Department of Biochemical Physiology and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
* Author for correspondence (e-mail: d.vanhoof{at}bio.uu.nl)
Accepted 21 August 2002
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Summary |
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Key words: Lipophorin, Low-density lipoprotein, LDL receptor, RAP, Transferrin, Endocytosis, Recycling
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Introduction |
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Uptake of LDL is mediated by the LDL receptor (LDLR), which is the
prototype for a large class of endocytic transmembrane receptors
(Brown et al., 1997;
Hussain et al., 1999
).
Endocytosis of LDL has been extensively investigated and shown to result in
the degradation of the complete lipoprotein particle in lysosomes
(Goldstein et al., 1985
;
Brown and Goldstein, 1986
;
Dunn and Maxfield, 1992
).
Recently, a receptor expressed by the fat body of Locusta migratoria
has been cloned and sequenced, and identified as a novel member of the LDLR
family (Dantuma et al., 1999
).
This insect lipophorin receptor (iLR) was shown to mediate endocytic uptake of
HDLp in transiently-transfected COS-7 cells. A characteristic feature of HDLp
is its functioning as a reusable shuttle both at rest and during flight
activity. Thus, the particle selectively loads and unloads lipids at target
tissues, without concomitant degradation of HDLp
(Van der Horst, 1990
;
Soulages and Wells, 1994
;
Ryan and Van der Horst, 2000
;
Van der Horst et al., 2001
;
Van der Horst et al., 2002
).
In apparent contrast to the concept of selective lipid uptake, however, during
developmental stages of larval and young adult locusts, receptor-mediated
endocytic uptake of HDLp in the fat body was demonstrated
(Dantuma et al., 1997
). These
authors additionally showed that incubation of fat body tissue with HDLp
resulted in uptake of lipids, however, without substantial degradation of the
apolipoprotein component. The involvement of an LDLR family member in
lipoprotein metabolism implies complete lysosomal degradation of HDLp which is
in disagreement with these findings. Thus far, the intracellular distribution
after internalization of HDLp mediated by iLR had not been investigated.
Therefore, the intriguing question remained to be answered whether this novel
iLR, in contrast to all other LDLR family members, is able to recycle its
ligand after internalization.
LDL, along with di-ferric transferrin (Tf), has been extensively used to
study intracellular transport of ligands that are internalized by
receptor-mediated endocytosis (Goldstein
et al., 1985; Brown and
Goldstein, 1986
; Mellman,
1996
; Mukherjee et al.,
1997
). Via clathrin-coated pits, the ligand-receptor complexes
enter the cell in vesicles that subsequently fuse with tubulo-vesicular
sorting endosomes. Due to mild acidification of the vesicle lumen, LDL
dissociates from its receptor, but Tf merely unloads its two iron-ions and
remains attached to the Tf receptor (TfR)
(Mellman, 1996
;
Mukherjee et al., 1997
). After
repeated fusions with endocytic vesicles, sorting endosomes become
inaccessible to newly internalized material. Whereas the released LDL
particles are retained in the sorting endosome, most of the remaining membrane
constituents (e.g. LDLR and TfR), enter the tubular extensions. The tubules
bud off and are delivered to the morphologically distinct endocytic recycling
compartment (ERC) (Yamashiro et al.,
1984
; Mayor et al.,
1993
; Mukherjee et al.,
1997
). Consequently, Tf accumulates in these large, long-lived,
juxtanuclear vacuoles and, eventually, exits the compartments with a t
of
7 minutes (Mayor et al.,
1993
; Ghosh et al.,
1994
). Sorting endosomes, however, mature into lysosomes in which
LDL particles are completely degraded
(Goldstein et al., 1985
;
Brown and Goldstein, 1986
;
Dunn et al., 1989
).
In the present study, CHO cell lines, in which the intracellular LDL and Tf
transport pathways are well characterized, were stably transfected with iLR
cDNA. These transfected cells were used to analyze the distribution and
sorting of internalized insect and mammalian ligands, simultaneously.
Multicolour imaging allowed visualization of multiple fluorescently-labeled
ligands after endocytic uptake with high temporal and spatial resolution.
