/25) to co-localize the GFP-tagged proteins
with the ER. A polyclonal antibody to BiP (binding protein) was raised in rabbits against the S. cerevisiae endoplasmic reticulum
protein Kar2p (BiP) by standard protocols using mature Kar2p
recombinant protein as an antigen. The primary antibodies were
counterstained with anti-rabbit tetramethylrhodamine B isothiocyanate
for BiP (1/200), or anti-mouse tetramethylrhodamine B isothiocyanate
(1/200) for HA. For nuclear staining, spheroplasts were pretreated with RNase (200 µg/ml) at 37 °C for 1 h, then stained with
propidium iodide (2 µg/ml) for 20 min. Cells were attached onto the
microscope slides with poly-L-lysine (Sigma), coverslips
were mounted on the slides using SlowFade (Molecular Probes). The cells
were then analyzed using a Leica TCS-NT confocal microscope with an
ArKr triple line laser, with green fluorescence collected in channel 1 (488 nm excitation, 530 ± 30 nm emission) and red fluorescence in
channel 2 (568 nm excitation, LPS90 nm).
Extraction of Recombinant GFP- and HA-Inp54p from
Yeast--
This extraction method is a slight modification of that as
described in Seedorf and Silver (25). Yeast cells expressing Inp54p
tagged with GFP or HA were harvested and resuspended in PBSM buffer
(phosphate-buffered saline, 5 mM MgCl2), 0.5 mM phenylmethylsulfonyl fluoride, and 3 µg/ml each of
leupeptin, aprotonin, pepstatin, and chymostatin. Glass bead lysis was
performed by vortexing and incubating on ice at 30-s intervals for 12 cycles, respectively. The lysate was centrifuged for 10 min at 4 °C,
the supernatant represented cytosolic fraction, the pellet was
resuspended in PBSM buffer with 1% Triton. After overnight Triton
extraction at 4 °C, the lysate was spun for 10 min to separate the
Triton-soluble and Triton-insoluble fractions. These were separated on
10% SDS-PAGE and transferred to polyvinylidene fluoride membranes
according to standard protocols (23), and immunoblotted using an
antibody specific for GFP (P. Silver) or HA (Covance). To determine the 5-phosphatase activity of Inp54p tagged with GFP or HA, the fusion protein was immunoprecipitated from the Triton-soluble fraction using
50 µl (50% v/v) of protein A-Sepharose, 0.8 µg of anti-GFP antibody (Roche Molecular Biochemicals) or 6 µg of anti-HA antibody (Covance), and 7 µg of rabbit anti-mouse immunoglobulin (DAKO) which
served as a linker. Immunoprecipitation was performed at 4 °C
overnight with gentle agitation. The protein A-Sepharose pellet was
washed 6 times with ice-cold Tris saline (20 mM Tris, pH
7.2, 150 mM NaCl), and the pellet used in
PtdIns(4,5)P2 5-phosphatase enzyme assays as described previously.
Protease Protection Assays on Yeast Expressing Recombinant HA- or
GFP-Inp54p--
Microsomes were extracted from yeast by differential
centrifugation fractionation according to Parlati et al.
(26) and Paddon et al. (27). The medium-speed microsomal
pellet, which was found to be enriched in ER markers, was treated with
proteinase K according to the methods described by Bascom et
al. (28). The control fractions (untreated) and proteinase
K-treated fractions were separated by 10% SDS-PAGE, and immunoblotted
with either an anti-HA antibody or anti-GFP antibody. The same
fractions were immunoblotted with anti-BiP antibody as a control to
ensure that the microsomal membranes were intact.
BPTI Secretion Assay--
W303 Inp54p Sequence Predicts for a C-terminal Tail-anchor--
Inp54p
is one of four inositol polyphosphate 5-phosphatases (5-phosphatases)
found in the yeast S. cerevisiae. Unlike the other
previously characterized yeast 5-phosphatases, Inp54p predicts for a
smaller molecular mass, 44 kDa, comprising a central 300-amino acid
5-phosphatase domain and no other defined signaling motif. However, a
hydropathic profile of Inp54p (scale = Kyte-Doolittle × Inp54p Localizes to the ER of the Cell--
To characterize the
intracellular localization of Inp54p, the protein was expressed as a
fusion protein with GFP under the control of a galactose-inducible
promoter in inp54 null mutant cells. Cells cultured in the
presence of raffinose, which does not induce production of the fusion
protein, demonstrated no fluoresence (Fig.
2A). Incubation of cells in
media supplemented with galactose resulted in expression of Inp54p-GFP
fluorescence in the perinuclear region, as shown by co-localization
with propidium iodide, which stains the nucleus (Fig. 2B).
Co-localization using an antibody directed against the yeast protein
Kar2p or BiP which localizes to the ER, demonstrated
co-localization with Inp54p-GFP expression. Peripheral and reticular
staining was also detected by both Inp54p-GFP fluorescence and the
anti-BiP antibody. To confirm that GFP did not play any role in
targeting the fusion protein to the ER, cells expressing the GFP alone
were induced and analyzed. GFP was detected throughout the entire cell
(Fig. 2B).
To ensure that the ER localization of Inp54p-GFP did not result from
overexpression, which may cause proteins to distribute aberrantly,
Inp54p-GFP was expressed under the control of its native promoter. The
entire INP54 open reading frame and 968 bp upstream of the
initiating ATG was amplified by PCR and cloned into a single copy yeast
expression vector pRS416-GFP, which lacks a promoter. The construct was
transformed into inp54 null mutant cells, which were grown
to mid-log phase and analyzed by confocal microscopy. As the expression
level was very low (results not shown) the signal was amplified using
an antibody specific for GFP. Inp54p-GFP expressed under the Inp54p
native promoter localized to a perinuclear ring-like structure,
comparable to that observed in Inp54p overexpressing cells (Fig.
2C), indicating that the ER localization of Inp54p is not
affected by the level of expression of the recombinant protein.
To further validate the intracellular distribution of Inp54p in yeast
cells and demonstrate that the position of the tag did not influence
the localization of the protein, a hemagglutinin (HA) tag was fused at
either the N or C terminus of Inp54p. Inp54p was amplified by PCR as
described under "Experimental Procedures," with the HA tag
incorporated in either the 5' or 3' oligonucleotide primer, and cloned
into the pPS1303H vector from which the GFP sequence had been removed.
Following induction, cells were fixed and stained with an antibody
specific for HA to visualize the fusion proteins. Fig. 2D
shows that Inp54p tagged with HA was expressed in a perinuclear
distribution consistent with ER localization, as observed with the
GFP-tagged protein (Fig. 2B). In addition, these results
demonstrate that the position of the tag at the N or C terminus did not
affect the localization of the Inp54 protein, as has been shown
previously for other ER tail-anchored proteins such as UBC6 (34) and
cytochrome b5 (33).
