 |
INTRODUCTION |
The multisubunit complex, oligosaccharyltransferase (1), catalyzes
the transfer of Glc3Man9GlcNAc2
from dolichyl pyrophosphate (Dol-P-P)1 to appropriate
asparagine residues during the co-translational N-glycosylation of nascent polypeptides in yeast and
mammalian cells (2-4). During the primary N-glycosylation
reaction, Dol-P-P is released on the luminal surface of the endoplasmic
reticulum (ER). In order for Dol-P-P to be re-utilized as a glycosyl
carrier lipid for additional rounds of lipid intermediate biosynthesis, it must be converted to dolichyl phosphate (Dol-P) and translocated to
the cytoplasmic leaflet of the ER (4). Although it cannot yet be
excluded that Dol-P-P, or perhaps Dol-P, diffuses transversely from the
luminal leaflet to the cytoplasmic face by a protein-mediated mechanism, it is more likely that it is dephosphorylated on the luminal
surface to form free dolichol that could more readily diffuse back to
the cytoplasmic leaflet. Cytoplasmically oriented dolichol would then
be re-phosphorylated by dolichol kinase (5, 6). Recent studies (7, 8)
have shown that the CWH8 gene in Saccharomyces
cerevisiae encodes a Dol-P-P phosphatase that actively converts
Dol-P-P to Dol-P and is also capable of dephosphorylating Dol-P at a
slower rate. Moreover, the yeast Dol-P-P phosphatase is recovered in
crude microsomal fractions, but its subcellular location has not been established.
Although there have been numerous reports (4, 7-13) that crude
microsomal fractions from a variety of mammalian cells contain enzymes
that can hydrolyze exogenous Dol-P-P, the identity, specificity, number, and exact subcellular location of the enzymes catalyzing the
hydrolysis of Dol-P-P have not been established. Recently, Inoue
et al. (14) have described a mouse brain cDNA, referred to as gene 2-23, with a high degree of homology to yeast
CWH8. The current study presents several lines of evidence
that mouse 2-23 encodes a mammalian homologue of the CWH8
gene product from yeast. In this report, this gene is referred to as
Dol-P-P phosphatase 1 (DOLPP1). The identification of the
mammalian homologue as a Dol-P-P pyrophosphate phosphatase is based on
its ability to complement defects in growth and protein
N-glycosylation in the cwh8
mutant (8, 15) and
to prevent the accumulation of Dol-P-P in the mutant cells. In
addition, expression of DOLPP1 modified with either a
carboxyl-terminal His6 tag in the yeast mutants or an amino-terminal extension encoding the T7 bacteriophage epitope in
Sf9 cells results in the appearance of a new polypeptide with an
apparent molecular mass of 27 kDa and a substantial increase in
the level of Dol-P-P phosphatase activity. The mouse enzyme exhibits a
marked preference for Dol-P-P over Dol-P and phosphatidic acid (PA), as
reported for the yeast enzyme.
The results described in this paper provide solid evidence that
DOLPP1 encodes the mammalian homologue of the Dol-P-P
pyrophosphate phosphatase encoded by the CWH8 gene in
S. cerevisiae, and represent the first experimental proof
that this enzyme is located in the ER. The possible function of this
novel lipid pyrophosphate phosphatase in the recycling of the glycosyl
carrier lipid in the ER of brain and other mammalian cells is discussed.
| |
EXPERIMENTAL PROCEDURES |
Materials--
Plasmid YEp352 containing the yeast gene
CWH8 coding sequence and 240 bp of 5'-flanking sequence was
a gift from Drs. Fabiana Fernandez and Markus Aebi (ETH, Zurich,
Switzerland). The cDNA designated as m2-23 was a gift
from Dr. S. Inoue (Tsukuba Research Institute, Banyu Pharmaceutical
Co., Ltd. Ibaraki Prefecture, Japan). Yeast strains W303-1A,
cwh8, and lpp1/dpp1 were the generous gifts from Dr. George Carman, Department of Food Science, Rutgers University, New Brunswick, NJ.
n-Octyl-
-D-glucopyranoside was purchased from
Calbiochem-Novabiochem. Triton X-100 was from Pierce.
Trichloroacetonitrile, tetrabutylammonium hydroxide, and anhydrous
acetonitrile were obtained from Aldrich. Nycodenz, dioleoylphosphatidic acid (lot number 129F8359), and diolein (D-8894) were obtained from
Sigma. Dolichol (C95) was a generous gift from Dr. M. Mizuno, Kuraray
Chemical Co. (Okayama, Japan). Carrier-free
[32P]phosphoric acid was purchased from American
Radiolabeled Chemicals (St. Louis, MO). Econo-safeTM
biodegradable scintillation counting mixture is a product of Research
Products International (Mount Prospect, IL). Yeast nitrogen base, yeast
extract, and BactoPeptone are products of BD Biosciences. Casamino
acids is a product of Fisher. Antibodies were obtained from the
following commercial sources and used according to instructions from
the supplier: rabbit anti-calnexin polyclonal antibody and mouse
anti-KDEL monoclonal antibody (StressGen Biotechnologies, Victoria,
British Columbia, Canada); mouse anti-KDEL receptor monoclonal
antibody, mouse anti--COP monoclonal antibody, horseradish peroxidase-conjugated sheep anti-rabbit IgG, horseradish
peroxidase-conjugated sheep anti-mouse IgG (Amersham Biosciences); and
fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and Texas
Red-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR).
Anti-CPY antiserum was generously provided by Dr. Neta Dean, State
University of New York, Stony Brook, NY. All other chemicals and
reagents were purchased from standard commercial sources.
Affinity-purified Rabbit Anti-Dolpp1p Antibodies and
Immunoblotting--
Three New Zealand White rabbits were each
immunized with 300 µg of a synthetic peptide corresponding to amino
acid residues 86-105 of mouse Dolpp1p coupled to keyhole limpet
hemocyanin as described previously (16). The antigen was injected
intradermally in Freund's complete adjuvant, and rabbits were boosted
three times at 3-week intervals with 300 µg of peptide in Freund's
incomplete adjuvant. An IgG fraction was prepared from preimmune and
immune serum by specific binding to protein A-Sepharose CL-4B (Amersham Biosciences). IgG was affinity-purified by specific binding to a column
consisting of the Dolpp1p peptide cross-linked to SulfoLink coupling
gel (Pierce). For analysis of DolPP1 expression by
immunoblotting, samples were analyzed by electrophoresis in 15%
SDS-PAGE gels and immunoblotting essentially as described (17). Filters
were blocked for 1 h with Sea Block (East Coast Biologics, North
Berwick, ME) and washed in PBS-T (0.25% (v/v) Tween 20 in
phosphate-buffered saline) followed by incubation for 2 h with
rabbit anti-Dolpp1p polyclonal antibody (1 µg/ml). The filters were
washed with PBS-T and incubated for 45 min in a solution containing
horseradish peroxidase-conjugated secondary antibody (sheep anti-rabbit
IgG, Amersham Biosciences) diluted 1:2500 in PBS-T. The filters were washed and developed using ECL chemiluminescence reagents (Amersham Biosciences).
