From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, IWK Health Centre, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
Received for publication, February 7, 2001, and in revised form, February 22, 2001
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ABSTRACT |
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Oxysterol-binding proteins (OSBPs)
are a family of eukaryotic intracellular lipid receptors. Mammalian
OSBP1 binds oxygenated derivatives of cholesterol and mediates sterol
and phospholipid synthesis through as yet poorly undefined mechanisms.
The precise cellular roles for the remaining members of the
oxysterol-binding protein family remain to be elucidated. In yeast, a
family of OSBPs has been identified based on primary sequence
similarity to the ligand binding domain of mammalian OSBP1. Yeast
Kes1p, an oxysterol-binding protein family member that consists of only the ligand binding domain, has been demonstrated to regulate the Sec14p
pathway for Golgi-derived vesicle transport. Specifically, inactivation
of the KES1 gene resulted in the ability of yeast to
survive in the absence of Sec14p, a
phosphatidylinositol/phosphatidylcholine transfer protein that is
normally required for cell viability due to its essential requirement
in transporting vesicles from the Golgi. We cloned the two human
members of the OSBP family, ORP1 and ORP2, with the highest degree of
similarity to yeast Kes1p. We expressed ORP1 and ORP2 in yeast lacking
Sec14p and Kes1p function and found that ORP1 complemented Kes1p
function with respect to cell growth and Golgi vesicle transport,
whereas ORP2 was unable to do so. Phenotypes associated with
overexpression of ORP2 in yeast were a dramatic decrease in cell growth
and a block in Golgi-derived vesicle transport distinct from that of ORP1. Purification of ORP1 and ORP2 for ligand binding studies demonstrated ORP1 and ORP2 did not bind 25-hydroxycholesterol but
instead bound phospholipids with both proteins exhibiting strong
binding to phosphatidic acid and weak binding to phosphatidylinositol 3-phosphate. In Chinese hamster ovary cells, ORP1 localized to a
cytosolic location, whereas ORP2 was associated with the Golgi apparatus, consistent with our vesicle transport studies that indicated
ORP1 and ORP2 function at different steps in the regulation of
vesicle transport.
Oxysterols are naturally occurring hydroxylated derivatives of
cholesterol that can affect lipid homeostasis through both transcriptional and post-translational mechanisms (1-3). One such
oxysterol, 25-hydroxycholesterol, was demonstrated to bind with high
specificity to a low abundance cytosolic receptor termed oxysterol-binding protein 1 (OSBP1)1 (4). Human OSBP1 was
predicted to encode a protein of 807 amino acids with an N-terminal
glycine/alanine-rich region and PH domain and a C-terminal ligand
binding domain. OSBP1 translocated from a cytoplasmic compartment of
the Golgi apparatus when cells were treated with 25-hydroxycholesterol
or in response to decreased plasma membrane cholesterol. The PH domain
was required for translocation of OSBP1 to the Golgi, and this
translocation directly correlated with alterations in sterol and
phospholipid homeostasis through both transcriptional and
post-translational mechanisms (5-7). The precise molecular mechanism
by which OSBP transduces these signals is currently unknown.
In yeast, a family of OSBP proteins has been identified based on
primary sequence similarity to the ligand binding domain of mammalian
OSBP1 (8-10). Genetic studies on yeast have provided some clues toward
understanding the biological function and mechanism of OSBPs. It was
demonstrated the Kes1p, an oxysterol-binding protein family member that
consists of only the ligand binding domain, was a negative regulator of
the SEC14 pathway for Golgi-derived vesicle transport (11).
Specifically, inactivation of the KES1 gene resulted in the
ability of yeast to survive in the absence of Sec14p, a protein that is
normally essential for cell viability (12-14). This regulation was
specific to Kes1p as inactivation of the genes of the remaining members
of the yeast OSBP gene family was unable to bypass the essential
function of Sec14p. Sec14p is a phosphatidylinositol
(PI)/phosphatidylcholine (PC) transfer protein, which in
vitro catalyzed transport of phospholipid monomers from one
membrane bilayer to another (14-16) and in vivo is
speculated to function as a diffusible sensor of PC levels that in turn
regulates lipid homeostasis for subsequent downstream management of
Golgi-derived vesicle trafficking and sorting (11, 17-21). Besides
KES1, inactivating mutations in several other structural
genes have been demonstrated to relieve the cell of it essential
requirement for Sec14p and these include the following: (i) each of the
enzymes in CDP-choline pathway for PC biosynthesis, and (ii)
SacIp, a PI-4-phosphate phosphatase (22, 23). Additional
experimentation demonstrated that increased expression of
KES1 in sec14ts yeast containing
inactivating mutations in any of the enzymes of the CDP-choline pathway
reversed the sec14 To understand more precisely the functions and underlying mechanisms of
mammalian OSBPs, we screened a human cDNA library and isolated the
two members of the human OSBP family that most resemble yeast Kes1p. We
purified the encoded proteins and demonstrated that neither could bind
25-hydroxycholesterol but instead bound phosphatidic acid (PA) with
high affinity and phosphatidylinositol-3-P (PI-3-P) with weak affinity.
Thus, these new proteins were termed oxysterol-binding protein-related
proteins, ORP1 and ORP2. Expression of ORP1 in yeast reversed the
bypass provided by inactivation of the KES1 gene in cells
lacking a functional Sec14p, whereas ORP2 expression was unable to do
so. Instead, overexpression of ORP2 resulted in the cessation of cell
growth in wild type yeast, and this correlated with a block in vesicle
transport within the Golgi. ORP2-mediated cell growth inhibition was
more extreme in cells lacking a functional Kes1p implying Kes1p/ORP1
shares a biological function with ORP2. Expression of ORP1- and
ORP2-green fluorescent protein (GFP) chimeras in Chinese hamster ovary
(CHO) cells localized ORP1 to a cytosolic compartment and ORP2 to the Golgi apparatus.