Incubation of iLR-transfected CHO cells with HDLp in combination with either
LDL or Tf initially resulted in colocalization of the insect lipoprotein with
LDL in sorting endosomes. However, in contrast to LDL that dissociates from
its receptor, HDLp is efficiently removed from these vesicles and, together
with iLR, accumulates in the Tf-positive ERC, as confirmed with
immunofluorescence. In addition to HDLp, iLR is capable of binding and
internalizing human receptor-associated protein (RAP), a ligand that is
structurally unrelated to lipoproteins. Like HDLp, this ligand is transported
to the ERC after receptor-mediated endocytosis. Similar to Tf, internalized
HDLp is re-secreted from the cells with a t of
13 minutes and
thereby escapes the lysosomal fate of endocytosed LDL particles. This provides
the first example of an LDLR homologue that, in contrast to all the other
family members, is able to recycle LDL-like lipoprotein upon receptor-mediated
endocytosis.
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Materials and Methods |
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Cell culture
The CHO cells were cultured in 75 cm2 polystyrene culture flasks
(Nunc Brand Products) with growth medium containing HAM's F-10 Nutrient
mixture, 10% heat inactivated fetal bovine serum, 100 U/ml penicillin G sodium
and 100 µg/ml streptomycin sulphate in 85% saline (Gibco BRL) at 37°C
in a humidified atmosphere of 5% CO2. Growth medium of
iLR-expressing cells was supplemented with 300 µg/ml G-418. For
fluorescence microscopy and confocal laser scanning microscopy, cells were
grown on 15 or 18-mm and 24-mm glass coverslips (Menzel-Gläser) in
12-wells (3.5 cm2/well) and 6-wells (9.6 cm2/well)
multidishes (Nunc Brand Products), respectively.
Generation of CHO cell lines stably expressing iLR
Wild-type CHO cells were grown to 40% confluency in 6-wells
multidishes and transfected for 20 hours with 5 µg of piLR-e plasmid
(Dantuma et al., 1999
) DNA in
2 ml serum free growth medium supplemented with 20 µl Lipofectin reagent
(Invitrogen Life Technologies) according to the supplier's protocol. The cells
were grown for 7-10 days in selective growth medium, containing 400 µg/ml
G-418, to obtain stably transfected cells. These cells were isolated by
limited dilution to generate monoclonal cell lines and checked for iLR
expression. Because variable levels of iLR expression were observed in the
different cell lines, we used a monoclonal CHO(iLR) cell line that showed the
highest expression level of iLR for the incubation experiments described in
this study.
Western blot analysis of CHO cell membrane extracts
Cells were harvested from 75 cm2 polystyrene flasks at 80%
confluency, resuspended in CHAPS buffer (20 mM HEPES, 124 mM NaCl, 4.7 mM KCl,
2.5 mM CaCl2, 2.5 mM Na2HPO4, 1.2 mM
MgSO4, 1 mM EDTA, 0.1 mM benzamidine, 1 µg/ml leupeptin, 1
µg/ml aprotinin, 1% CHAPS), incubated for 10 minutes on ice, and spun down
at 15,000 g for 10 minutes at 4°C. Supernatant was diluted
with 1 volume glycerol and soluble membrane proteins were either heated for 5
minutes at 95°C in Laemmli buffer
(Laemmli, 1970
) or directly
dissolved in Laemmli buffer with 0.1% SDS prior to separation by SDS-PAGE on a
10% polyacrylamide gel. The separated membrane proteins were transferred to
PVDF membrane (Millipore) by western blotting and incubated with
rabbit-anti-iLR (1:2000) or rabbit-anti-LDLR antibodies (1:5000) as indicated
for 2 hours, followed by 1 hour AP-GAR incubation. Hybridized AP-GAR was
visualized by incubating the blot in TSM buffer, containing 100 mM Tris-HCl,
100 mM NaCl, 10 mM MgAc2, 50 µg/ml p-nitro blue tetrazolium
chloride (NBT; Boehringer Mannheim), 25 µg/ml
5-bromo-4-chloro-3-indoyl-phosphate p-toluidine (BCIP; Roche Diagnostics), pH
9.0.