Preparation of Radiolabeled Phosphorylated Lipid
Substrates--
[32P]PA, [32P]Dol-P,
[
,-32P]Dol-P-P, and [-32P]Dol-P-P
were chemically synthesized in anhydrous acetonitrile from the
appropriate lipid using [32P]tetrabutylammonium phosphate
and trichloroacetonitrile as described by Danilov et al.
(18). Unlabeled polyisoprenyl phosphates were synthesized using
tetrabutylammonium phosphate. Synthetic substrates were purified by
preparative thin layer chromatography on Baker Si250 thin layer plates
developed in
CHCl3/CH3OH/H2O/NH4OH
(65:35:6:1, v/v).
Recombinant DNA Methods--
Plasmid and genomic DNA
preparation, restriction enzyme digestion, and DNA ligations were
performed by standard methods (19). Transformation of yeast (20) and
Escherichia coli (19) were performed as described. The
annealing temperature for the PCR was 55 °C, and extension times
were typically 2.0 min at 72 °C. PCRs were routinely run for a total
of 30 cycles. DNA sequencing reactions were performed by Davis
Sequencing, LLC (Davis, CA). For blot hybridization of RNA, total RNA
was isolated from mouse tissues as described (21). Blots were
pre-hybridized for 30 min at 65 °C in Rapid-Hyb buffer (Amersham
Biosciences), followed by hybridization with a random hexamer-primed
32P-labeled mouse cDNA fragment corresponding to the
entire 717-bp coding region of DolPP1 for 3 h at 65 °C. The
blots were washed in 2× SSC (1× SSC = 150 mM NaCl
and 15 mM sodium citrate (pH 7)), 0.1% SDS at ambient
temperature for 30 min, followed by stringent washes in 0.5× SSC,
0.1% SDS at 65 °C for 15 min and in 0.1 × SSC, 0.1% SDS at
65 °C for 15 min.
Yeast Culture--
Yeast cultures were grown at 30 °C in
either 1% yeast extract, 2% BactoPeptone, and 2% dextrose or in
yeast nitrogen base (6.7 g/liter), 50 mM sodium succinate
(pH 5.0), 2% dextrose, amino acids (25 mg/liter), and purine and
pyrimidine bases (25 mg/liter) as required to meet auxotrophic
requirements for selective growth. Yeast transformants were screened on
2% agar plates containing 6.7 g/liter yeast nitrogen base, 0.5%
casamino acids, 50 mM sodium succinate (pH 5.0), 2%
dextrose, and 25 mg/liter adenine or leucine as required. The yeast
strains used in this study and their respective genotypes are contained
in Table I.
Expression of Mus musculus DOLPP1 and DOLPP1-His6 in
Yeast--
A DNA fragment containing 240 bp upstream of the coding
sequence of the yeast CWH8 gene was amplified by PCR using
the appropriate oligonucleotide primers (forward primer,
5'-ACTGAATTCGGGTTTCTGGGAAAAC-3'; reverse primer,
5'-CCCGGTACCGATATGATCCGATTCAAAACA-3') and plasmid YEp352
containing the CWH8 gene (8, 15) as template. The forward
and reverse primers were designed to contain altered bases to insert
flanking EcoRI and KpnI restriction sites to
facilitate cloning (underlined above). The resulting PCR product was
digested with EcoRI and KpnI and ligated into
similarly prepared YEp352 (22) to generate a plasmid containing the
putative yeast CWH8 promoter sequence, YEp352/yPro. A DNA
fragment containing the entire murine 2-23 coding sequence flanked by
KpnI and BamHI restriction sites (underlined in
sequences below) was amplified by PCR using the appropriate
oligonucleotide primers (forward,
5'-GGTCTCCGGGTACCATGGCAGCGGA-3'; reverse,
5'-ACGGGATCCTCACTGCAGTTTTGTT-3') and a cDNA
containing the mouse 2-23 gene obtained from Tsukuba Research
Institute, Banyu Pharmaceutical Co., Ltd. (Tsukuba, Ibaraki, Japan), as
template. The PCR product was digested with
KpnI/BamHI to yield a 0.74-kb DNA fragment. This
fragment was then ligated into the KpnI/BamHI sites of plasmid YEp352/yPro to form the plasmid YEp352/yPro-DOLPP1. The correct DNA sequence of YEp352/yPro-DOLPP1 was confirmed by direct
sequencing using the M13 primer sites in YEp352. This construct was
then transformed into the pertinent yeast strains for the expression of
the mouse DOLPP1 gene according to Schiestl and Gietz
(20).
DOLPP1 carrying a carboxyl-terminal His6 tag was
prepared by PCR using appropriate oligonucleotide primers (forward,
5'-ACTGAATTCGGGTTTCTGGGAAAAC-3'; reverse,
5'-TCTAAGCTTTCAGTGGTGGTGGTGGTGGTGCTGCAGTTTTGTT-3') and pBS/yPro-DOLPP1 cDNA as template. The PCR product was digested with
EcoRI/HindIII (underlined in sequences above) and
ligated into similarly prepared YEp352. The construct was transformed into the pertinent yeast strains for the expression of
DOLPP1-His6 according to Schiestl and Gietz
(20). Yeast microsomal fractions for the routine analysis of lipid
phosphatase activities were prepared exactly as described previously
(8). Assays of Dol-P-P, Dol-P, and PA phosphatase activities were
performed as described previously (9, 23).
Measurement of Cellular Levels of Dol-P and Dol-P-P in Yeast
Cells--
Yeast cells (1 liter) were grown in YPD containing 2%
dextrose to 1-4 A600 and collected by
centrifugation at 1,000 × g, 20 min. The cells were
resuspended in distilled water, sedimented again, resuspended in
distilled water to 200 A600/ml, and incubated for 30 min at 30 °C in 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 containing 1 mg/ml lyticase (Sigma).