To confirm that Inp54p-GFP and HA-Inp54p were functional proteins,
following induction in galactose-containing media, recombinant fusion
proteins were extracted from yeast cells with Triton X-100, immunoprecipitated with antibodies specific for GFP or HA, and assayed
for PtdIns(4,5)P2 5-phosphatase activity as described under
"Experimental Procedures." Immunoprecipitated GFP displayed no
activity against PtdIns(4,5)P2. Both Inp54p-GFP and
HA-Inp54p were able to hydrolyze PtdIns(4,5)P2 confirming
that the constructs encoded functional proteins (results not shown).
Inp54p Attaches to the Endoplasmic Reticulum Membrane with the Bulk
of the Protein Oriented toward the Cytoplasm--
The C terminus of
tail-anchored proteins inserts into the cytoplasmic surface of the
endoplasmic reticulum or mitochondrial membrane with the N-terminal
domain located in the cytoplasm (33, 37, 39-41). Inp54p lacks a signal
sequence suggesting the enzyme does not represent an integral
endoplasmic reticulum protein, but post-translationally associates with
this compartment. The membrane orientation of Inp54p in the endoplasmic
reticulum was determined by a protease protection assay. Cells
expressing HA-Inp54p, or Inp54p-GFP, were subjected to differential
centrifugation to isolate the ER-enriched microsomal membrane fraction
(26). Recombinant Inp54p association with the membrane fraction was
sensitive to alkaline treatment using 0.1 M
NaCO3, pH 11.5 (results not shown), therefore it is not an
integral membrane protein. Inp54p associates tightly with the membrane,
however, since treatment with 0.5 and 1 M NaCl failed to
release recombinant Inp54p from membranes (results not shown).
ER-enriched microsomes were incubated in the presence or absence of
Proteinase K for 1 or 2 h at 4 °C, followed by centrifugation to isolate the microsomal pellet. Proteins were separated by 10% SDS-PAGE and immunoblotted using an antibody specific for either HA or
GFP. Following a 1-h incubation with Proteinase K, virtually all of the
Inp54p was degraded (Fig. 3, A
and B) indicating that the bulk of the protein was oriented
toward the cytoplasm of the cell. To confirm the integrity of the
microsomal membranes, the same fractions were blotted with an antibody
directed against the ER lumenal protein BiP. Proteinase K treatment had
no effect on the integrity of BiP indicating that the microsomal
membranes were intact (Fig. 3, A and B). It was
noteworthy that similar results were observed for Proteinase K
treatment of Inp54p regardless of whether the tag was at the N terminus
(HA) or the C terminus (GFP) of the protein suggesting that the
C-terminal tail of Inp54p may loop back into the cytosol. Previous
studies of the tail-anchored protein cytochrome
b5 have demonstrated that the addition of a C-terminal tag does not prevent the transmembrane tail from inserting correctly into the membrane (33).
The C-terminal Hydrophobic Domain of Inp54p Mediates Localization
to the ER--
To determine whether the C-terminal hydrophobic domain
mediates ER localization a series of C-terminal Inp54p truncation
mutants were constructed and expressed in inp54 null mutant
yeast. Previous studies have demonstrated removal of the hydrophobic
tail of tail-anchored proteins results in the relocalization of the
truncated protein to the cytoplasm (42-47).
Mutant Inp54p331 contained the 5-phosphatase domain from
amino acids 1-331 and lacked the entire hydrophobic C-terminal 53 amino acids. The second mutant, Inp54p353, lacked amino
acids 354-384. These residues are predicted to have a transmembrane topology by a Kyte-Doolittle hydrophobicity plot analysis.
Inp54p371 lacks the leucine-rich area spanning the last 13 amino acids (residues 372-384), which is the minimal hydrophobic
domain in the C terminus. All mutant sequences were amplified by PCR
using appropriate primers as described under "Experimental
Procedures," and cloned into the pPS1303 vector. Expression of the
mutant fusion proteins was induced by incubating the cells in
galactose-containing media for 4 h and cells were examined live by
confocal microscopy. All three mutant proteins were expressed in the
cytosol although Inp54p371-GFP still showed some ER
staining, but to a much lesser degree than that observed in the
full-length protein (Fig. 4A).
This indicates that the C-terminal tail of Inp54p mediates ER
localization and that the last 13 amino acids are required to maintain
this localization.
To confirm the cytosolic localization of the mutant fusion proteins,
yeast cells were fractionated into cytosol, Triton-soluble and
Triton-insoluble fractions as outlined under "Experimental Procedures." Proteins were separated by SDS-PAGE and immunoblotted with an antibody specific for GFP (Fig. 4B). Full-length
Inp54p384-GFP which localized to the ER, was present
intact, as a 71-kDa protein consistent with the predicted molecular
mass of the recombinant protein, predominantly in the
Triton-soluble fraction, with only a very small amount in the
Triton-insoluble fraction. In contrast, the three Inp54p truncation
mutants were expressed predominantly in the cytosol. Occasionally, a
small, variable proportion of the mutant protein was detected in the
Triton-soluble and Triton-insoluble fractions. Minimal proteolysis of
recombinant mutant proteins was detected. These studies collectively
indicate that the Inp54p C-terminal domain is critical for mediating
membrane association, specifically to the endoplasmic reticulum.
To confirm that the 5-phosphatase catalytic function is not regulated
by the C-terminal hydrophobic domain, the activity of purified
recombinant full-length Inp54p384 versus mutant
Inp54p331 which lacks the entire C-terminal hydrophobic
domain was determined. Recombinant wild-type versus
C-terminal mutant (His)6-tagged Inp54p were expressed in
Escherichia coli, and purified using Talon metal affinity
chromatography and assayed for PtdIns(4,5)P2 5-phosphatase activity. No significant difference in PtdIns(4,5)P2
5-phosphatase activity was detected between full-length
Inp54p384 or mutant Inp54p331 (Fig.
4C) confirming that the C-terminal leucine-rich domain does
not play a role in regulating enzyme activity.
The Last 13 Amino Acids of Inp54p Constitute the ER Localizing
Sequence--
Previous studies have demonstrated that C-terminal
hydrophobic domains are sufficient to direct heterologous proteins to
specific subcellular compartments (34, 38, 48). To determine whether the last 13 amino acids of Inp54p (372) comprise the ER targeting region, a series of leucine-rich domain (LRD) mutant constructs were
generated, encoding the C-terminal 13, 12, 11, and 10 amino acids of
Inp54p fused to GFP (designated LRD13, LRD12, LRD11, and LRD10,
respectively). Following induction of the recombinant fusion proteins,
expressing cells were analyzed by confocal microscopy. LRD13 was
sufficient to localize GFP to the ER (Fig.