Incubation of CHO cells with fluorescently-labeled ligands
LDL and HDLp (1 mg/ml) were fluorescently labeled in PBS with 50 µl/ml
DiI in DMSO (3 µg/µl) at 37°C under continuous stirring for 16 hours
and 3 hours, respectively. HDLp and RAP (1 mg/ml) were labeled with 20
µl/ml OG dissolved in DMSO (1 µg/µl) at room temperature under
continuous stirring for 1 hour according to the manufacturer's instructions.
Fluorescently-labeled lipoproteins were purified with Sephadex G-25 PD-10
columns (Amersham Pharmacia Biotech) to replace the PBS by incubation medium
containing 10 mM HEPES, 50 mM NaCl, 10 mM KCl, 5 mM CaCl2, 2 mM
MgSO4, pH 7.4. OG-RAP was dialyzed against incubation medium using
standard cellulose membrane (Medicell International). For endocytic uptake,
CHO cells were incubated with 10 µg/ml DiI-LDL, 25 µg/ml OG-HDLp, 3.6
µg/ml OG-RAP and 25 µg/ml TMR-Tf as indicated for 15 minutes at 37°C
or 30 minutes at 18°C. Cells were rinsed in incubation medium and either
directly fixed in 4% paraformaldehyde diluted in PBS for 30 minutes at room
temperature, or chased in growth medium at 37°C for variable time periods.
When indicated, nocodazole (5 µM) or monensin (25 µM) was added to the
medium prior to, as well as during the chase.
Immunofluorescence
Fixed cells were washed twice with PBS buffer and permeabilized with PBS
buffer supplemented with 1.0 mg/ml saponin (PBSS) for 5 minutes at room
temperature. The cells were subsequently incubated with PBSS containing 50 mM
glycin for 10 minutes and 5% BSA for 30 minutes at room temperature. The cells
were blocked twice for 5 minutes with 0.1% cold water fish gelatin in PBSS
(PBSSG) at room temperature and incubated with corresponding primary
antibodies (1:500) for 1 hour at 37°C. After rinsing four times for 5
minutes with PBSSG at room temperature, the samples were processed for
indirect immunofluorescence by incubation with Cy5-GAR for 30 minutes at
37°C and rinsed an additional four times with PBSSG.
Microscopy and image processing
Coverslips with fixed cells were mounted in Mowiol supplemented with
anti-fade reagent (DABCO) and examined on a fluorescence Axioscop microscope
(Zeiss) with a Hg HBO-50 lamp and a Plan-Neofluar 100x/1.30 oil lens.
Using FITC/TRITC filters, digital images were acquired with a DXM 1200 digital
camera and ACT-1 version 2.00 software (Nikon Corporation).
To image living cells, we mounted the coverslips in a temperature-controlled aluminium chamber and incubated the cells at 37°C in growth medium supplemented with 1 µl/ml 1 mM LT, where indicated. Confocal multicolour images of cells were acquired using a Leica TCS-NT confocal laser scanning-system on an inverted microscope DMIRBE (Leica Microsystems) with a PL APO 40x/1.25-0.75 oil lens (Leica Microsystems) and an argon-krypton laser as excitation source. Emission of OG, excited with the 488 laser line, was detected using a 530/30 nm (RSP 580) bandpass filter. DiI, TMR and LT were excited with the 568 nm laser line and detected using a 600/30 nm (RSP 660) bandpass filter. The 647 nm laser line was used to excite Cy5 and emission was detected with a 665 nm longpass filter.
Images were processed using Scion Image beta version 4.0.2 (Scion Corporation) and PaintShop pro 7.00 (Jasc Software) software. SigmaPlot for Windows 4.00 (SPSS Inc) was used to generate surface fluorescence intensity mesh plots. To quantitate the relative intensity of fluorescently-labeled ligand in cells, the average brightness of pixels in manually defined areas covering the cells was determined using the Scion Image software. The digital data of more than 200 individual cells per data point were processed using Microsoft Excel 2000 (Microsoft Corporation) and plotted using the SigmaPlot software.