The yeast spheroplasts were sedimented at 1,000 × g
for 20 min; the supernatant was decanted, and the pellet was mixed
vigorously with 40 volumes of CHCl3/CH3OH (2:1, v/v). Dol-32P and Dol-P-32P (5,000 cpm of each)
were added as recovery markers, and the insoluble material was
sedimented in a clinical centrifuge. The organic layer was removed and
reserved on ice. The insoluble pellet was sequentially extracted twice
with 5 ml of CHCl3/CH3OH (2:1 v/v) and twice
with 5 ml of CHCl3/CH3OH/H2O
(10:10:3, v/v). The organic extracts were combined, taken to dryness by
rotary evaporation under reduced pressure at 30 °C, and then
deacylated in 1 ml of toluene/CH3OH (1:1, v/v) containing
0.1 M KOH on ice for 30 min. Following deacylation, the
reaction was neutralized with acetic acid, diluted with 5 ml of
CHCl3/CH3OH (2:1 v/v), and partitioned with 1 ml of water. The aqueous layer was discarded, and the organic phase was
subsequently partitioned twice with 1 ml of
CHCl3/CH3OH/H2O (3:48:47, v/v). The
organic phase was dried under a stream of N2 gas and
redissolved in 0.2 ml of
CHCl3/CH3OH/H2O (10:10:3, v/v).
Dol-P and Dol-P-P in the organic extracts were separated by ion
exchange chromatography on a 20-ml column of DEAE-650M (Toyopearl,
Supelco, Bellefonte, PA) equilibrated with
CHCl3/CH3OH/H2O (10:10:3, v/v) and
eluted with a linear gradient of 0 to 0.2 M ammonium
acetate (60 ml). The fractions containing Dol-P and Dol-P-P were pooled
separately and supplemented with CHCl3 and water to give a
final composition of
CHCl3/CH3OH/H2O (3:2:1, v/v). The phases were separated by a brief centrifugation, and the aqueous layer
was discarded. The organic layer was then partitioned once with 1/5
volume of CHCl3/CH3OH/H2O (3:48:47,
v/v), taken to dryness under a stream of N2 gas, and
redissolved in a small volume of CHCl3/CH3OH/H2O (10:10:3, v/v).
Aliquots representing equivalent portions of the initial extracts were
spotted on 10 × 20-cm Baker Si250 Silica Gel thin layer plates
and developed in
CHCl3/CH3OH/H20/NH4OH (65:35:6:1,v/v). Following chromatography, the thin layer plates were
allowed to air dry and were visualized by staining with anisaldehyde (24). The migration positions of radioactive standards were determined
with a Bioscan System 200 Imaging Scanner (Bioscan Inc. Washington,
D. C.).
Expression of T7-DOLPP1 in Insect Cells--
A DNA fragment
containing the coding sequence of the mouse 2-23 gene flanked by
KpnI and HindIII and a 5'-extension encoding the
T7 bacteriophage epitope was obtained by PCR (forward primer, 5'-GGGGTACCATGGCTAGCATGACTGGTGGACAGCAAATGGGTATGGCAGCGGACGGA-3'; reverse primer, 5'-TCTAAGCTTTCACTGCAGTTTTGTTCCA-3') using
the cDNA plasmid containing the mouse 2-23 coding sequence as a
template. The PCR product was digested with
KpnI/HindIII (underlined in sequences above),
ligated into the same restriction enzyme sites of the pFASTBAC1
baculovirus vector, and expressed in insect Sf9 cells using the
Bac-to-Bac Baculovirus Expression System (Invitrogen). Sf9 cells
were routinely grown at 27 °C in Sf900 II SFM (Invitrogen) containing penicillin (50 units/ml) and streptomycin (50 µg/ml) (25).
Cells were infected with a viral multiplicity of 5-10 and harvested
after 48 h. To prepare a microsomal fraction, Sf9 cells
were sedimented by centrifugation at 1,000 × g for 10 min, resuspended in 20 ml of ice-cold 10 mM HEPES (pH 7.0)
and 0.25 M sucrose per ml of packed cells, and lysed by
sonication (four 15-s pulses) with a Kontes Micro Ultrasonic Cell
Disruptor at 40% full power. Unbroken cells and cellular debris were
removed by centrifugation at 1,000 × g for 10 min at
4 °C. Microsomes were then sedimented (100,000 × g
for 10 min) in a Beckman TL-100 tabletop ultracentrifuge and
resuspended at a membrane protein concentration of ~10 mg/ml in 10 mM HEPES (pH 7.0), 0.25 M sucrose for further analysis.
Expression of pCHA7/DOLPP1 in COS Cells--
To
construct a Dolpp1p transient expression plasmid for mammalian COS
cells, the GenBankTM expressed sequence tag division was
searched for clones homologous to S. cerevisiae Cwh8p,
yielding IMAGE clone 1974625, GenBankTM accession number
AI876279. (This clone was obtained independently of gene 2-23 sequence
described above.) Both strands of the cDNA were sequenced, which
revealed that the clone was incomplete at the 5' end. Therefore, a
5'-RACE procedure was employed using 1 µg of mouse total brain
mRNA and a SMART RACE cDNA Amplification Kit
(Clontech Laboratories, Inc., Palo Alto, CA).
Briefly, Moloney murine leukemia virus-reverse transcriptase
Superscript II (Invitrogen) was used to perform the reverse
transcription according to the supplier's protocol. PCR was performed
using the supplied universal primer mix, a gene-specific primer
(5'-CTGGGAATGGCTGGAGGGCATCCCG-3'), and a nested primer
(5'-CGCCCTGATTCAGTGCCAGTCCCCC-3') to obtain the 5' terminus of
mouse DOLPP1 cDNA. The PCR was performed using a
PerkinElmer Life Sciences GeneAmp System 9600 using the Advantage 2 PCR
kit (Clontech Laboratories, Inc., Palo Alto, CA)
under conditions suggested by the supplier. The resulting fragment was
subcloned into the plasmid pGEM-T-Easy (Promega, Madison, WI),
sequenced, and shown to be complete at the 5' end. The entire open
reading frame was then amplified from total mouse brain cDNA using
primers corresponding to sequences within the 5'-RACE product and mouse expressed sequence tag IMAGE clone 1974625 (5'-
ATGGCAGCGGACGGACAGTGC-3' and 5'-TCACTGCAGTTTTGTTCCAAG-3'). PCR
conditions for the amplification consisted of the following: 95 °C,
5 min, one cycle; 94 °C, 30 s, 68bto 58 °C ("touch down"
protocol), 30 s, 72 °C, 1 min for 10 cycles; 94 °C, 30 s, 58 °C, 30 s, 72 °C, 1 min for 25 cycles; 72 °C, 10 min. The resulting PCR product was subcloned into pCHA7 (26) using
SalI and BamHI restriction sites at the 5' and 3' ends of the insert, respectively. The construct was verified by sequencing, and the open reading frame was found to be 100% identical to GenBankTM AB030189, which was deposited subsequently
(14).
Simian COS-7L cells were maintained in Dulbecco's modified Eagle's
medium (high glucose), supplemented with 10% (v/v) fetal bovine serum,
100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml
amphotericin B. Cells were plated on Day 0 in 100-mm dishes and were
transiently transfected on Day 1 using 6 µg of DNA (pCHA7/DOLPP1) and
18 µl of FuGENE 6 reagent (Roche Molecular Biochemicals) per plate.