5). In contrast, LRD12, LRD11, and LRD10
fused to GFP predominantly demonstrated diffuse cytoplasmic staining,
although minimal ER staining was still detected. This indicates that
the last 13 amino acids of Inp54p constitute the minimum ER targeting
motif, and while the shorter sequences contain some targeting
information, it is not as efficient as LRD13.
Substitution of a Single Charged Amino Acid in the Tail-anchor
Domain Has No Effect on Localization--
Several models for the
insertion of the C-terminal targeting domain of tail-anchored proteins
to the membrane have been proposed. It has been suggested that the
absolute sequence of the tail is not important, rather, the amino acid
composition in terms of hydrophobicity and length of the domain dictate
the intracellular localization (34, 49, 50). Furthermore, several
studies indicate that charged amino acids within the tail-anchor are
the determinants of specific subcellular location to the endoplasmic reticulum versus mitochondria. Kuroda et al. (48)
demonstrated that charged amino acids within the last 10 amino acids of
two isoforms of cytochrome b5 (outer
mitochondrial membrane b5 and ER
b5) determined their localization in the cell.
Mutation of the positively charged amino acids (Arg137 and
Lys144) to noncharged residues altered the localization of
outer mitochondrial membrane b5 from the
mitochondria to the ER. Mutation of the negatively charged residue
(Asp134) to a neutral Ala or a positive Lys in ER
b5 redirected this protein from the ER to the mitochondria.
Examination of the hydrophobic tail of Inp54p, in particular the last
13 amino acids at the extreme C terminus, revealed a single charged
residue (Lys) at position 382, and no other charged amino acids. To
investigate whether this residue plays a role in ER targeting,
Lys382 was mutated to a neutral alanine. An HA tag was
fused to the N terminus of Inp54p(K382A) and the mutant recombinant
5-phosphatase expressed in the inp54 null mutant strain. As
can be seen from Fig. 6, mutation of
Lys382 to Ala did not affect the ER localization of the
protein. The ER isoform of cytochrome b5 has 2 charged residues at the C-terminal tail, an arginine and an aspartic
acid. The negatively charged aspartic acid (Asp134)
mediates ER localization, whereas the positively charged arginine (Arg128) demonstrated no role in localization (48). Since
Lys382 is the only charged residue in the tail of Inp54p,
the "charged amino acids as determinant of intacellular
localization" model cannot be applied in this case. A more detailed
analysis of the 13-residue tail-anchor of Inp54p has to be performed to
delineate the targeting information, and is the subject of ongoing
studies.
Null Mutation of inp54 Results in Increased Secretory Capacity
Compared with Wild-type Cells--
In mammalian cells
PtdIns(4,5)P2 recruits ADP-ribosylation factor to the Golgi
membrane where GTP-bound ADP-ribosylation factor engages coatomer
proteins and stimulates phospholipase D, followed by vesicle budding
(2). PtdIns(4)P and PtdIns(4,5)P2 have been proposed to act
as receptors for the coatomer complex COPII, which coats vesicles
budding from the ER (51). PtdIns(4,5)P2 is also required
for fusion of secretory granules with the plasma membrane in exocytosis
and clathrin-mediated endocytosis (4). To date, PtdIns(4,5)P2 has not been shown to be directly involved in
regulating vesicular transport from the ER in yeast. However, its
precursor PtdIns(4)P is required for normal secretion in S. cerevisiae. Mutation of the PtdIns 4-kinase Pik1p results in
impaired secretion from the Golgi and abnormal Golgi morphology (52,
53).
Defects in endocytosis have been noted in the yeast SacI
domain-containing 5-phosphatases, Inp51p, Inp52p, and Inp53p
null mutants, however, secretion was reported to be normal (16). The
functional role of Inp54p in yeast is as yet unclear and although null
mutation of inp54 is not lethal, no other phenotype has been described. inp54 null mutant was generated as described
under "Experimental Procedures" and the morphology of the null
mutant strain compared with wild-type. Electron microscopy analysis of inp54 null mutant did not reveal any significant phenotype
that differed from the wild-type (results not shown). The growth of inp54 null mutant was tested on media supplemented with
either 0.9 M NaCl or 1.4 M sorbitol. The degree
of viability on hyperosmotic media was the same for wild-type and null
mutant strains (results not shown). Unlike double null mutants of the
SacI domain-containing 5-phosphatases which have abnormal
actin and chitin organization (16), inp54 null mutant
demonstrated normal actin patches and chitin deposition as compared
with the wild-type, assessed by staining cells with phalloidin or
calcofluor, respectively, and analysis by confocal microscopy (results
not shown).
Inp54p is a PtdIns(4,5)P2 5-phosphatase which localizes on
the cytosolic face of the ER. PtdIns(4,5)P2 and PtdIns(4)P
have been demonstrated to enhance binding of coatomer complexes to liposomes suggesting that these phosphoinositides are important in
protein transport from the ER, possibly by promoting vesicle budding
(51). Since proteins that are to be secreted have to be synthesized in
the ER and transported further along the secretory pathway, Inp54p may
also contribute indirectly to the regulation of secretion. Therefore,
we investigated the role of Inp54p in secretion from the ER. We
utilized a reporter protein assay which measures the amount of BPTI
secreted from the cell (30, 54). Wild-type yeast and cells with null
mutation of inp51, inp52, inp53, or
inp54 were transformed with an expression plasmid containing BPTI under the control of a galactose promoter. Cells were grown in the
presence of galactose for 0, 16, 24, and 48 h at 30 °C and the
media containing secreted BPTI was collected. The level of BPTI present
in the media was determined by measuring the inhibition of trypsin
cleavage of a synthetic substrate L-BAPNA
(N
Since accumulation of high levels of BPTI within the cell reduces its
viability, we tested the viability of inp54 null mutant when
BPTI was expressed continuously in the cell. A previous report has
shown that the mammalian 180-kDa ribosome receptor that induces membrane proliferation in yeast could rescue cells expressing high
amounts of BPTI by increasing secretion up to 4-fold (54). Wild-type
and inp54 null mutant cells transformed with the BPTI plasmid were plated in 10-fold serial dilutions onto complete minimal
media supplemented with either glucose or galactose and incubated at
30 °C for 2 days. Both wild-type and inp54 null mutant strains grew inefficiently (up to 10 The results of the studies described here demonstrate that the
yeast PtdIns(4,5)P2 5-phosphatase Inp54p is a C-terminal
tail-anchored protein that localizes to the cytoplasmic surface of the
ER where it may play a role in the regulation of secretion. Evidence
for the ER localization of this novel 5-phosphatase is suggested by the
presence of the C-terminal hydrophobic domain, which is consistent with
a tail-anchor motif comprising a stretch of hydrophobic residues at the
extreme C terminus. Many studies indicate C-terminal tail-anchored proteins localize to either the mitochondria or the endoplasmic reticulum (reviewed in Ref. 36). We have demonstrated that recombinant Inp54p expressed as a fusion protein with either GFP or HA co-localizes with the ER-specific protein BiP, both when the protein is
overexpressed, or expressed under the control of its native promoter.