Media and Reagents--
Standard molecular biology methods,
yeast genetic techniques, and transformation methods were used (24,
25). Yeast complex medium supplemented to a final glucose concentration
of 2% (YPD) and synthetic minimal media have been described (25).
Tran35S-labeled methionine/cysteine mixture was purchased
from PerkinElmer Life Sciences. Goat anti-rabbit and anti-mouse
conjugated antibodies were from Bio-Rad. The enhanced chemiluminescence
kit was from Amersham Pharmacia Biotech. All phospholipids were
products of Avanti Polar Lipids, except the phosphoinositides that were
purchased from both Matreya and Biomol. Similar results were obtained
using lipids from either source. Pure protein kinase C Yeast Strain Construction--
The yeast wild type strains
w303-1a (a ura3-1 his3-11, 15 leu2-3, 112 trp1-1 ade2-1
can1-100), w303-1A ( Isolation of a Full-length Human ORP1 and ORP2 cDNAs--
A
tBLASTn search of the expressed sequence tag (EST) data base revealed
five separate groups of cDNAs that were similar to yeast Kes1p and
human OSBP1. Two of these groups consisted of cDNAs predicted to
contain only the ligand binding domain. One group contained only
partial cDNAs, and a full-length ORP1 cDNA was isolated from an
oligo(dT)-primed human brain cDNA library (Life Technologies, Inc.)
using the Gene Trapper positive selection system (Life Technologies,
Inc.). A 25-mer oligonucleotide was synthesized based on the most 5'
EST DNA sequence, and this oligonucleotide was used to enrich for novel
ORP1 cDNAs. A total of 1000-2000 colonies from the enriched
cDNA library were screened using a 32P-labeled
oligonucleotide designed from the partial cDNA sequence. Positive
clones with large inserts were subsequently sequenced by automated
dye-termination DNA sequencing. A full-length ORP1 cDNA was
isolated subsequent to several rounds of enrichment and colony
hybridization using oligonucleotides designed from the emerging
upstream novel DNA sequences. The second OSBP homologue that contained
only the ligand binding domain, ORP2, was obtained full-length from the
Kazusa DNA Research Institute (Kisarazu, Japan).
Construction of ORP1 and ORP2 Expression Vectors--
The full
open reading frames of human ORP1 and ORP2 were amplified by PCR using
Vent DNA polymerase (New England Biolabs). After additional incubation
with Taq DNA polymerase (Life Technologies, Inc.) to add
deoxyadenosine tails, the PCR products were subcloned into
pcDNA2.1-Topo (Invitrogen). The integrity of the entire ORP1 and
ORP2 amplified sequences was confirmed by DNA sequencing. The open
reading frames were subsequently subcloned into the yeast pESC-URA
expression vector (Invitrogen). The pESC-URA vector contains the
GAL1 promoter upstream of the open reading frames for
regulated expression of human ORP1 and ORP2 proteins by substituting
galactose for glucose as carbon source. The open reading frames were
also subcloned into the mammalian expression vector pEGFP-N1
(CLONTECH) resulting in the addition of the coding
sequence for GFP in frame with the 3' end of ORP1 and ORP2. The open
reading frames were also subcloned into the Escherichia coli
expression vector pET23b (Novagen) resulting in the addition of a
His6 tag to the C terminus of the expressed protein.
Yeast Growth Assays--
For the yeast growth assay, a single
colony from each of the yeast strains was grown overnight in defined
media containing the appropriate nutrients to ensure plasmid
maintenance. Yeast cell concentration was estimated by measuring
absorbance at 600 nm, and the identical number of cells was removed
from each culture. These cells were washed in minimal media containing
2% galactose substituted as carbon source; a series of 1:4 dilutions
were made, and 1 µl of each dilution was spotted onto minimal media
agar plates containing glucose and/or galactose and the appropriate nutrients to ensure plasmid maintenance. Cells were incubated at either
25 or 37 °C for 3-4 days.
Carboxypeptidase Y (CPY) Processing Assay--
The CPY assay was
carried out essentially as described (27). Yeast cells were grown in
defined media containing the required nutrients to ensure plasmid
maintenance, but 2% galactose was substituted as carbon source (to
induce human ORP1 or ORP2 expression). Cells were back inoculated to an
absorbance at 600 nm of 0.4 and grown for 6-8 h. Aliquots of cells
(absorbance at 600 nm of 3.0) were resuspended in fresh media and
incubated at 37 °C for 60 min. Cells were then labeled with
[35S]methionine/cysteine for 10 min, and then unlabeled
methionine and cysteine were added to the reaction media (subsequent to
10 min of radiolabeling) to final concentrations of 0.5% each. The cells were incubated for an additional 0-60 min, and the labeling reaction was terminated by transferring samples to tubes containing 10 mM NaF in ice-cold phosphate-buffered saline. Cells were
disrupted by agitation with glass beads and cellular proteins after
pelleting cell debris by centrifugation in a microcentrifuge at 14,000 rpm for 20 min. CPY was immunoprecipitated from the supernatant using a
polyclonal antibody (kindly provided by Dr. Scott Emr, University of
California, San Diego) and protein A-Sepharose (Amersham Pharmacia Biotech). The immunoprecipitated proteins were resolved using 8%
SDS-polyacrylamide gel electrophoresis, and the gel was exposed to
x-ray film for subsequent development.