The cells were harvested 48 h post-transfection into ice-cold
phosphate-buffered saline and pelleted at low speed. Cell pellets were
resuspended in a homogenization buffer containing 50 mM
Tris-HCl (pH 7.5), 0.25 mM sucrose, 5 mM EDTA,
and 5 mM 2-mercaptoethanol and sonicated briefly. Nuclei
and intact cells were removed by centrifugation at 500 × g for 5 min at 4 °C. The postnuclear supernatant was
further centrifuged at 100,000 × g for 1 h at
4 °C in a Beckman Optima TLX ultracentrifuge. The resulting supernatant (S100) was reserved, and the membrane pellet (P100) was
washed once and resuspended in homogenization buffer for immunoblotting or in 10 mM HEPES (pH 7.0), 0.25 M sucrose at a
concentration of 10 mg of protein per ml for analysis of enzyme activity.
Subcellular Localization of Dolpp1p--
COS-7L cells (six
100-mm dishes) were transfected using pCHA7/DOLPP1 as described above
and washed twice in a buffer consisting of 10 mM
triethanolamine, 10 mM acetic acid, 250 mM
sucrose, 1 mM EDTA, and 1 mM dithiothreitol.
Cells were harvested with a rubber policeman in 800 µl of buffer
containing 10 µg/ml each of chymostatin, leupeptin, and pepstatin and
homogenized by 15 passages through a 25-gauge needle on a 1-ml syringe.
Nuclei and intact cells were removed by microcentrifugation at
1,200 × g for 5 min at 4 °C. The postnuclear
supernatant was loaded on preformed Nycodenz gradients prepared exactly
as described (27). Briefly, linear Nycodenz gradients were prepared for
the Beckman SW 41 Ti rotor from initial discontinuous gradients (24, 19, 15, and 10% Nycodenz in 10 mM Tris-HCl (pH 7.4), 3 mM KCl, and 1 mM EDTA) that were allowed to
diffuse in a horizontal position for 45 min at room temperature and
then centrifuged for 4 h at 37,000 rpm in a Beckman L8-M
ultracentrifuge to generate a nonlinear density gradient profile. The
postnuclear supernatant was loaded on top of the gradients and
centrifuged for 1.5 h at 37,000 rpm. Fifteen fractions were
collected, and aliquots of each fraction were subjected to
electrophoresis on SDS-PAGE gels. The distribution of Dolpp1p, calnexin
(ER marker), KDEL receptor (Golgi marker), and -COP (Golgi marker)
in the gradients was determined by immunoblotting using ECL
chemiluminescence reagents (Amersham Biosciences).
Immunofluorescence--
COS-7L cells were grown on 12-mm
diameter coverslips and transfected with pCHA7/DOLPP1 using FuGENE 6 as
described above. At 48 h post-transfection, the cells were fixed
for 15 min in 4% formaldehyde and permeabilized for 10 min with 0.05%
Triton X-100 at room temperature. The cells were doubly stained using the rabbit anti-Dolpp1p antibody and either the anti-KDEL monoclonal antibody or anti-KDEL receptor monoclonal antibody. The secondary antibodies used were fluorescein isothiocyanate-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. Immunofluorescence was visualized under a Zeiss Axiophot
immunofluorescence microscope.
Preparation of Sealed Microsomal Vesicles and Estimation of
Vesicle Integrity--
Sealed microsomal vesicles were prepared from
the pertinent yeast strains and evaluated for integrity as described
previously (8, 28).
General Analytical Methods--
Protein was determined by the
method of Rodriquez-Vico et al. (29) using a commercial
protein assay reagent (BCA, Pierce) and bovine serum albumin as
standard. Lipid-phosphorus analysis was according to Bartlett (30).
Radioactivity was measured by liquid scintillation spectrometry in a
Packard Tri-Carb TR-2100 Liquid Scintillation Analyzer (Packard
Instrument Co., Meriden, CT) in the presence of Econo-Safe
Biodegradable Counting Mixture (Research Products International, Mount
Prospect, IL).
| |
RESULTS |
Mammalian Cells Express a Homologue of the Yeast CWH8
Gene--
Recently, Inoue and co-workers (14) identified and sequenced
64 cDNA clones from a mouse brain cDNA library that suppress bacterial growth when expressed in E. coli. One of these
clones (GenBankTM accession number AB030189) has a high
degree of homology to the yeast CWH8 gene
(GenBankTM accession number NP_011550), an ER
Dol-P-P phosphatase with a luminally oriented active site (7, 8). Fig.
1 compares the deduced amino acid
sequences of yeast Cwh8p and the predicted amino acid sequences from
the mouse brain cDNA (designated Dolpp1p). The sequences (Fig. 1,
panel A) are 29.8% identical and 49.4% strongly similar.
In addition, Dolpp1p contains the consensus lipid-phosphate phosphatase
motif (Fig. 1, panel B) defined by Stukey and Carman (31),
suggesting that Dolpp1p is the mammalian homologue of yeast CWH8.
View larger version (41K):
[in this window]
[in a new window]
|
Fig. 1.
Amino acid sequence alignment of proteins
encoded by yeast CWH8 and mouse
DOLPP1, and comparison of Dolpp1p with the consensus
lipid phosphate phosphatase motif. Alignment (panel A)
was performed using the ClustalW program available through Pole
Bio-Informatique Lyonnais. Gaps in the sequences are
indicated by dashes. Identical amino acid residues are
shaded dark gray; similar residues are light
gray. Dolpp1p contains the consensus lipid phosphate phosphatase
motif (panel B) reported by Stukey and Carman (31).
|
|
The DOLPP1 mRNA was found to be widely expressed in
various mouse tissues as assessed by RNA blotting (Fig.
2). A single transcript migrating at 2.0 kb was observed in all tissues examined, with highest expression in
brain, kidney, lung, and intestine and low but detectable levels in
liver, spleen, and uterus. The broad tissue distribution is consistent
with the ER Dol-P-P phosphatase having an essential function in the
lipid intermediate pathway for protein N-glycosylation in
all mammalian cells.
View larger version (58K):
[in this window]
[in a new window]
|
Fig. 2.