We have shown inp54 null mutant cells demonstrate increased
secretion, consistent with a functional role for Inp54p in the ER.
All four yeast 5-phosphatases hydrolyze PtdIns(4,5)P2
forming PtdIns(4)P. The necessity for four 5-phosphatases in yeast to regulate cellular concentrations of PtdIns(4,5)P2 may in
part be explained by the localization of each 5-phosphatase to specific membrane compartments. However, this has yet to be shown. Recently our
laboratory has demonstrated that Inp52p and Inp53p are localized diffusely throughout the cell excluding the nucleus and translocate to
cortical actin patches following hyperosmotic stress (21). The
intracellular localization of Inp51p has not been delineated, as the
enzyme is extensively proteolyzed when expressed as a recombinant protein (21, 55). The localization of Inp54p to the ER is the first
evidence of the targeting of a yeast 5-phosphatase to a specific
subcellular compartment in the resting cell. To date no mammalian
5-phosphatase has been localized to the ER, although the Lowe's
protein has been identified in both the Golgi and the lysosomal
compartment (56, 57), and a novel 72-kDa 5-phosphatase has been
localized to the cytoplasmic surface of the Golgi, where the enzyme is
proposed to regulate phosphoinositide-mediated vesicular trafficking
(58).
Inp54p localizes to the ER of yeast cells by the C-terminal hydrophobic
tail comprising the last 13 amino acids of the protein. Deletion of the
Inp54p hydrophobic tail resulted in a cytoplasmic distribution of the
protein, consistent with previous observations that the C terminus of
tail-anchored proteins contains the membrane targeting information (38,
45, 47, 48, 59, 60). The last 13 amino acids of Inp54p also direct GFP
to the ER. The length of the hydrophobic tail is a critical factor in
determining intracellular localization. While shorter hydrophobic
domains of 10-12 amino acids have been demonstrated to target
synaptobrevin and cytochrome b5 to the ER (59,
61), domains of greater than 21 amino acids generally result in plasma
membrane targeting (34, 50). Lengthening the hydrophobic tail of the ER
protein Ufe1p from 16 to 24 amino acids, by the insertion of eight
hydrophobic residues, resulted in the protein being mislocalized to the
plasma membrane (50). Increasing the length of the C-terminal membrane
anchor of UBC6 from 17 to 21 amino acids caused mistargeting of the
protein from the ER to the Golgi, whereas a further increase to 26 amino acids resulted in expression of the protein at the plasma
membrane (34). Based on these previous studies, the short 13-amino acid
targeting sequence of Inp54p would be consistent with the ER
localization of the protein.
The exact mechanism by which C-terminal anchored proteins are targeted
to specific membranes remains unclear. Several studies have shown that
the targeting of various isoforms of synaptobrevin to the ER requires
ATP, but is independent of the Sec61p/SRP pathway, relying instead on
an as yet uncharacterized receptor system (37, 40, 62). The C-terminal
tails of VAMP-2 and Ufe1p form an amphipathic helix critical for
localization of the proteins to the ER (50, 63). Mutation of the
residues forming the polar face of the helix of Ufe1p to leucine
abolished ER targeting of the protein (50). Mapping of the C-terminal
hydrophobic sequence of Inp54p to a helical wheel reveals that the
lysine, tyrosine, and serine are predicted to line one face of the
helix, however, this would not be predicted to form an amphipathic
face. Mutation of the positively charged lysine residue at position 382 had no effect on the localization of the protein, as has been shown for mutation of the arginine residue within the C terminus of cytochrome b5 (48).
We predict Inp54p is orientated on the ER membrane with the bulk of the
protein located on the cytoplasmic side of the ER membrane. Location of
the tag did not affect the cytoplasmic orientation of Inp54p, or its
intracellular localization. Previous studies have shown that C-terminal
tags attached to cytochrome b5 and synaptobrevin
did not affect their insertion or orientation in the membrane (33, 37,
41). The topology of tail-anchored proteins in the membrane is
predicted to mimic classical transmembrane segments, whereby the tail
spans the width of the membrane bilayer, with only a few residues on
the other side of the membrane (32). At only 13 amino acids in length,
the hydrophobic region of Inp54p is too short to span the membrane as
typically integral membrane proteins contain a hydrophobic span of 20 amino acids (64). We have also demonstrated that although Inp54p
membrane association is resistant to salt extraction, it is sensitive
to alkaline treatment, indicating that it is not an integral membrane
protein. Since a GFP tag fused to the extreme C terminus of Inp54p was
degraded by proteinase K, it is most likely that the C terminus of
Inp54p is also exposed to the cytoplasm. Thus, we propose the
hydrophobic tail of Inp54p dips into the membrane to form a hairpin
loop configuration whereby both the N and C termini are located in the
cytosol (Fig. 8). As Inp54p membrane
association is sensitive to high salt treatment, it is also possible
that Inp54p could form a tight noncovalent interaction with an ER
membrane protein. Protein-tyrosine phosphatase 1B has been proposed to
adopt such a membrane topology (47).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
List of constructs and oligonucleotide primers used in this study
-D-galactoside for 2 h
at 23 °C. Following induction, cells were pelleted and soluble
proteins were extracted in 1/10 volume of buffer B (20 mM
Tris, pH 8, 250 mM NaCl) supplemented with 12 mM
-mercaptoethanol, 1 mM
phenylmethylsulfonyl fluoride, 2 µg/ml aprotonin, 2 µg/ml leupeptin, 1 mM benzamidine, and 1% Triton X-100 at
4 °C overnight with gentle agitation. Triton extracts were
centrifuged at 15,000 × g for 15 min then the 20-ml
supernatant was incubated with 2.5 ml of Talon resin
(CLONTECH) with gentle agitation in a 50-ml tube
for 4 h at 4 °C. Following incubation, the resin was poured into a column and washed with 20 column volumes of buffer B. Bound proteins were eluted with 4 column volumes of buffer B at pH 6.5 supplemented with 100 mM imidazole and 700-µl fractions
were collected. 50 µl of the starting material, flow-through, and the
eluted fractions were analyzed by 12% SDS-PAGE (22), and either
visualized by Coomassie Brilliant Blue staining or transferred to
polyvinylidene fluoride membranes according to standard protocols (23)
and immunoblotted using monoclonal antibodies to the
(His)6-tag (Silenus). Immunoblots were developed using
enhanced chemiluminescence (ECL) reagent (PerkinElmer Life Sciences)
according to the manufacturer's instructions. The protein
concentration in Coomassie-stained gels was quantitated using
densitometry by comparison with a standard amount of protein loaded on
the gel.