Incubation of CHO cells with 125I-labeled ligands
HDLp was labeled with 125I[iodine] using iodine monochloride
according to McFarlane (McFarlane,
1958), resulting in a specific labeling activity of 85 and 236
cpm/ng HDLp. 125I-RAP was prepared using chloramine-T according to
Rodenburg et al. (Rodenburg et al.,
1998
), resulting in a specific labeling activity of
45,000
cpm/ng protein. Two experiments were performed in duplicate, using wild-type
CHO and CHO(iLR) cells that were cultured in 12-well plates and grown to
70% confluency. The cells were incubated for 45 minutes at 37°C in
incubation medium containing 25 µg/ml 125I-HDLp or 83 ng/ml (2.1
nM) 125I-RAP without monensin, followed by an additional 15 minutes
in the presence of 25 µM monensin. The cells were placed on ice, washed
twice with cold wash buffer, containing 150 mM NaCl, 50 mM Tris-HCl, 2% BSA,
pH 7.4, and subsequently lysed and dissolved in 0.1 N NaOH. The radioactivity
of samples was determined with a Tri Carb 2300 TR liquid scintillation
analyzer (Packard) in Emulsifier Safe liquid scintillation fluid (Packard) and
a maximal counting time of 10 minutes per sample. To determine the total cell
protein per well, cells were washed thrice with 4°C HEPES buffer and
incubated for 4 hours at 4°C in a lysis buffer, containing 50 mM Tris-HCl
(pH 7.7), 150 mM NaCl, 0.1 mM benzamidine, 1 mg/ml leupeptin, 1 mg/ml
aprotinin, and 1% NP40. Protein concentrations were determined using the
colorimetric detergent compatible protein assay (Bio-Rad).
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Results |
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|
iLR mediates uptake of HDLp and human RAP in stably transfected CHO
cells
To investigate the functional ligand-binding specificity of iLR and LDLR,
iLR-transfected cells were incubated with fluorescently-labeled ligands in a
buffer that was supplemented with HEPES (i.e. incubation medium) to retard the
transit of internalized ligands at the early endosomal stage
(Sullivan et al., 1987). Upon
15 minutes of incubation at 37°C with DiI-labeled human LDL (DiI-LDL),
numerous cytoplasmic vesicles distributed throughout CHO(iLR) cells could be
observed (Fig. 2A). Such a
punctate staining pattern, indicative for receptor-mediated endocytosis, was
absent in ldlA(iLR) cells (Fig.
2B). This indicates that LDL uptake is exclusively accomplished by
the endogenous LDLR, and not a result of aspecific endocytosis via iLR. A
comparable particulate pattern was observed in iLR-transfected cells incubated
with DiI-labeled HDLp (DiI-HDLp) (Fig.
2C), however, not in non-transfected cells
(Fig. 2D). DiI is a fluorescent
lipid homologue that incorporates in the lipid moiety of lipoproteins. To
confirm the concomitant endocytic uptake of the protein component of the
lipoprotein, HDLp was labeled covalently with the amine-reactive fluorescent
probe Oregon Green (OG). Analogous incubation experiments with OG-labeled HDLp
(OG-HDLp) led to a similar endocytic uptake as could be visualized by DiI-HDLp
(Fig. 2E,F). These data suggest
that the lipid uptake mediated by iLR is a result of HDLp internalization
rather than a selective lipid-transfer mechanism occuring at the cell surface.
To verify that the internalized lipoproteins are localized in endosomes after
a 15 minutes incubation period at 37°C, the uptake experiments were
repeated for 30 minutes at 18°C. Intracellular distribution of endocytosed
ligands stagnates at a temperature of 18°C or below, preventing lysosomal
degradation of ligands and recycling of receptors
(Sullivan et al., 1987
). The
endocytic vesicle patterns of CHO(iLR) cells incubated at either temperature
were indistinguishable (Fig.