RNA blot hybridization of DOLPP1
RNA from mouse tissues. Total RNA (20 µg) from various
tissues was subjected to electrophoresis and transferred to a nylon
membrane. Hybridization was performed using a 32P-labeled
DOLPP1 cDNA probe (upper panel) followed by
washing as described under "Experimental Procedures." The filter
was exposed to Amersham Biosciences Hyperfilm MP film with an
intensifying screen at  85 °C for 4 days. The integrity of RNA in
each lane was demonstrated by stripping and reprobing the filter with a
32P-labeled cDNA probe encoding mouse cyclophilin A
(lower panel).
|
|
Overexpression of Dolpp1p Corrects Defects in Growth Rate and
N-Glycosylation of CPY in the cwh8 Mutant--
The mutant yeast
strain, cwh8, grows at an abnormally slow rate and
exhibits a defect in protein N-glycosylation (8, 15). To
determine whether expression of mammalian DOLPP1 corrects
the growth deficiency of the cwh8 yeast
strain, the DOLPP1 gene was subcloned into YEp352
under the control of the putative yeast CWH8 promoter and
transformed into cwh8. The effect on rate of growth in
complete media was then assessed. As shown in Table II, the cell density of wild type yeast
cultures typically doubles approximately every 2 h, whereas the
doubling time (T2×) for cwh8 was
~4 h. Overexpression of DOLPP1 in cwh8
reduces the doubling time to 3 h, similar to the effect of
transformation with YEp352 containing the yeast CWH8 gene
(T2× = 2.5 h).
View this table:
[in this window]
[in a new window]
|
Table II
Overexpression of either DOLPP1 or CWH8 partially restores the normal
rate of growth in the cwh8 yeast mutant
Yeast strains were grown at 30 °C in 1% yeast extract, 2%
Bacto-Peptone, and 2% dextrose (YPD) to an A600 of
~0.1 OD/ml. A600 measurements were collected at
hourly intervals. Doubling times (T2×) were
calculated using the formula: T2× = (T · log2)/log(AT1/AT0).
Where AT0 is the A600 of
the culture at the start of the culture period,
AT1 is the A600 at some
later time, and T is the total elapsed time.
|
|
As reported previously (8, 15), mutation of the CWH8 gene in
yeast results in defects in protein N-glycosylation, as assessed by N-glycosylation of carboxypeptidase Y (CPY)
(Fig. 3). Although SDS-PAGE of wild type
yeast extracts revealed a single protein band corresponding to mature,
fully N-glycosylated CPY (Fig. 3, lane 1),
extracts from the cwh8 mutant contain several bands
corresponding to underglycosylated isoforms of CPY (Fig. 3, lane
2). (The migration positions of CPY lacking 1-4 oligosaccharide chains are indicated by the arrows.) Expression of YEp352
carrying either the DOLPP1 gene or the yeast CWH8
gene restored full glycosylation to CPY (Fig. 3, lanes 3 and
4, respectively). The conclusion that the faster migrating
bands represent underglycosylated isoforms of CPY was confirmed by the
observation that digestion with endoglycosidase H converted all of the
protein bands into the fastest migrating isoform (data not shown).
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Overexpression of DOLPP1
cDNA corrects hypoglycosylation of CPY in the yeast
cwh8 mutant. Total cell extracts
from the various yeast strains were separated by SDS-PAGE and analyzed
by Western blotting using -CPY serum as described previously (56).
Lane 1, wild type; lane 2, cwh8;
lane 3, cwh8/DOLPP1; and
lane 4, cwh8/CWH8. The
positions of mature CPY and underglycosylated isoforms are
indicated.
|
|
Overexpression of Either Dolpp1p or Cwh8p Reverses the
Accumulation of Dol-P-P in the cwh8 Mutant--
Abnormally high
levels of Dol-P-P accumulate in cwh8 yeast cells, due to
a lack of the Dol-P-P phosphatase activity responsible for the
hydrolysis of the Dol-P-P released during the transfer of the precursor
oligosaccharide from
Glc3Man9GlcNAc2-P-P-Dol to nascent
polypeptides in the lumen of the ER (8). To determine whether
overexpression of Dolpp1p in cwh8 will restore normal Dol-P-P levels, total lipid extracts were prepared from either wild
type, cwh8 plus YEp352, cwh8 plus
YEp352/yPro-DOLPP1, or cwh8 plus YEp352/CWH8 yeast cells
and analyzed for Dol-P-P (Fig. 4,
panel A, lanes 1-4). As reported earlier (8), the lipid extracts from cwh8 cells (Fig. 4, panel A, lane
2), contained a prominent anisaldehyde-positive compound with the
chromatographic mobility of standard Dol75-P-P. In contrast
to this result, virtually no Dol-P-P was detected in extracts from wild
type cells (Fig. 4, panel A, lane 1) or from
cwh8 yeast cells expressing either the DOLPP1
gene (Fig. 4, panel A, lane 3) or the CWH8 gene
(Fig. 4, panel A, lane 4). To confirm the identity of the
anisaldehyde-positive compound as Dol-P-P, this lipid was
quantitatively converted to a product chromatographically identical to
Dol75-P by mild acid hydrolysis (2 h at 80 °C in
CHCl3, CH3OH, 2 N HCl, 10:10:3,
v/v). It should be noted that the levels of Dol-P in the various yeast strains were not significantly different (Fig. 4, panel B, lanes 1-4).
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 4.
Overexpression of Dolpp1p corrects Dol-P-P
accumulation in the yeast cwh8
mutant. Dol-P-P was extracted from either wild type
(lane 1), cwh8 (lane 2),
cwh8/DOLPP1 (lane 3), or
cwh8/CWH8 yeast strains and chromatographed on
Baker Si250 Silica Gel thin layer plates as described under
"Experimental Procedures." Dol-P-P was visualized by anisaldehyde
staining (24).
|
|
Increase in Dol-P-P Phosphatase Activity Upon Expression of Dolpp1p
in lpp1/dpp1 Yeast or Sf9 Insect
Cells--
The results described above indicate that the DOLPP1
gene encodes a functional homologue of the yeast Dol-P-P
phosphatase. To provide more direct biochemical evidence that Dolpp1p
functions as a Dol-P-P phosphatase, microsomal fractions were prepared
from the lpp1/dpp1 yeast mutant strain
after transformation with YEp352/yPro-DOLPP1, and the rate of
hydrolysis of Dol-P-P was assayed. The
lpp1/dpp1 strain was chosen as the parental
strain for this study in order to minimize the nonspecific phosphatase activities encoded by LPP1 and DPP1 (7, 8, 13).
Microsomes from lpp1/dpp1/DOLPP1
were found to enzymatically dephosphorylate Dol-P-P at a markedly
higher rate than microsomes from the
lpp1/dpp1 (Fig.
5, panel A) parental strain.
Preliminary studies determined that the Dol-P-P phosphatase activity
encoded by DOLPP1 was active in the presence of
n-octyl glucoside and slightly less sensitive to
inactivation by Triton X-100 than Cwh8p. Divalent cations were not
required for activity for the mammalian enzyme (data not shown).
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 5.