Purification of ORP1 and ORP2 Proteins--
ORP1 and ORP2 in the
pET23b plasmids were transformed into BL21(DE3) pLysS E. coli and transformant-selected on medium containing 25 µg/ml
chloramphenicol and 50 µg/ml ampicillin at 37 °C. Transformants were grown in the same medium to an absorbance at 600 nm of 0.6, and
transcription of ORP1 and ORP2 was induced by the addition of
isopropyl-1-thio- Production of Antibodies to ORP1 and ORP2--
The human ORP1
coding sequence for the C-terminal 100 amino acids was fused in frame
to the coding region for glutathione S-transferase in the
E. coli expression vector pGEX-3X (Amersham Pharmacia
Biotech). The fusion protein was expressed in E. coli and
purified using glutathione resin as directed by the manufacturer, and
the purified fusion protein was used for antibody generation. ORP2
antibodies were raised against the peptide CQERRGDHLRKAKLDEDSGKADSD coupled to keyhole limpet hemocyanin (New England Peptide). The purified GST-ORP1 protein chimera and the ORP2-coupled peptide were
injected into rabbits subcutaneously by the Dalhousie University Carleton Animal Care staff for the production of ORP1 and ORP2 antibodies.
Lipid Ligand Binding Assay--
Pure ORP1 and ORP2 proteins and
COS-7 extracts of overexpressed recombinant proteins were analyzed for
25-hydroxycholesterol binding exactly as described (4). Standard
phospholipid binding assays were performed as described (29) by
immobilizing 100 pmol of pure phospholipid on Hybond-C nitrocellulose
membranes (Amersham Pharmacia Biotech). Blots were blocked with
Tris-buffered saline containing 3% fatty acid-free bovine serum
albumin. Pure protein preparations of ORP1, ORP2, or protein kinase
C Cell Culture, Transfections, and Fluorescence
Microscopy--
CHO-K1 cells were cultured in Dulbecco's modified
Eagle's medium containing 5% fetal calf serum and 34 µg/ml proline.
Cells were seeded onto glass coverslips and transfected with pEGFP-ORP1 or pEGFP-ORP2 using LipofectAMINE (Life Technologies, Inc.). Thirty hours after transfection cells were fixed in 10 mM sodium
phosphate (pH 7.4), 225 mM NaCl, and 2 mM
MgCl2 (PBS) containing 3% (w/v) formaldehyde for 15 min at
room temperature. Following two washes in PBS containing 5 mM ammonium chloride, cells were permeabilized in PBS
containing 0.05% (v/v) Triton X-100 for 10 min at room temperature,
washed twice with PBS containing 1% (w/v) fatty acid-free bovine serum
albumin, and incubated for 15 min at room temperature in PBS containing
1% fatty acid-free bovine serum albumin. To stain the Golgi apparatus,
cells were incubated in PBS containing 1% fatty acid-free bovine serum
albumin containing 10 µg/ml Lens culinaris (8) lectin
coupled to Texas Red (EY Laboratories) for 1 h at room
temperature. Cells were washed twice with PBS containing 1% fatty
acid-free bovine serum albumin and mounted on microscope slides with
90% glycerol, 50 mM Tris-HCl (pH 9.0), and 2.5%
1,4-diazadicyclo-(2,2,2)-octane. To stain the endoplasmic reticulum,
cells were incubated in PBS containing 1% fatty acid-free bovine serum
albumin containing mouse monoclonal antibodies to protein disulfide
isomerase (Stressgen) at a 1:500 dilution followed by a goat anti-mouse
secondary antibody coupled to Texas Red (1:4000). To disrupt the Golgi
apparatus, cells were treated with brefeldin A (2 µg/ml) for 30 min
prior to the fixation step.
Isolation of Human ORP1 and ORP2 cDNAs--
The
KES1 gene of yeast had been previously isolated based on the
observation that inactivating mutations of KES1 resulted in
cells that were able to bypass the essential requirement of a PC/PI
transfer protein, Sec14p (17). Sec14p is necessary for vesicle
trafficking competence from the late Golgi to the cell surface and
yeast vacuole (functional equivalent of the mammalian lysosome)
(12-14). This role in vesicle trafficking was demonstrated to be
specific to Kes1p as genetic inactivation of the other members of the
yeast OSBP family were unable to relieve yeast cells of the requirement
for a functional Sec14p (11). The only characterized mammalian OSBP is
OSBP1, a protein that consists of a ligand binding domain similar to
that of Kes1p but also has a large N-terminal extension containing a PH
domain, a Gly/Ala-rich region. OSBP1 translocates to the Golgi
apparatus from a cytoplasmic vesicular compartment and has been
demonstrated to regulate flux through the sterol and phospholipid
biosynthetic pathways through as yet unknown mechanisms (3,
5-8).
We searched the human expressed sequence tags (EST) data base using
sequences from the ligand binding region of human OSBP1 and yeast Kes1p
for mammalian ESTs that were predicted to contain only the ligand
binding domain portion of the OSBP family. Two different classes of
OSBP encoding cDNAs were identified. An alignment of one class of
EST sequences with similarity to the ligand binding region of OSBP1 and
Kes1p demonstrated that there were no full-length cDNAs
represented. We employed a positive cDNA selection system to screen
libraries for a full-length human cDNA. After several rounds of
library enrichment, colony hybridization, restriction analysis, and
cDNA walking, we isolated a new member of the OSBP family of
proteins that we termed ORP1 (OSBP-related Protein, GenBankTM accession number AF274714). DNA sequencing
revealed an insert of 3.3 kilobase pairs in size that contained an
internal 1.3-kilobase pair open reading frame. Stop codons were found
in all three reading frames upstream of the predicted initiator Met
codon. The second member of the OSBP family that contained only the
ligand binding domain contained ESTs that were predicted to be full
length. One of these was obtained from the Kazusa DNA Research
Institute (Kisarazu, Japan) and was sequenced in its entirety
(GenBankTM accession number AY028168).