, inp51, inp52, and
inp53 null mutant strains were transformed with
YEplac181-GalBPTI plasmid containing a leucine nutritional marker. This
was constructed by amplifying the Gal promoter and BPTI coding regions
from pEB316U, using oligonucleotides listed in Table II and cloning the
PCR product into the EcoRI/HindIII site of
YEplac181 vector (29). W303
and the inp54 mutant strain were transformed with a BPTI expression plasmid, pEB316U, containing a
uracil nutritional marker. Yeast cells expressing BPTI were grown at
30 °C overnight in complete minimal media lacking either leucine or
uracil, supplemented with 2% glucose. The next day, cultures were
diluted 1/200 in raffinose containing media, grown overnight until they
reached 107 cells per ml, and induced with galactose for
the specified time frames. The amount of BPTI secreted in the culture
media was quantified according to the method described by Parekh
et al. (30). A rescue experiment was done to investigate
whether
inp54 mutant could remove a sufficient
amount of BPTI from the intracellular space and maintain viability when
grown continuously on galactose-supplemented media. Since accumulation
of high levels of BPTI is toxic, continuous induction would be lethal
to the cells. Raffinose cultures of yeast cells were spotted in 10-fold
serial dilutions in 5-µl aliquots onto complete minimal agar plates
supplemented with either 2% glucose or galactose.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1)
indicates that the region spanning amino acids 372-384 is strongly
hydrophobic, and is highly rich in leucine residues (Fig. 1A). To identify other
5-phosphatases containing a similar C-terminal hydrophobic tail we
performed tBLASTn and PSI-BLAST (31) searches of the nonredundant
protein and nucleotide data bases at the NCBI. In all searches the
filter excluding low compositional complexity regions was turned off.
We were unable to identify any 5-phosphatase in any other organism that
contained a similar hydrophobic anchor sequence in its C terminus.
However, we noted that the C terminus of Inp54p strongly resembled that
found in many C-terminal tail-anchored proteins (Fig. 1B).
Tail-anchored proteins are a class of integral membrane proteins which
lack any N-terminal targeting sequence and insert into membranes via a
single C-terminal hydrophobic sequence (32-36). These proteins, which
include cytochrome b5, UBC6, Bcl2, and numerous
SNARE proteins have been identified in both mammalian cells and
S. cerevisiae (32, 37). A comparison of the C-terminal
hydrophobic domain of Inp54p versus previously characterized
tail-anchored proteins is shown in Fig. 1B. Tail-anchored proteins are predominantly localized on the membranes of the
endoplasmic reticulum and/or mitochondria, with the bulk of the protein
oriented toward the cytoplasm (33, 34, 36-38).
View larger version (32K):
[in a new window]
Fig. 1.
A, hydropathic profile of Inp54p
calculated using the Kyte-Doolittle x-1 scale. In this plot, a
value of 2 indicates strong hydrophobicity corresponding to a
transmembrane topology. The leucine-rich sequence of the C-terminal 13 amino acids is shown. B, table showing the C-terminal
portions of several known tail-anchored proteins. The hydrophobic tail
which serves as a membrane anchor is highlighted in gray.
The following abbreviations are used: Inp54p (Inp54p;
Saccharomyces cerevisiae, GenBank accession number
NP_014576), PTN1_HUMAN (protein-tyrosine phosphatase 1B; Homo
sapiens, P18031), BCL2_HUMAN (apoptosis regulator BCL-2; H. sapiens, P10415), SYB1_HUMAN (synaptobrevin 1; H. sapiens, P23763), VAMP_1B (synaptobrevin 1 isoform Vamp-1B;
H. sapiens, AAC28336), SYB2_HUMAN (synaptobrevin 2; H. sapiens, P19065), SNC1_Yeast (synaptobrevin homolog 1; S. cerevisiae, P31109), Ufe1p (t-SNARE; S. cerevisiae,
AAC13730), CYB5_HUMAN (cytochrome b5; H. sapiens, P00167), and CAA73117 (cytochrome
b5; Rattus norvegicus, mitochondrial
isoform, CAA73117).
View larger version (65K):
[in a new window]
Fig. 2.
Intracellular localization of
Inp54p-GFP. INP54 was cloned in-frame with GFP under the
control of a galactose-inducible promoter and transformed into an
inp54 null mutant strain. A, yeast were grown in
the presence of raffinose to inhibit the production of the Inp54p-GFP
fusion protein, and analyzed by confocal microscopy. The expression of
GFP under the same conditions is included as a control. Bar
indicates 5 µm. B, expression of recombinant GFP, or
Inp54p-GFP was induced following incubation for 4 h in
galactose-containing media. The cells were fixed and stained with
propidium iodide to visualize the nucleus, or an antibody directed
against the Kar2p protein (BiP) in the endoplasmic reticulum. Cells
induced for 2 h, which represent a lower level of expression, are
also shown. Bar indicates 5 µm. C, cells
expressing Inp54p-GFP under the control of the native INP54
promoter were grown to mid-log phase, fixed, and stained with an
anti-GFP antibody. Bar indicates 5 µm. D,
expression of Inp54p tagged with HA at either the N or C terminus was
induced by growing the cells in galactose-containing media for 4 h. The cells were fixed and stained with anti-HA antibody to visualize
the HA-tagged Inp54p. Bar indicates 5 µm.
View larger version (38K):
[in a new window]
Fig. 3.
Inp54p attaches to the endoplasmic reticulum
membrane with the bulk of the protein oriented toward the
cytoplasm. Cells expressing HA-Inp54p (A) or Inp54p-GFP
(B) were subjected to differential centrifugation to isolate
ER-rich microsomes. Microsomes were treated with proteinase K for 1 or
2 h at 4 °C, then centrifuged at 25,000 × g to
obtain supernatant (S) and microsomal (M)
fractions. Proteins were separated by 10% SDS-PAGE and immunoblotted
with anti-HA (A) or anti-GFP antibodies (B).