Time course of Dol-P-P hydrolysis
(panel A) and comparison of Dol-P-P, Dol-P, and PA as
substrates (panel B) for phosphatase activity in
microsomal fractions from
lpp1/dpp1/DOLPP1
yeast. For the time course shown in panel A,
reaction mixtures for the determination of Dol-P-P phosphatase
contained yeast microsomes (10 µg of protein) from either
lpp1/dpp1/DOLPP1 ( ) or
lpp1/dpp1 ( ) strain, 10 mM
EDTA, 50 mM sodium citrate/sodium phosphate (pH 7.0), 0.6%
OG, and 20 µM [-32P]Dol-P-P in a total
volume of 0.02 ml. Following incubation at 30 °C for the indicated
periods, the amount of 32Pi released was
determined as described previously (9, 23). For the comparison of the
different phospholipid substrates (panel B), reaction
mixtures contained yeast microsomes from
lpp1/dpp1/DOLPP1 (10 µg of
protein), 10 mM EDTA, 50 mM sodium
citrate/sodium phosphate (pH 7.0), 0.6% OG, and the indicated
concentration of either [-32P]Dol-P-P (),
[32P]Dol-P (), or [32P]PA ( ) in a
total volume of 0.02 ml. After 2 min at 30 °C, the amount of
32Pi released was determined as described
previously (9, 23).
|
|
To examine the specificity of Dolpp1p, yeast microsomes from various
yeast strains were assayed for Dol-P-P, Dol-P, or PA phosphatase
activity. As seen in Table III, the
overexpression of either Dolpp1p or Cwh8p in the
lpp1/dpp1 yeast strain results in a large
increase of Dol-P-P phosphatase activity and a very small increase in
Dol-P phosphatase activity. No increase in PA phosphatase was
observed.
View this table:
[in this window]
[in a new window]
|
Table III
Dol-P, Dol-P-P, and PA phosphatase activities assayed in microsomes
from various yeast strains
Phosphatase reaction mixtures contained microsomes (20 µg of protein)
from the indicated yeast strain, 10 mM EDTA, 0.6% OG, 25 mM citrate-sodium phosphate (pH 7), and 20 µM
[-32P]Dol-P-P, [32P]Dol-P, or [32P]PA
in a total of 0.02 ml. Following incubation for 2 min at 30 °C, the
rate of dephosphorylation of each substrate was assayed as described
previously (9,23).
|
|
The substrate concentration curves depicted in Fig. 5 (panel
B) show that Dol-P-P () is rapidly dephosphorylated by Dolpp1p, whereas Dol-P () and PA () are relatively poor substrates over the entire concentration range tested. This comparison indicates that
Dolpp1p exhibits a relatively high affinity for Dol-P-P (apparent Km <10 µM). When
[,-32P]Dol-P-P was used as substrate in Dol-P-P
phosphatase assays, approximately equal quantities of
32Pi and [32P]Dol-P were formed
during the initial rate phase of the reaction, indicating that Dolpp1p
is indeed a novel lipid pyrophosphate phosphatase exhibiting high
specificity for Dol-P-P.
Expression of an amino-terminally tagged T7-DOLPP1 construct
in the insect cell Sf9-baculovirus system yielded both
T7-immunoreactive protein and increased enzymatic
activity (Fig.
6, panel A, and Table
IV). When a Dolpp1p construct containing
a carboxyl-terminal histidine tag was expressed in the
lpp1/dpp1 yeast strain, a protein of the
expected Mr (28 kDa) was observed (Fig. 6,
panel B) with a corresponding increase in Dol-P-P
phosphatase activity (data not shown). Similarly, these results
indicate that the mammalian DOLPP1 cDNA reconstitutes
Dol-P-P phosphatase activity not only in yeast but also in insect cells
and that small extensions at the amino and carboxyl termini of the
protein produced no gross effects on enzyme activity. Although stable
detergent-solubilized preparations have been obtained, attempts to
purify the Dolpp1p in an active form from baculovirus-infected insect
cell membranes have been unsuccessful to date.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 6.
Expression of T7 and His6-tagged
constructs of Dolpp1p phosphatase in Sf9 and yeast cells.
Extracts from Sf9 insect cells 2 days after transfection with
either control virus (panel A, lane 1) or after transfection
with pFASBac/DOLPP1-T7 (panel A, lanes 2 and
3, two separate isolates) were probed with -T7 antiserum.
Extracts from cwh8 yeast cells following transformation
with either YEp-352 (panel B, lane 1),
YEp352/HIS6-DOLPP1 (panel B, lane 2),
or lpp1/dpp1 yeast following transformation
with YEp352/HIS6-DOLPP1 (panel B, lane
3) were probed with -His antiserum.
|
|
View this table:
[in this window]
[in a new window]
|
Table IV
Dol-P-P, Dol-P, and PA phosphatase activities assayed in microsomes
from insect Sf9 and COS cell lines expressing Dolpp1p
Phosphatase reaction mixtures contained microsomes from either
Sf9 cells (20 µg of protein) or COS cells (10 µg of protein)
following transformation with either empty vector or vector carrying
DOLPP1, 10 mM EDTA, 0.6% OG, 25 mM
citrate-sodium phosphate (pH 7), and 20 µM either
[-32P]Dol-P-P, [32P]Dol-P, or [32P]PA
in a total of 0.02 ml. Following incubation for 5 min at 30 °C, the
rates of dephosphorylation of each substrate were assayed as described
previously (9,23). Two independent experiments using
COS cells are shown.
|
|
Expression and Subcellular Localization of Dolpp1p in COS
Cells--
Although mutations in the CWH8 gene affect the
rate of lipid intermediate synthesis and protein
N-glycosylation in the ER, the precise subcellular location
of the yeast enzyme has not been firmly established. To explore the
properties of the DOLPP1 expressed in mammalian cells, the
full-length mouse brain DOLPP1 cDNA was inserted into
the eukaryotic expression vector pCHA7 to yield the plasmid
pCHA7/DOLPP1, and expression in COS cells was detected by enzyme assay
and immunoblotting. When microsomes from transfected cells were
assayed, a large (12-16-fold) increase in Dol-P-P phosphatase activity
was seen compared with identical microsomal fractions from cells
transfected with the empty vector alone (Table IV). It is noteworthy
that no concomitant increase in Dol-P or PA dephosphorylation was
observed, corroborating the specificity for Dol-P-P.
Soluble (S100) and particulate (P100) fractions from
DOLPP1-transfected COS cells were also analyzed by SDS-PAGE
and immunoblotting using a rabbit polyclonal antibody raised against a
Dolpp1p peptide (Fig. 7). This analysis
revealed a single band in the P100 fraction (Fig. 7, lane 4)
at an Mr of 27 in accord with the molecular mass of Dolpp1p predicted from the DOLPP1 cDNA sequence (27.1 kDa). No bands were visible in the S100 fraction or the fractions from the vector-transfected cells (Fig. 7, lanes 1-3).