The ORP1 open reading frame codes for a protein of 437 amino acids with
a calculated molecular mass of 46 kDa, whereas ORP2 predicts a
468-amino acid protein with a molecular mass of 53 kDa (Fig.
1). The proteins do not contain any
predicted membrane spanning domains, nor were there any recognizable
sorting sequences or functional motifs.
Ability of ORP1 and ORP2 to Reverse kes1
The ORP1 and ORP2 open reading frames were placed under control of the
GAL1 promoter. This results in repression of transcription from the
GAL1 promoter when yeasts are grown on glucose-containing media, but upon switching to galactose-containing media the promoter is
derepressed and protein expression occurs. The
GAL1::ORP1 and GAL1::ORP2
plasmids were introduced into wild type and sec14ts
kes1::HIS3 yeast cells. The expression
level of the ORP1 and ORP2 proteins correlated with the amount of
galactose present in the medium with highest protein levels, as
detected by Western blot, in cells grown in 85-100% galactose as
carbon source (Fig. 2C). We
were unable to obtain Western blot data for ORP2 expression in 100%
galactose as overexpression of ORP2 at this level resulted in the
cessation of cell growth (Fig. 2A). However, the expression level of ORP2 grown in 85% galactose was similar to that of ORP1 grown
in 100% galactose, so comparisons of their ability to complement loss
of function of yeast KES1 could be performed.
If ORP1 or ORP2 could complement KES1 then expression of
ORP1 or ORP2 in sec14ts
kes1
Another interesting observation from these studies was that the
cessation of cell growth due to high level overexpression of ORP2 in
yeast (grown on 100% galactose) was more apparent in yeast that
contained a non-functioning KES1 gene even at 25 °C, a
temperature where Sec14p is functioning. This enhancement of phenotype
in cells lacking a functional Kes1p implies that Kes1p/ORP1 and ORP2
likely affect a similar biological function.
ORP1 and ORP2 Affect Carboxypeptidase Y (CPY) Processing and
Transport--
To test if the ORP1- and ORP2-dependent
growth phenotypes influenced Golgi-derived vesicle transport and to
ensure that the alteration in growth observed upon ORP1 expression in
sec14ts kes1::HIS3
yeast was indeed due to re-establishment of vesicle transport, we
examined the effect of ORP1 and ORP2 expression on CPY protein
processing. Normally, newly synthesized CPY protein is modified by
N-glycosylation in the endoplasmic reticulum (P1 form) and
is subsequently transported to the Golgi where CPY is further modified
by core glycosylation (P2 form). The P2 form of CPY traverses the Golgi
and is packaged into a vesicle destined for the vacuole (lysosome)
where it is processed to its mature form.
In wild type yeast cells, after a 60-min chase of
[35S]labeled methionine/cysteine into CPY, most of the
labeled CPY protein was fully processed to the mature form (Fig.
3). In contrast, when the
sec14ts cells were shifted to the non-permissive
temperature of 37 °C, there was a marked accumulation of the P1 and
P2 forms of the CPY protein, indicating decreased CPY processing and
thus decreased vesicle transport. The normal CPY secretion pattern can
be restored by a mutation in the kes1 gene in the
sec14ts yeast (sec14ts
kes1::HIS3) (Fig. 3). Expression of
ORP1 in sec14ts yeast containing the inactivated
kes1
As we had observed that induced overexpression of ORP2 (using 100%
galactose) eventually resulted in cessation of cell growth, we tested
whether CPY processing was blocked upon induction of ORP2 expression.
Indeed, overexpression of ORP2 resulted in a marked delay in CPY
processing from its P1 and P2 forms to its mature form (Fig.
4). This is consistent with the
hypothesis that Kes1p/ORP1 and ORP2 affect the same biological process,
in this case vesicle trafficking, that was derived from our observation that cell growth inhibition due to overexpression of ORP2 was more
pronounced in cells lacking functional Kes1p/ORP1.
Purification of ORP1 and ORP2 Proteins and an Assessment of Their
Lipid Ligands--
The ligand binding specificity for members of the
OSBP protein family has only been determined for mammalian OSBP1 (4, 28). The ligand binding domain of OSBP1 binds oxygenated derivatives of
cholesterol with a preference for 25-hydroxycholesterol. We expressed
His6-tagged versions of ORP1 and ORP2 in E. coli
and purified the proteins using metal affinity column chromatography. We were unable to detect binding to 25-hydroxycholesterol using the
pure protein or the protein overexpressed in COS-7 or CHO cells (4).
However, this was not entirely unexpected as inactivating or
overexpressing the KES1 gene did not affect sterol
metabolism, and altering rates of sterol metabolism were unable to
affect cell growth or vesicle trafficking in an
Sec14p-dependent manner. Phospholipid metabolism is
intimately linked with Sec14p function as Sec14p itself is a
PI/PC-binding protein, and all of the other known genes that alter
growth and vesicle trafficking in yeast lacking a functional Sec14p
code for proteins that either (i) decrease PC synthesis, (ii) alter the
turnover of PC via phospholipase D, or (iii) affect phosphatases and
kinases that alter the phosphate composition of polyphosphate
phosphoinositols (13, 14, 17-23). Thus, we examined whether ORP1 or
ORP2 could bind various phospholipids (Figs.
5A and
6) (29). Lipids were spotted onto
nitrocellulose filters, and either pure ORP1 or ORP2 was added.