Molecular weight markers are shown on the left. The same
fractions were probed with an anti-BiP antibody to confirm the
integrity of the microsomal membranes. Untreated microsomes are
included as a control.
View larger version (21K):
[in a new window]
Fig. 4.
The C terminus of Inp54p is required for ER
localization. A, mutant constructs of INP54
with C-terminal deletions were generated as described under
"Experimental Procedures." A schematic representation of each
construct is shown on the left with the full-length
Inp54p384 included for comparison. The subscript on each
construct refers to the last amino acid in the Inp54p mutant proteins.
Expression of Inp54p-GFP C-terminal deletion mutants was induced by
incubation with galactose for 4 h and following fixation the cells
were analyzed by confocal microscopy. Bar indicates 5 µm.
B, yeast cells expressing full-length or mutant Inp54p-GFP were fractionated into cytosol,
Triton-soluble, and Triton-insoluble fractions as described under
"Experimental Procedures." The fractions were separated by 10%
SDS-PAGE and immunoblotted with an anti-GFP antibody. C, E. coli cultures harboring recombinant full-length
(His)6-Inp54p384 or mutant
(His)6-Inp54p331 were induced with 0.1 mM isopropyl- -D-thiogalactopyranoside for
2 h at 23 °C. The induced cultures were extracted with 1%
Triton X-100 overnight, and the soluble extract was purified by Talon
resin immobilized metal affinity chromatography as described under
"Experimental Procedures." 50 µl of eluted fractions were
separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue.
Peak eluted fractions 6 and 7 for each recombinant wild-type or mutant
Inp54p were assayed for PtdIns(4,5)P2 5-phosphatase
activity in triplicate.
View larger version (55K):
[in a new window]
Fig. 5.
The C-terminal leucine-rich tail of Inp54p is
sufficient to localize GFP to the endoplasmic reticulum. GFP was
fused to LRD13, LRD12, LRD11, and LRD10, which represent the last 13, 12, 11, and 10 amino acids of the Inp54p C terminus, respectively. A
schematic representation of each construct is shown on the
left with the numbers referring to the amino acid
position in the full-length protein. Following a 4-h induction, cells
expressing the LRD-GFP fusion proteins were viewed live using
laser-scanning confocal microscopy. Bar indicates 5 µm.
View larger version (46K):
[in a new window]
Fig. 6.
Intracellular localization of Inp54p (K382A)
does not differ from the wild-type protein. Expression of Inp54p
(K382A) fused to the C terminus of HA was induced, and cells fixed and
stained with anti-HA antibody as described under "Experimental
Procedures," and analyzed by confocal microscopy. Wild-type HA-Inp54p
is included for comparison. A schematic representation of each
construct is shown on the left and the introduced mutation
indicated in bold and underlined. Bar
indicates 5 µm.
-benzoylarginine-p-nitroanilide),
as previously described (30). Similar levels of BPTI were secreted into
the media at all time points assayed for wild-type cells, the
inp51, inp52, and inp53 single null
mutant strains (Fig. 7). The results are
consistent with studies by Singer-Kruger et al. (16) who
showed no abnormalities in secretion in the inp51, inp52,
and inp53 mutants. In contrast, deletion of INP54
resulted in a marked increase in secretion, ~2-3-fold higher after
48 h induction (Fig. 7), suggesting that Inp54p plays a role in
the negative regulation of secretion.
View larger version (13K):
[in a new window]
Fig. 7.
Secretion of a reporter protein is increased
in inp54 null mutant cells. A plasmid containing
BPTI under the control of a galactose-inducible promoter was
transformed into wild-type, inp51, inp52, inp53, or
inp54 null mutant strains. Yeast cells were incubated for 0, 16, 24, and 48 h in galactose-containing media to induce
production of BPTI secreted into the media. The amount of BPTI at each
time point was calculated in triplicate and averaged as described under
"Experimental Procedures." Results shown are one representative
experiment of three. , wild-type strain;
, inp51 null
mutant;
, inp52 null mutant;
, inp53 null
mutant; and
, inp54 null mutant.
4 dilution) on
galactose plates, when BPTI was produced constitutively. Viability was
103-fold less compared with cells grown on glucose plates
(up to 10
7 dilution), in which BPTI synthesis was
repressed. There was no significant difference in viability between the
wild-type and inp54 mutant when BPTI was expressed
continuously (data not shown), indicating that the increased secretion
in the inp54 null mutant strain was not sufficient to rescue
the cells from BPTI-induced growth arrest.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (11K):
[in a new window]
Fig. 8.
Possible models of Inp54p topology on the ER
membrane. The C-terminal hydrophobic tail may "dip" into the
membrane and loop back into the cytoplasm (A).
Alternatively, the tail may associate tightly with a receptor/binding
protein, where it binds noncovalently in a "hairpin loop" mode
(B) (adapted from Frangioni et al. (47)).
The localization of Inp54p on the cytoplasmic surface of the ER would
place the enzyme in an optimal position to regulate PtdIns(4,5)P2 levels on vesicles budding from the ER. In
mammalian cells PtdIns(4,5)P2 binds ADP-ribosylation factor
which results in the recruitment of coatomer proteins and activation of
phospholipase D, followed by vesicle budding from the Golgi membrane
(65-67). In yeast, the PtdIns 4-kinase Pik1p is essential for normal
Golgi morphology and Golgi-plasma membrane trafficking (52, 53, 68).
Pik1p-dependent generation of PtdIns(4)P regulates the transport of vesicles destined for the cell surface and normal secretion (52). Pik1p kinase activity is also required for normal endocytic transport of Ste6p from the plasma membrane to the vacuole (53). In mammalian cells PtdIns 4-kinase plays a role in secretion of
granules from chromaffin cells (69). As pik1 null mutants demonstrate decreases in both PtdIns(4)P and PtdIns(4,5)P2,
a role for PtdIns(4,5)P2 in regulating secretion cannot be
excluded (53). While PtdIns(4,5)P2 has not been shown to
play a definitive role in vesicular trafficking from the ER, our
results would suggest the increased secretion observed in
inp54 null mutants is mediated by the regulation of
PtdIns(4,5)P2 in this compartment.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. Susan Brown for critically reading the manuscript, Dr. Trevor Lithgow for helpful discussions, and A. P. Wijayanthi and Sarah Ellis for electron microscopy. Confocal images were obtained at the Biomedical Confocal Imaging Facility of Monash University.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Australian Research Council Grant 9606077.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.
§ Contributed equally to the results of this article.
¶ Recipient of a International Postgraduate Research Scholarship and Monash Graduate Scholarship.