View larger version (40K):
[in this window]
[in a new window]
|
Fig. 7.
Dolpp1p expressed in COS cells is
membrane-associated. COS-7L cells were transfected with pCHA7
(lanes 1 and 3) or pCHA7/DOLPP1 (lanes
2 and 4) and harvested 48 h later. Cells were
sonicated briefly and centrifuged at 100,000 × g to
obtain a soluble fraction (S100, lanes 1 and
2) and a particulate fraction (P100, lanes
3 and 4). A single band appears exclusively in the
particulate fraction (lane 4) as visualized by
immunoblotting using a rabbit anti-Dolpp1p peptide antibody. The
visualized band corresponds to the predicted molecular mass of Dolpp1p
(27 kDa).
|
|
To determine further the subcellular location of Dolpp1p, transfected
COS cells were fractionated on nonlinear Nycodenz gradients, using a
method previously developed to provide effective separation of ER and
Golgi complex proteins (27). As shown in Fig.
8, >95% of the immunoreactivity
sedimented to the bottom of the gradient (upper panel),
co-sedimenting with calnexin, a well established ER marker. A much
smaller amount was found at the top of the gradient co-fractionating
with the Golgi markers, KDEL receptor and -COP (Fig. 8). The minor
immunoreactivity in Golgi shown in Fig. 8 was not observed in every
experiment and seemed to correlate with higher levels of Dolpp1p
expression.
View larger version (49K):
[in this window]
[in a new window]
|
Fig. 8.
Subcellular localization of Dolpp1p on
Nycodenz density gradients. COS-7L cells were transfected with
pCHA7/DOLPP1 and cell homogenates separated on preformed Nycodenz
gradients. Fractions along the gradient were analyzed by immunoblotting
using anti-Dolpp1p anti-peptide antibodies and other markers as
indicated. Marker proteins were calnexin (ER membrane marker), -COP
(Golgi marker), and KDEL receptor (Golgi marker). A predominantly ER
localization for Dolpp1p was observed (top panel). The
results illustrated in this figure are representative of three separate
experiments.
|
|
When COS cells overexpressing DOLPP1 were examined by
immunofluorescence using a specific Dolpp1p antibody, a reticular and nuclear envelope pattern characteristic of ER localization was observed
(Fig. 9). This pattern essentially
completely overlapped with the ER marker (KDEL, upper panel)
and showed no overlap with a Golgi marker (KDEL receptor, lower
panel).
View larger version (22K):
[in this window]
[in a new window]
|
Fig. 9.
ER localization of Dolpp1p by
immunofluorescence in fixed and permeabilized COS-7L cells.
COS-7L cells were transfected with pCHA7/DOLPP1 and
simultaneously stained with anti-Dolpp1p antibodies (green,
A and D) and one of two markers, anti-KDEL
antibody (ER marker, red, B and C) or
anti-KDEL receptor (Golgi marker, red, E and
F). Dolpp1p (green) shows a reticulated and
nuclear envelope pattern typical of ER localization, an impression that
is confirmed by the overlapped images (merge, C).
Dolpp1p staining was distinct from Golgi staining (F). Weak
nuclear labeling was evident in some preparations and was deemed
nonspecific because it appeared in the presence of either anti-Dolpp1p
or non-immune antibodies (not shown). Experiment shown is one of two
yielding similar results.
|
|
Topological Orientation of the Active Site of Dolpp1p--
To
determine whether the active site of Dolpp1p faces the lumen of the ER
or the cytoplasm, protease-sensitivity studies were conducted on the
Dol-P-P phosphatase activity in intact and unsealed ER vesicles from
the lpp1/dpp1/DOLPP1 yeast
strain. When detergent-disrupted ER vesicles (Table
V) were treated with trypsin, the Dol-P-P phosphatase activity was extensively inactivated (74.8% of control). However, the phosphatase activity was found to be relatively resistant to proteolysis in intact ER vesicles (87.5% of control). Separate pilot experiments employing glucosidase I/II latency (28) established that the sealed vesicles used in this experiment were >98% intact throughout the time of incubation and that 0.2% Triton X-100 was sufficient to unseal >95% of the yeast ER vesicles. Thus, it appears that the mammalian Dol-P-P phosphatase has a trypsin-sensitive site,
perhaps part of the reactive site of the enzyme, that is luminally
oriented, as in the yeast enzyme (Fig.
10).
View this table:
[in this window]
[in a new window]
|
Table V
Effect of trypsinization on Dol-P-P phosphatase activity in intact and
disrupted microsomal vesicles from
lpp1/dpp1/DOLPP1 yeast
Sealed yeast microsomal vesicles (10 µg of membrane protein) from
lpp1/dpp1/DOLPP1 yeast were
preincubated for 60 min at 21 °C with or without 0.2% Triton X-100
in the presence or absence of trypsin (10 µg/ml) in a reaction
mixture containing 1 mM MgCl2, 10 mM
sodium-HEPES (pH 7.4), 0.25 M sucrose. Proteolysis was
stopped by the addition of 5 mM phenylmethylsulfonyl
fluoride, and Dol-P-P phosphatase activity was determined as described
under "Experimental Procedures." Microsomal vesicles were >98%
intact as determined by glucosidase I/II latency as described
previously (28).
|
|
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 10.
Predicted topological arrangement of
mammalian Dolpp1p and yeast Cwh8p in the ER. The topology of
potential transmembrane domains of Dolpp1p was predicted using the
TMPred transmembrane helices prediction algorithm (43). Identical and
highly conserved residues in Dolpp1p and Cwh8p are shaded
black and gray, respectively.
|
|
| |
DISCUSSION |
When Glc3Man9GlcNAc2 is
transferred from the glycosyl carrier lipid to appropriate asparagine
residues in nascent proteins, Dol-P-P is formed on the luminal
monolayer of the ER. This luminally oriented Dol-P-P must be converted
to Dol-P and return to the cytoplasmic leaflet of the ER in order to
participate in subsequent rounds of lipid intermediate biosynthesis
(4). The CWH8 gene in S. cerevisiae has recently
been shown to encode a Dol-P-P-specific pyrophosphate phosphatase that
was proposed to function in a model for the recycling of the carrier
lipid (7, 8). This paper describes the identification and
characterization of a mammalian homologue of the yeast CWH8
gene, DOLPP1, from a mouse brain cDNA library.
Although there have been numerous reports (7-10, 13, 32-41)
describing Dol-P-P/Dol-P phosphatase activities in crude microsomal fractions from various mammalian tissues, the exact number, location, and specificity have not been rigorously defined. Previous studies have
been complicated by an inability to study the enzymatic properties of
these proteins independent of the activities of other contaminating phosphatases. Expression of Dolpp1p in the
lpp1/dpp1 yeast strain has allowed us to
characterize the brain Dol-P-P phosphatase in the absence of other
endogenous lipid phosphatase activities hydrolyzing Dol-P-P and Dol-P.