Antibodies to ORP1 and ORP2 were used to detect protein binding to
specific lipids. Both ORP1 and ORP2 strongly bound PA and weakly bound cardiolipin and PI-3-P (Figs. 5A and 6). Others (29) have
observed that this method agrees with the phospholipid binding
specificity observed using mixed micelle and liposome protocols. We
included protein kinase C Intracellular Location of ORP1 and ORP2--
Mammalian OSBP1 has
been localized by immunofluorescence and was found in a vesicular
cytoplasmic compartment. OSBP1 can be induced to translocate to the
Golgi apparatus upon treatment of cells with 25-hydroxycholesterol (8)
or by removal of cholesterol or sphingomyelin from the plasma membrane
of cells in culture (3, 5-7). Yeast Kes1p has yet to be localized
through microscopic means but has been localized through subcellular
fractionation and was found in various particulate and soluble
subcellular fractions (11). Human ORP1 and ORP2 were expressed in CHO
cells as chimeras with GFP to assess their subcellular location.
Western blots using GFP antibodies indicated full-length ORP1-GFP, and
ORP2-GFP chimeras were made, and there was no sign of proteolytic
products (data not shown), so the signal observed through microscopy is
not due to partial chimeras or free GFP. Treatment of cells with
brefeldin A, which collapses the Golgi into the endoplasmic reticulum,
resulted in the relocation of ORP2-GFP but not ORP1-GFP, implying ORP2 is Golgi-localized (Fig. 7A).
Consistent with this result was the overlapping immunofluorescence of
ORP2 with the Golgi-specific L. culinaris lectin (Fig.
7B). The GFP-ORP1 chimera was mainly found diffused in the
cytosol and was occasionally found in small amounts in the nucleus but
did not localize with the endoplasmic reticulum marker protein
disulfide isomerase (Fig. 6B).
OSBP1 is the prototypical member of a family of proteins that
share a common ligand binding region near their C termini. Some members
of the OSBP protein family consist almost entirely of the ligand
binding region, whereas others possess N-terminal extensions containing
a variety of motifs including PH domains, ankyrin-binding motifs, and a
Gly/Ala-rich region (8, 10, 11). OSBP1 contains an N-terminal PH domain
and Gly/Ala-rich region. OSBP1 has been cloned, purified, and
demonstrated to bind oxygenated derivatives of cholesterol with varying
degrees of efficiency (4, 8, 28). Upon ligand binding, OSBP1
translocates from a cytosolic compartment to the Golgi apparatus (3,
5-8, 33). Translocation of OSBP1 to the Golgi apparatus is associated
with pleiotropic alterations in the metabolism of several lipids (3,
5-8), although the precise mechanism by which OSBP1 affects lipid
metabolism as well as how these alterations affect the biology of the
cell are still unclear. Work in yeast has indicated that one member of
the OSBP family, encoded by the yeast KES1 gene, may
participate in vesicle trafficking (11). A conditional lethal
temperature-sensitive allele of SEC14
(sec14ts), an essential PC/PI transfer protein
required for vesicle transport from the Golgi, was used to search for
genes whose inactivation resulted in the ability of cells to restore
Golgi-derived vesicle transport and the associated reparation of cell
growth when challenged with a non-functioning Sec14p (17). Inactivating
mutation in several yeast genes was found to bypass the requirements
for SEC14 and to date these include the following: (i) a
yeast member of the OSBP family, KES1, (ii) each of the
enzymes for PC synthesis through the CDP-choline pathway, and (iii) a
PI-4-P phosphatase encoded by SAC1 (17, 22, 23). No other
member of the yeast OSBP family was able to bypass the normally
essential requirement for SEC14 indicating this ability is
specific to KES1 function (11).
In our study we report the isolation of two novel human cDNAs, ORP1
and ORP2, coding for new members of the mammalian OSBP protein family.
Similar to yeast Kes1p, ORP1 and ORP2 encode proteins that consist
almost entirely of an OSBP-like ligand binding domain and possess no
other obvious motifs or targeting signals. Expression of human ORP1,
but not ORP2, in yeast containing a sec14ts allele
and an inactivated KES1 gene reimposed the
sec14ts phenotype resulting in cell death due to
decreased ability to transport vesicles from the Golgi at the
non-permissive temperature for the sec14ts allele.
High level overexpression of ORP2 in yeast resulted in growth
cessation, and the growth defect was more pronounced when the
KES1 gene was inactivated implying that Kes1p/ORP1 and ORP2 affect a similar cellular process. Consistent with this prediction was
a correlation between ORP2 overexpression with an inability to
facilitate vesicle trafficking, as monitored by CPY processing, along
the Golgi-mediated pathway. Also consistent with a role for ORP1 and
ORP2 in vesicle trafficking through the Golgi pathway were our
immunofluorescence experiments that position ORP2 in the Golgi
apparatus and ORP1 in a cytosolic compartment.
ORP1 and ORP2 were purified, and ligand binding studies were unable to
demonstrate binding to sterols (4, 8, 28). This was not entirely
surprising as the sterol pathway in yeast does not alter known
Kes1p-mediated events, and inactivation of the KES1 gene did
not alter sterol synthesis in yeast (11). However, phospholipid
metabolism is intimately associated with Kes1p biology, so ORP1 and
ORP2 binding toward a variety of phospholipids was tested. Both ORP1
and ORP2 strongly bound PA, with weak binding toward CL and PI-3-P also
observed. PA is the product of phospholipase D, an enzyme that affects
SEC14-mediated transport from the Golgi in yeast (21, 34)
and appears to regulate vesicle formation in mammalian cells (35-37).