To whom correspondence should be addressed: Monash University,
Dept. of Biochemistry and Molecular Biology, Wellington Road, Clayton
Victoria 3800, Australia. Tel.: 61-3-9905-1245; Fax: 61-3-9905-4699; E-mail: Christina.Mitchell@med.monash.edu.au.
Published, JBC Papers in Press, December 14, 2000, DOI 10.1074/jbc.M010471200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: PtdIns(4, 5)P2, phosphatidylinositol (4,5)-bisphosphate; 5-phosphatase, inositol polyphosphate 5-phosphatase; PtdIns(3)P, phosphatidylinositol (3)-phosphate; PtdIns(4)P, phosphatidylinositol (4)-phosphate; PtdIns(3, 5)P2, phosphatidylinositol (3,5)-bisphosphate; BPTI, bovine pancreatic trypsin inhibitor; BiP, binding protein; LRD, leucine-rich domain; bp, base pair(s); ORF, open reading frame; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; GFP, green fluorescent protein; ER, endoplasmic reticulum; HA, hemagglutinin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | De, Camilli, P., Emr, S. D., McPherson, P. S., and Novick, P. (1996) Science 271, 1533-1539[Abstract] |
2. | Toker, A. (1998) Curr. Opin. Cell Biol. 10, 254-261[CrossRef][Medline] [Order article via Infotrieve] |
3. | Hinchliffe, K. A., Ciruela, A., and Irvine, R. F. (1998) Biochim. Biophys. Acta 1436, 87-104[Medline] [Order article via Infotrieve] |
4. | Martin, T. F. (1998) Annu. Rev. Cell Dev. Biol. 14, 231-264[CrossRef][Medline] [Order article via Infotrieve] |
5. | Odorizzi, G., Babst, M., and Emr, S. D. (2000) Trends Biochem. Sci 25, 229-235[CrossRef][Medline] [Order article via Infotrieve] |
6. | Janmey, P. A. (1994) Annu. Rev. Physiol. 56, 169-191[CrossRef][Medline] [Order article via Infotrieve] |
7. | Gilmore, A. P., and Burridge, K. (1996) Nature 381, 531-535[CrossRef][Medline] [Order article via Infotrieve] |
8. | De Corte, V., Gettemans, J., and Vandekerckhove, J. (1997) FEBS Lett. 401, 191-196[CrossRef][Medline] [Order article via Infotrieve] |
9. | Ren, X. D., and Schwartz, M. A. (1998) Curr. Opin. Genet. Dev. 8, 63-67[CrossRef][Medline] [Order article via Infotrieve] |
10. | Majerus, P. W. (1996) Genes Dev. 10, 1051-1053[CrossRef][Medline] [Order article via Infotrieve] |
11. | Mitchell, C. A., Brown, S., Campbell, J. K., Munday, A. D., and Speed, C. J. (1996) Biochem. Soc. Trans. 24, 994-1000[Medline] [Order article via Infotrieve] |
12. | Srinivasan, S., Seaman, M., Nemoto, Y., Daniell, L., Suchy, S. F., Emr, S., De Camilli, P., and Nussbaum, R. (1997) Eur. J. Cell Biol. 74, 350-360[Medline] [Order article via Infotrieve] |
13. |
Stolz, L. E.,
Huynh, C. V.,
Thorner, J.,
and York, J. D.
(1998)
Genetics
148,
1715-1729 |
14. | McPherson, P. S., Garcia, E. P., Slepnev, V. I., David, C., Zhang, X., Grabs, D., Sossin, W. S., Bauerfeind, R., Nemoto, Y., and De Camilli, P. (1996) Nature 379, 353-357[CrossRef][Medline] [Order article via Infotrieve] |
15. |
Guo, S.,
Stolz, L. E.,
Lemrow, S. M.,
and York, J. D.
(1999)
J. Biol. Chem.
274,
12990-12995 |
16. |
Singer-Kruger, B.,
Nemoto, Y.,
Daniell, L.,
Ferro-Novick, S.,
and De Camilli, P.
(1998)
J. Cell Sci.
111,
3347-3356 |
17. | Raucher, D., Stauffer, T., Chen, W., Shen, K., Guo, S., York, J. D., Sheetz, M. P., and Meyer, T. (2000) Cell 100, 221-228[Medline] [Order article via Infotrieve] |
18. |
Winzeler, E. A.,
Shoemaker, D. D.,
Astromoff, A.,
Liang, H.,
Anderson, K.,
Andre, B.,
Bangham, R.,
Benito, R.,
Boeke, J. D.,
Bussey, H.,
Chu, A. M.,
Connelly, C.,
Davis, K.,
Dietrich, F.,
Dow, S. W.,
El Bakkoury, M.,
Foury, F.,
Friend, S. H.,
Gentalen, E.,
Giaever, G.,
Hegemann, J. H.,
Jones, T.,
Laub, M.,
Liao, H.,
Davis, R. W.,
et al..
(1999)
Science
285,
901-906 |
19. | Jones, J. S., and Prakash, L. (1990) Yeast 6, 363-366[Medline] [Order article via Infotrieve] |
20. | Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1991) Current Protocols in Molecular Biology , John Wiley and Sons, Inc., New York |
21. |
Ooms, L. M.,
McColl, B. K.,
Wiradjaja, F.,
Wijayaratnam, A. P. W.,
Gleeson, P.,
Gething, M.-J.,
Sambrook, J.,
and Mitchell, C. A.
(2000)
Mol. Cell. Biol.
20,
9376-9390 |
22. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
23. | Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 4350-4354[Abstract] |
24. | Franzusoff, A., Redding, K., Crosby, J., Fuller, R. S., and Schekman, R. (1991) J. Cell Biol. 112, 27-37[Abstract] |
25. |
Seedorf, M.,
and Silver, P. A.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
8590-8595 |
26. |
Parlati, F.,
Dominguez, M.,
Bergeron, J. J.,
and Thomas, D. Y.
(1995)
J. Biol. Chem.
270,
244-253 |
27. | Paddon, C., Loayza, D., Vangelista, L., Solari, R., and Michaelis, S. (1996) Mol. Microbiol. 19, 1007-1017[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Bascom, R. A.,
Srinivasan, S.,
and Nussbaum, R. L.
(1999)
J. Biol. Chem.
274,
2953-2962 |
29. | Gietz, R. D., and Sugino, A. (1988) Gene (Amst.) 74, 527-534[CrossRef][Medline] [Order article via Infotrieve] |
30. | Parekh, R., Forrester, K., and Wittrup, D. (1995) Protein Expression Purif. 6, 537-545[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402 |
32. | Kutay, U., Hartmann, E., and Rapoport, T. A. (1993) Trends Cell Biol. 3, 72-75[CrossRef] |
33. | Kuroda, R., Kinoshita, J., Honsho, M., Mitoma, J., and Ito, A. (1996) J. Biochem. (Tokyo) 120, 828-833[Abstract] |
34. |
Yang, M.,
Ellenberg, J.,
Bonifacino, J. S.,
and Weissman, A. M.