2G,H), which strongly suggests that HDLp is transferred to sorting
endosomes after receptor-mediated endocytosis. Uptake of fluorescently-labeled
HDLp could be reduced with an equimolar concentration, and almost completely
inhibited with a tenfold excess of unlabeled HDLp
(Fig. 2I). This indicates that
labeled and unlabeled HDLp compete for the same binding site. Therefore, it is
most unlikely that the interaction between HDLp and iLR is altered by the
covalently-bound OG label. From these experiments, we conclude that LDL uptake
is restricted to endogenous LDLR-expressing cells and that HDLp uptake is
exclusively mediated by iLR.
|
RAP has been shown to inhibit the binding of lipoproteins to LDLR family
members, such as LDLR-related protein (LRP), very low-density lipoprotein
receptor (VLDLR) and megalin (Herz et al.,
1991; Kounnas et al.,
1992
; Battey et al.,
1994
), but has only weak affinity for LDLR itself
(Medh et al., 1995
). RAP
serves as a molecular chaperone to assist the folding of several LDLR family
members and prevents premature ligand interaction in the endoplasmic reticulum
(Bu and Schwartz, 1998
;
Bu and Marzolo, 2000
). As
expected, when CHO(iLR) cells were incubated with DiI-LDL and an equimolar
concentration of human RAP, endocytosis of LDL was not significantly reduced
(Fig. 3A). However, endocytic
uptake of HDLp could be completely prevented by an equimolar concentration of
RAP (Fig. 3B). Inhibition of
HDLp endocytosis by RAP indicates that iLR binds HDLp in the prevalent
lipoprotein-binding manner, namely via its cysteine-rich ligand-binding domain
(Dantuma et al., 1999
).
Additionally, the observation that a 1:1 ratio of RAP to OG-HDLp is sufficient
to completely inhibit HDLp endocytosis suggests that, in comparison to HDLp,
RAP has a higher affinity for iLR. Moreover, these data suggest that RAP is a
ligand of iLR and, thus, could also be internalized by the insect receptor. To
obtain evidence for this latter issue, we incubated CHO cells with OG-labeled
RAP (OG-RAP) for 30 minutes at 18°C which resulted in a perinuclear
vesicle distribution (Fig. 3C).
Although the staining pattern appeared different from that observed in
CHO(iLR) cells incubated with HDLp, endocytic uptake of RAP was clearly
evident. Minor amounts of RAP could also be detected in endocytic vesicles of
wild-type CHO cells (Fig. 3D),
which is likely due to the expression of endogenous LRP and VLDLR. However,
the fluorescence intensity of these vesicles was much lower in comparison to
iLR-transfected cells, thus the majority of intracellular RAP in CHO(iLR)
cells is endocytosed by iLR. The observation that, in addition to HDLp, RAP is
also a ligand of iLR is in excellent agreement with iLR being an LDLR family
member.
|
Mammalian and insect lipoproteins follow distinct intracellular
routes
Receptor-bound LDL is rapidly delivered to sorting endosomes upon
endocytosis by mammalian cells (Ghosh et
al., 1994; Mellman,
1996
; Mukherjee et al.,
1997
). The results of the incubation experiments at 18°C
(Fig. 2H) suggest that HDLp and
LDL are internalized and transferred to the same vesicles. To investigate
whether HDLp accumulates in these tubulo vesicular endosomes, CHO(iLR) cells
were incubated at 18°C with OG-HDLp in incubation medium supplemented with
DiI-LDL. There was significant colocalization of HDLp
(Fig. 4A) with LDL-containing
endocytic vesicles (Fig. 4B,C) that were distributed throughout the cell, which supports the assumption that
HDLp accumulates in sorting endosomes after endocytic uptake.
|
In sorting endosomes, LDL dissociates from LDLR due to mild luminal
acidification after which the ligand is degraded in lysosomes. The receptor,
however, is transported back to the cell surface via the ERC for additional
uptake of extracellular LDL (Mellman,
1996; Mukherjee et al.,
1997
). By observing living cells with confocal laser scanning
microscopy, we were able to visualize the sorting of mammalian and insect
lipoproteins simultaneously, directly after endocytic uptake. CHO(iLR) cells
were preincubated with OG-HDLp and DiI-LDL for 15 minutes at 37°C and
subjected to a chase in growth medium without fluorescently-labeled ligands
(chase medium) for an additional 30 minutes at 37°C. Within 10 minutes, a
large amount of HDLp concentrated in the juxtanuclear area
(Fig. 4D,E) in which LDL was
almost completely absent (Fig.