Genetic evidence in yeast indicates that the regulation of vesicle
transport from the Golgi by SEC14 is negatively regulated by
phospholipase D and possibly KES1/ORP1. Our observation that
human ORP1 and ORP2 have the capacity to bind PA implies that this
binding may be an important requirement for their regulation of vesicle
trafficking from the Golgi. ORP1 and ORP2 also bound the lipids CL and
PI-3-P, although with a much lower affinity than for PA. The CL binding
is likely not associated with in vivo ORP1 and ORP2 function
as this lipid is found almost exclusively in the mitochondria, and
there are no known links between ORP1, ORP2, KES1, or
SEC14 and transport to the mitochondria. However, the PI-3-P
binding may be associated with ORP1 and ORP2 phenotypes as the
synthesis of PI-3-P is required for vacuolar targeting from the Golgi
in yeast, and its conversion to PI-3,5-P is required to maintain
appropriate vacuole size (32, 39). The goal of future studies
will be to determine what ligand binding requirements are necessary for
ORP1 and ORP2 to fulfill their roles in vesicle trafficking and cell
growth regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
bypass afforded by each of
these mutations (11). Therefore, Kes1p likely counterbalances Sec14p
function, with both genes normally required to prevent toxicity of the
CDP-choline pathway and Sac1p to Golgi-derived vesicle transport.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purchased from Biomol.
ura3-1 his3-11, 15 leu2-3, 112 trp1-1 ade2-1 can1-100), and the sec14ts strain
CTY1-1A (a ura3-52
his3-200 lys2-801
sec14ts) have been described (17, 26). CTY1-1A was
crossed with w303-1A, and haploid progeny were isolated to create a
sec14ts yeast strain that can grow on
media-containing galactose as the main carbon source, CMY102 (a
ura3 his3 trp1 leu2 sec14ts GAL+). A
KES1 one-step gene disruption vector was constructed in
pBluescript II SK+ (Stratagene) by introducing a HIS3
genomic fragment into the EcoRI site of the KES1
gene. The recombinant DNA fragment was linearized with ApaI
and EcoRV and transformed into yeast strain CMY102 to create
strain CMY136 (a ura3 his3 trp1 leu2 sec14-1ts
kes1::HIS3 GAL+).
Recombinants were initially identified based on histidine prototrophy and the ability to grow at 37 °C. The
kes1::HIS3 genomic disruption was confirmed by
Southern analysis and PCR (24, 25).
-D-galactopyranoside for 2 h.
Cells were lysed with lysozyme and purified with Talon
(CLONTECH) resin using imidazole buffers as
described by the manufacturer. Purified protein were dialyzed overnight
versus phosphate-buffered saline at 4 °C. SDS-PAGE
analysis and Coomassie staining of the purified protein preparations
estimated a purity of greater than 95% homogeneity.
(10 pmol/ml) were allowed to bind to the immobilized lipids by
incubating the lipid blots with the purified proteins in the presence
of Tris-buffered saline containing 3% bovine serum albumin at 4 °C
for 12 h. Blots were washed with blocking buffer containing 0.1%
Tween 20 (w/v) and incubated with ORP1, ORP2, or protein kinase C
primary antibodies (1:1000) in blocking buffer for 1 h at room
temperature, washed twice, and incubated for 1 h with secondary
antibodies (1:10,000) coupled to horseradish peroxidase, washed six
times with blocking buffer containing 0.1% Tween 20 (w/v), and
subsequently developed using the ECL system (Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (45K):
[in a new window]
Fig. 1.
Alignment of human ORP1 and ORP2 predicted
amino acid sequences. A, domain comparison and amino
acid sequence identity (in the ligand binding domain compared with
OSBP1) for relevant members of the OSBP protein family. B,
amino acid sequence alignment of human ORP1 and ORP2 as predicted using
the CLUSTAL alignment tool. Identical amino acid residues are indicated
with dark shading, and similar amino acid residues are
indicated with light shading. The corresponding cDNAs
are deposited in GenBankTM under accessions numbers
AF274714 and AY028168.
-mediated
Bypass of the sec14ts Cell Growth
Phenotype--
SEC14 is an essential yeast gene that codes
for a PI/PC transfer protein necessary for vesicle trafficking from the
late Golgi to the cell surface and the vacuole (lysosome) (12-14,
17-21). Yeast cells with a temperature-sensitive SEC14
allele (sec14ts) grow normally at 25 °C but die
at the restrictive temperature of 37 °C (17). The requirement of
SEC14 for cell survival can be bypassed by inactivating the
KES1 gene (11, 17), and hence sec14ts
kes1
yeast can grow at 37 °C due to the
reestablishment of secretory competence, although the precise mechanism
by which loss of Kes1p function can accomplish
sec14
bypass has yet to be identified.
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Fig. 2.
ORP1 and ORP2 expression in yeast and their
ability to complement KES1 and affect yeast cell
growth. ORP1 and ORP2 expression is under control of the inducible
GAL1 promoter. A, the indicated yeast strains
were grown overnight in defined media containing glucose as carbon
source and with appropriate nutrients to ensure plasmid maintenance.
Yeast cell concentration was estimated by measuring absorbance at 600 nm, and identical numbers of cells were removed from each culture.
Cells were washed twice with minimal media containing 2% galactose
(w/v) substituted as carbon source, and a series of 1:4 dilutions were
made with 1 µl of each dilution spotted onto minimal media agar
plates containing 2% galactose and the appropriate nutrients to ensure
plasmid maintenance. Cells were incubated at either 25 or 37 °C for
4-6 days. B, each dilution was spotted onto minimal media
agar plates containing both galactose and glucose as carbon sources
(85% galactose) at a final sugar concentration of 2% (w/v).