(1997)
J. Biol. Chem.
272,
1970-1975 |
35. |
da Fonseca, F. G.,
Wolffe, E. J.,
Weisberg, A.,
and Moss, B.
(2000)
J. Virol.
74,
7508-7517 |
36. | Wattenberg, B., and Lithgow, T. (2001) Traffic 2, 66-71[CrossRef][Medline] [Order article via Infotrieve] |
37. | Kutay, U., Ahnert-Hilger, G., Hartmann, E., Wiedenmann, B., and Rapoport, T. A. (1995) EMBO J. 14, 217-223[Abstract] |
38. | Egan, B., Beilharz, T., George, R., Isenmann, S., Gratzer, S., Wattenberg, B., and Lithgow, T. (1999) FEBS Lett. 451, 243-248[CrossRef][Medline] [Order article via Infotrieve] |
39. | Kim, P. K., Janiak-Spens, F., Trimble, W. S., Leber, B., and Andrews, D. W. (1997) Biochemistry 36, 8873-8882[CrossRef][Medline] [Order article via Infotrieve] |
40. | Lan, L., Isenmann, S., and Wattenberg, B. W. (2000) Biochem. J. 349, 611-621[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Pedrazzini, E.,
Villa, A.,
Longhi, R.,
Bulbarelli, A.,
and Borgese, N.
(2000)
J. Cell Biol.
148,
899-914 |
42. | Dailey, H. A., and Strittmatter, P. (1978) J. Biol. Chem. 253, 8203-8209[Abstract] |
43. | Carmichael, G. G., Schaffhausen, B. S., Dorsky, D. I., Oliver, D. B., and Benjamin, T. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3579-3583[Abstract] |
44. | Markland, W., Cheng, S. H., Oostra, B. A., and Smith, A. E. (1986) J. Virol. 59, 82-89[Medline] [Order article via Infotrieve] |
45. |
Chen-Levy, Z.,
and Cleary, M. L.
(1990)
J. Biol. Chem.
265,
4929-4933 |
46. | Masaki, R., Yamamoto, A., and Tashiro, Y. (1994) J. Cell Biol. 126, 1407-1420[Abstract] |
47. | Frangioni, J. V., Beahm, P. H., Shifrin, V., Jost, C. A., and Neel, B. G. (1992) Cell 68, 545-560[Medline] [Order article via Infotrieve] |
48. |
Kuroda, R.,
Ikenoue, T.,
Honsho, M.,
Tsujimoto, S.,
Mitoma, J. Y.,
and Ito, A.
(1998)
J. Biol. Chem.
273,
31097-31102 |
49. |
Pedrazzini, E.,
Villa, A.,
and Borgese, N.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
4207-4212 |
50. | Rayner, J. C., and Pelham, H. R. (1997) EMBO J. 16, 1832-1841[Abstract] |
51. | Matsuoka, K., Orci, L., Amherdt, M., Bednarek, S. Y., Hamamoto, S., Schekman, R., and Yeung, T. (1998) Cell 93, 263-275[Medline] [Order article via Infotrieve] |
52. |
Hama, H.,
Schnieders, E. A.,
Thorner, J.,
Takemoto, J. Y.,
and DeWald, D. B.
(1999)
J. Biol. Chem.
274,
34294-34300 |
53. |
Audhya, A.,
Foti, M.,
and Emr, S. D.
(2000)
Mol. Biol. Cell
11,
2673-2689 |
54. |
Becker, F.,
Block-Alper, L.,
Nakamura, G.,
Harada, J.,
Wittrup, K. D.,
and Meyer, D. I.
(1999)
J. Cell Biol.
146,
273-284 |
55. |
Stolz, L. E.,
Kuo, W. J.,
Longchamps, J.,
Sekhon, M. K.,
and York, J. D.
(1998)
J. Biol. Chem.
273,
11852-11861 |
56. | Olivos-Glander, I. M., Janne, P. A., and Nussbaum, R. L. (1995) Am. J. Hum. Genet. 57, 817-823[Medline] [Order article via Infotrieve] |
57. |
Zhang, X.,
Hartz, P. A.,
Philip, E.,
Racusen, L. C.,
and Majerus, P. W.
(1998)
J. Biol. Chem.
273,
1574-1582 |
58. |
Kong, A. M.,
Speed, C. J.,
O'Malley, C. J.,
Layton, M. J.,
Meehan, T.,
Loveland, K. L.,
Cheema, S.,
Ooms, L. M.,
and Mitchell, C. A.
(2000)
J. Biol. Chem.
275,
24052-24064 |
59. | Mitoma, J., and Ito, A. (1992) EMBO J. 11, 4197-4203[Abstract] |
60. | Linstedt, A. D., Foguet, M., Renz, M., Seelig, H. P., Glick, B. S., and Hauri, H. P. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5102-5105[Abstract] |
61. |
Whitley, P.,
Grahn, E.,
Kutay, U.,
Rapoport, T. A.,
and von Heijne, G.
(1996)
J. Biol. Chem.
271,
7583-7586 |
62. |
Kim, P. K.,
Hollerbach, C.,
Trimble, W. S.,
Leber, B.,
and Andrews, D. W.
(1999)
J. Biol. Chem.
274,
36876-36882 |
63. | Grote, E., Hao, J. C., Bennett, M. K., and Kelly, R. B. (1995) Cell 81, 581-589[Medline] [Order article via Infotrieve] |
64. |
Rapoport, T. A.
(1991)
FASEB J.
5,
2792-2798 |
65. | Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. (1993) Cell 75, 1137-1144[Medline] [Order article via Infotrieve] |
66. |
Palmer, D. J.,
Helms, J. B.,
Beckers, C. J.,
Orci, L.,
and Rothman, J. E.
(1993)
J. Biol. Chem.
268,
12083-12089 |
67. |
Randazzo, P. A.
(1997)
J. Biol. Chem.
272,
7688-7692 |
68. | Walch-Solimena, C., and Novick, P. (1999) Nat. Cell Biol. 1, 523-525[CrossRef][Medline] [Order article via Infotrieve] |
69. | Wiedemann, C., Schafer, T., and Burger, M. M. (1996) EMBO J. 15, 2094-2101[Abstract] |
70. | Wilsbach, K., and Payne, G. S. (1993) EMBO J. 12, 3049-3059[Abstract] |