The comparisons reported in Tables III and IV show that expression of
Dolpp1p in either yeast, insect Sf9 cells, or COS cells results
in a substantial and highly specific increase in Dol-P-P phosphatase
activity. The experiment described in Fig. 5 reveals that Dolpp1p
exhibits a marked preference for Dol-P-P (Km <10
µM), similar to Cwh8p (~25 µM) when
expressed in the lpp1/dpp1 yeast double
mutant. Reliable kinetic data for Dol-P and PA could not be calculated
due to the extremely low rates of dephosphorylation of these substrates
catalyzed by Dolpp1p.
Recently, lipid phosphate phosphohydrolases (LPPs) have been an active
subject of research due to their role in potential signaling pathways
(31, 42). The type 2 LPP isoforms share extensive sequence similarity,
are Mg2+-independent,
N-ethylmaleimide-insensitive, and show little substrate specificity hydrolyzing a variety of lipid-phosphate substrates, including phosphatidic acid, lysophosphatidic acid, ceramide
1-phosphate, sphingosine 1-phosphate, and diacylglycerol pyrophosphate
(42). DOLPP1 shares some properties with mammalian LPPs but
shows only modest sequence homology (~10%) with this family of
phosphohydrolases and differs markedly in its high degree of
specificity for Dol-P-P.
DOLPP1 shares a high degree of sequence identity with
CWH8 and is shown to be a functional homologue in the
cwh8 mutant yeast strain. This conclusion is based on the
following observations that overexpression of mouse DOLPP1
in cwh8 yeast cells: 1) complements the growth defect; 2)
corrects the deficiency in protein N-glycosylation; and 3)
reverses the accumulation of Dol-P-P resulting from defective Dol-P-P
phosphatase activity. Moreover, as noted above, expression of the
DOLPP1 gene in the lpp1/dpp1
yeast strain, Sf9 insect cells, or mammalian COS cells produces
large increases in Dol-P-P phosphatase activity, indicating that
Dolpp1p is an essential component of the brain enzyme. The presence of
a consensus lipid phosphatase motif within the amino acid sequence and
the observations that overexpression of DOLPP1 in yeast and
Sf9 cells produces dramatic increases in the level of Dol-P-P
phosphatase activity strongly suggest that Dolpp1p contains the
catalytic site. Whether other components may be needed for full
activity is a question that will only be answered upon successful
solubilization, isolation, and reconstitution of the recombinant enzyme
in active form.
Although the effects of mutations in the CWH8 gene on
protein N-glycosylation, lipid intermediate synthesis, and
the accumulation of Dol-P-P are consistent with the yeast enzyme being
located in the ER, these observations are only indirect support for
this conclusion. The immunofluorescence microscopy and subcellular fractionation studies described in this report represent the first direct evidence that Dolpp1p is located in the ER in COS cells. In
addition, protease sensitivity studies indicate that the mammalian Dol-P-P phosphatase has a critical domain that is luminally oriented. The relationship of this enzyme to the Dol-P-P phosphatase activity previously reported (9, 10) to be enriched in the Golgi compartment in
brain is not clear. However, it seems likely that unrelated lipid-phosphate phosphatases with broad substrate specificity and
overlapping subcellular distribution may have obscured the proper
localization of this activity. In this regard, other researchers have
reported that Dol-P-P phosphatase activity is found in the plasma
membrane in rat liver (32) or throughout the plasma membrane, rough ER,
smooth ER, Golgi, and lysosomal fractions in mouse L-1210 cells (33). A
small and variable proportion (<5%) of Dolpp1p was seen in the Golgi
upon overexpression in COS cells. This result raises the possibility
that the enzyme might be partially localized in a Golgi compartment
under some conditions. However, it is clear from these studies that the
DOLPP1 gene product is predominantly localized in the ER,
the site of lipid intermediate biosynthesis, and protein
N-glycosylation.
The specificity, location in the ER, and luminally oriented active site
of Dolpp1p meet all the criteria for a role in the recycling of Dol-P-P
released during primary protein N-glycosylation reactions on
the luminal surface. There is substantial evidence that cellular levels
of Dol-P are a rate-controlling factor in the regulation of lipid
intermediate biosynthesis (44-50). The induction of
cis-isoprenyltransferase activity correlates closely with the onset of developmental increases in Dol-P and
Glc3Man9GlcNAc2-P-P-Dol biosynthesis (51, 52), suggesting that de novo synthesis is an important source of Dol-P for lipid intermediate synthesis. However,
considering the impressive effect (~80% reduction in Glc3Man9GlcNAc2-P-P-Dol synthesis)
produced by mutations in CWH8 (8, 15), it seems likely that
recycling of Dol-P-P plays an important role in maintaining Dol-P
supplies for
Glc3Man9GlcNAc2-P-P-Dol biosynthesis. An important future goal will be to determine whether the
Dol-P formed via the recycling of Dol-P-P exists in a separate pool to
initiate new rounds of lipid intermediate synthesis or intermixes with
the pool acquired by the de novo pathway.
The hypothetical models for Dolpp1p and Cwh8p, based on the TMpred
program (43), indicate that the proteins contain four transmembrane
helices and are topologically identical (Ref. 8, Fig. 10). The
transmembrane domains (TMD) and the large luminal domains between TMD 1 and 2 are highly conserved in the two proteins. The high degree of
conservation in the TMDs suggests that these domains may be critical to
the function of Dolpp1p phosphatase and could be involved in the
interaction with the polyisoprenyl moiety of Dol-P-P. The two putative
luminal domains in Dolpp1p and Cwh8p have a significantly higher degree
of homology than the cytoplasmic domains and may form part of the
reactive site. Studies are in progress to determine the precise domains
forming the active site of the enzyme.
The identification of the DOLPP1 gene is an important
development, with implications for the inherited metabolic diseases in
humans that occur due to defects in the synthesis of the
dolichol-linked oligosaccharide donor required for protein
N-glycosylation. These disorders are collectively referred
to as congenital disorders of glycosylation (CDG) (53-55). Considering
the wide variety of enzymes that have already been described as causing
CDG, and the severity of the protein N-glycosylation
deficiency in the yeast CWH8 mutants, it seems inevitable
that a human CDG related to human DOLPP1 deficiency will be
discovered. The accumulation of Dol-P-P in the luminal leaflet
resulting from defects in Dolpp1p could block protein
N-glycosylation by end product inhibition and interfere with
other ER functions by causing biophysical alterations due to anionic
microheterogeneities. The description of this mammalian cDNA should
facilitate the clinical diagnosis of the underlying metabolic disorder
in patients of this type.
,
TOP
ABSTRACT
INTRODUCTION