C, expression of ORP1 and ORP2 protein was induced by the
presence of galactose. Galactose levels (shown at the
bottom) are presented as percentage of the total (20 g/liter) with glucose comprising the remainder. Identical amounts of
yeast cellular protein were separated by SDS-PAGE, and Western blots
were used to detect ORP1 and ORP2 proteins using the antibody
versus either GST-ORP1 or the ORP2 peptide as described
under "Experimental Procedures."
cells would result in cells that could no
longer survive at 37 °C, the non-permissive temperature for the
sec14ts allele. We observed that
sec14ts kes1::HIS3 yeast cells
transfected with ORP1 grew normally at 25 °C but did not grow at
37 °C, whereas those expressing ORP2 (grown on 85% galactose) grew
at both 25 and 37 °C (Fig. 2, A and B). To
ensure that expression of ORP1 did not confer a temperature-sensitive growth defect in and of itself, ORP1 was expressed in wild type yeast
cells. The wild type cells grew normally at both 25 and 37 °C (Fig.
2). These results indicated that ORP1 expression reversed the bypass of
the sec14ts allele afforded by inactivation of the
KES1 gene and thus provided evidence that ORP1 may be the
mammalian functional counterpart to yeast Kes1p.
gene resulted in an inhibition of CPY
secretion and a re-institution of the block in vesicle transport
provided by the sec14ts allele, consistent with our
observation that ORP1 also complemented KES1-dependent growth defects (Fig. 2). To
ensure ORP1 specificity for KES1 functional complementation,
ORP1 was expressed in wild type yeast, and CPY processing was examined.
The CPY secretion pattern in the cells transfected with ORP1 was
similar to untransfected cells or cells transfected with the vector
control (Fig. 3). These results indicated that ORP1 complemented
KES1 by reimposing Kes1p function with respect to
Golgi-derived vesicle transport and cell growth. Expression of ORP2 in
medium containing 85% galactose (the galactose amount required for
efficient ORP2 protein expression but at levels of ORP2 that do not
affect wild type yeast cell growth) did not affect CPY processing in
either wild type or sec14ts
kes1::HIS3 yeast at the permissive or
non-permissive temperature for the sec14ts allele.
Thus, the CPY processing data were consistent with the ability of ORP1,
but not ORP2, to complement Kes1p-related cell growth phenotypes and
indicate that ORP1 phenocopies both the growth and vesicle trafficking
defects associated with loss of function of Kes1p.
View larger version (51K):
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Fig. 3.
Effect of ORP1 and ORP2 on CPY
processing. ORP1 and ORP2 expression is under control of the
inducible GAL1 promoter. Yeast cells were grown in defined
media containing the required nutrients to ensure plasmid maintenance
with 100% galactose as carbon source (to induce human ORP1) and 85%
galactose, 15% glucose to induce ORP2 expression. Cells were
back-inoculated to an absorbance at 600 nm of 0.4 and grown for 6-8 h.
Aliquots of cells (absorbance at 600 nm of 3.0) were resuspended in
fresh media and incubated at 37 °C for 60 min. The indicated yeast
strains were grown overnight in defined media containing glucose as
carbon source and with appropriate nutrients to ensure plasmid
maintenance. Yeast cell concentration was estimated by measuring
absorbance at 600 nm, and identical numbers of cells were removed from
each culture. Cells were washed twice with minimal media containing 2%
galactose, and yeast cells were labeled with
[35S]methionine/cysteine for 10 min and then chased with
50 µM unlabeled methionine/cysteine for 0-60 min. CPY
immunoprecipitation and detection was performed as described under
"Experimental Procedures." P1 represents the ER form of
CPY; P2 represents the Golgi form; and M is the
mature vacuolar CPY.
View larger version (21K):
[in a new window]
Fig. 4.
Effect of overexpression of ORP2 on CPY
processing. ORP2 expression is under control of the inducible
GAL1 promoter. Yeast cells were grown in defined media
containing the required nutrients to ensure plasmid maintenance with
100% galactose as carbon source to induce ORP2 expression. Cells were
back-inoculated to an absorbance at 600 nm of 0.4 and grown for 6-8 h.
Aliquots of cells (absorbance at 600 nm of 3.0) were resuspended in
fresh media and incubated at 37 °C for 60 min. The indicated yeast
strains were grown overnight in defined media containing glucose as
carbon source and with appropriate nutrients to ensure plasmid
maintenance. Yeast cell concentration was estimated by measuring
absorbance at 600 nm, and identical numbers of cells were removed from
each culture. Cells were washed twice with minimal media containing 2%
galactose, and yeast cells were labeled with
[35S]methionine/cysteine for 10 min and then chased with
50 µM unlabeled methionine/cysteine for 0-60 min. CPY
immunoprecipitation and detection was performed as described under
"Experimental Procedures." P1 represents the ER form of
CPY; P2 represents the Golgi form; and M is the
mature vacuolar CPY.
as a specificity control (30, 31), and we observed the expected high degree of binding versus
phosphatidylserine, with weaker binding toward PI-4,5-P2,
and little to no binding toward either PA or PC (Fig.
5B).
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Fig. 5.
Ligand binding specificities of purified ORP1
and ORP2 proteins. A, ligand binding specificity of
ORP1 and ORP2. B, ligand binding specificity of protein
kinase C . ORP1 and ORP2 were expressed from pET23b plasmids
transformed into BL21(DE3) E. coli. ORP1 and ORP2 protein
production was induced by the addition of
isopropyl-1-thio-
-D-galactopyranoside for 2 h at
37 °C. Cells were lysed and proteins purified with Talon
(CLONTECH) resin using imidazole buffers as
described by the manufacturer. Purified proteins were dialyzed
overnight versus phosphate-buffered saline at 4 °C.
Phospholipids (100 pmol) were immobilized on nitrocellulose membranes,
and the membranes were blocked with Tris-buffered saline containing 3%
fatty acid-free bovine serum albumin. Pure protein preparations of
ORP1, ORP2, or protein kinase C
(10 pmol/ml) were allowed to bind to
the immobilized lipids by incubating the lipid blots with the purified
proteins in the presence of Tris-buffered saline containing 3% bovine
serum albumin at 4 °C for 12 h. Blots were washed with blocking
buffer containing 0.1% (w/v) Tween 20 and incubated with ORP1, ORP2,
or protein kinase C
primary antibodies (1:1000) in blocking buffer
for 1 h at room temperature, washed, and incubated for 1 h
with secondary antibodies (1:10,000) coupled to horseradish peroxidase
for subsequent development. CL, cardiolipin; DAG,
diacylglycerol; PE, phosphatidylglycerol;
PI-3,4-P2, phosphatidylinositol
3,4-bisphosphate; PI-3,4,5-P3,
phosphatidylinositol 3,4,5-triphosphate; PS,
phosphatidylserine; SM, sphingomyelin.
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[in a new window]
Fig. 6.
Concentration dependence of ligand binding by
purified ORP1 and ORP2 proteins. A, substrate
concentration dependence of ORP1 and ORP2 binding to phospholipids. The
1st lane on the left was spotted with 150 pmol of
each lipid, and subsequent lanes contain 1:1 serial dilutions such that
the last lane contains 2.33 pmol of lipid. Protein amount
used for the assay was 10 pmol/ml. B, protein concentration
dependence of ORP1 and ORP2 binding to phospholipids. In the 1st
lane on the left 5 pmol/ml of protein was used in the
assay followed by 1:1 dilutions such that the last lane
contains 0.08 pmol/ml of purified protein. Phospholipid amount used in
each assay was 150 pmol. Phospholipids were immobilized on
nitrocellulose membranes, and the membranes were blocked with
Tris-buffered saline containing 3% fatty acid-free bovine serum
albumin. Pure protein preparations of ORP1 or ORP2 were allowed to bind
to the immobilized lipids by incubating the lipid blots with the
purified proteins in the presence of Tris-buffered saline containing
3% bovine serum albumin at 4 °C for 12 h. Blots were washed
with blocking buffer containing 0.1% (w/v) Tween 20 and incubated with
ORP1 or ORP2 primary antibodies (1:1000) in blocking buffer for 1 h at room temperature, washed, and incubated for 1 h with
secondary antibodies (1:10,000) coupled to horseradish peroxidase for
subsequent development. CL, cardiolipin;
PI-3,4-P2, phosphatidylinositol
3,4-bisphosphate.
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Fig. 7.
Intracellular locations of ORP1 and
ORP2 in Chinese hamster ovary cells. A, effect of
brefeldin A (BFA) on the intracellular location of ORP1 and
ORP2. B, co-localization of ORP1 and ORP2 with specific
organelle markers. CHO-K1 cells were cultured in Dulbecco's modified
Eagle's medium containing 5% fetal calf serum and 34 µg/ml proline.
Cells were transfected with pEGFP-ORP1 or pEGFP-ORP2, and 30 h
after transfection cells were fixed in 10 mM sodium
phosphate (pH 7.4), 225 mM NaCl, and 2 mM
MgCl2 (PBS) containing 3% formaldehyde for 15 min at room
temperature. Cells were washed twice with PBS containing 5 mM ammonium chloride, permeabilized with PBS containing
0.05% Triton X-100, washed twice with PBS containing 1% fatty
acid-free bovine serum albumin, and nonspecific epitopes blocked with
PBS containing 1% fatty acid-free bovine serum albumin. To stain the
Golgi apparatus, cells were incubated in PBS containing 1% fatty
acid-free bovine serum albumin containing 10 µg/ml L. culinaris (LcH) lectin coupled to Texas Red for 1 h at room temperature. To stain the endoplasmic reticulum, cells were
incubated in PBS containing 1% fatty acid-free bovine serum albumin
containing mouse monoclonal antibodies to protein disulfide isomerase
(PDI) (Stressgen) at a 1:500 dilution followed by a goat
anti-mouse secondary antibody coupled to Texas Red (1:4000). Cells were
washed twice with PBS containing 1% fatty acid-free bovine serum
albumin and mounted on microscope slides with 90% glycerol, 50 mM Tris-HCl (pH 9.0), and 2.5%
1,4-diazadicyclo-(2,2,2)-octane. To disrupt the Golgi apparatus, cells
were treated with brefeldin A (2 µg/ml) for 30 min prior to the
fixation step.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Harold Cook and David Byers (Dalhousie University) for helpful discussions during the course of these studies. We gratefully acknowledge the excellent technical assistance of Robert Zwicker and Gladys Keddy in cell culture.
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FOOTNOTES |
---|
* This work was supported by Canadian Institutes of Health Research operating grant and scholarship (to C. R. M.), a Canadian Institutes of Health Research/Rx&D (Glaxo Wellcome and Smith Kline & French Laboratories) fellowship (to Y. X.), a Heart and Stroke Foundation of Canada operating grant, and a Canadian Institutes of Health Research scientist award (to N. D. R.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF274714 and AY028168.
Present address: Dept. of Cell Biology and Anatomy, University of
Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
§ Both authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 902-494-7066; Fax: 902-494-1394; E-mail: cmcmaste@is.dal.ca.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M101204200
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ABBREVIATIONS |
---|
The abbreviations used are: OSBP, oxysterol-binding protein; ORP, oxysterol-binding protein-related protein; PC, phosphatidylcholine; PA, phosphatidic acid; PI, phosphatidylinositol; PI-4-P, phosphatidylinositol 4-phosphate; PI-4, 5-P, phosphatidylinositol 4,5-bisphosphate; PI-3-P, phosphatidylinositol 3-phosphate; GFP, green fluorescent protein; CPY, carboxypeptidase Y; EST, expressed sequence tag; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.
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REFERENCES |
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