4F,G). To investigate whether these vesicles were late endosomes
or lysosomes, the membrane permeable probe, LysoTracker Yellow (LT), a weakly
basic amine that selectively accumulates in cellular compartments with low
luminal pH [i.e. lysosomes (Griffiths et
al., 1988
)], was added to the chase medium
(Fig. 4H,I). As shown in
Fig. 4J, there was almost no
colocalization of HDLp with LT, and in areas where there was apparent overlap,
the size and shape of the structures appeared different
(Fig. 4K-M). This result
implies that HDLp is not destined to be degraded via the classic LDL pathway.
Together, these results confirm that, in contrast to LDL, internalized HDLp is
not destined for lysosomal degradation.
The microtubule-depolymerising agent nocodazole was used to investigate
whether this perinuclear targeting was microtubule dependent. Depolymerization
of microtubules has little effect on endocytosis, however,
microtubule-dependent transport of internalized material is inhibited
(Jin and Snider, 1993).
Incubation of CHO(iLR) cells with DiI-LDL and OG-HDLp in the presence of 5
µM nocodazole followed by a chase for 30 minutes with an equal
concentration of nocodazole resulted in the formation of enlarged LDL-labeled
vesicles that were localized peripherally in the cells
(Fig. 4N). A similar
distribution of endocytic vesicles was observed when HDLp or RAP was used
(Fig. 4O,P, respectively).
Although HDLp- and RAP-containing vesicles appeared smaller in size and their
fluorescence intensity less in comparison to LDL-containing vesicles, these
data indicate that transit of iLR-bound ligands (i.e. HDLp and RAP) is
microtubule dependent.
HDLp and RAP are transported to the ERC by iLR
To determine whether HDLp is translocated to the juxtanuclear localized
ERC, we used Tf which converges in the ERC after endocytic uptake due to the
durable association with TfR (Yamashiro et
al., 1984; Mayor et al.,
1993
). CHO(iLR) cells that were incubated with OG-HDLp and
tetramethyl-rhodamine-labeled Tf (TMR-Tf), and subjected to a chase, show that
HDLp is translocated to the ERC within 10 minutes
(Fig. 5, left panel). Despite a
small portion of individual vesicles that remained dispersed throughout the
cell, the majority of HDLp colocalized with Tf in the ERC
(Fig. 5, middle and right
panel) from where molecules eventually exit the cell
(Yamashiro et al., 1984
).
However, the gathering of HDLp in the ERC appeared slightly slower in
comparison to the rapid transport of Tf, which is most likely the result of
different sorting rates.
|
Additional evidence for the transport of HDLp to the ERC was obtained from
experiments with monensin, a carboxylic ionophore which disrupts the route of
recycling receptors (e.g. LDLR and TfR) by preventing the receptors from
returning to the cell surface and thereby causing them to reside within the
ERC (Basu et al., 1981;
Stein et al., 1984
). A
concentration of 25 µM monensin appeared sufficient to interrupt receptor
recycling and trap internalized receptors of CHO(iLR) cells that were
preincubated with DiI-LDL or OG-HDLp, and chased for an additional 30 minutes.
Monensin did not significantly affect lysosomal targeting of LDL
(Fig. 6A), however, HDLp
accumulated in the juxtanuclear area (Fig.
6B). OG fluorescence observed in the ERC represents either
undegraded OG-HDLp or OG released from degraded OG-HDLp. To confirm the
concomitant transport of the non-exchangeable apolipoprotein matrix of HDLp
with the fluorescent label OG to the ERC, we used antibodies against apoLp-I
and -II to immunolocalize the proteins. Cells were fixed after preincubation
with OG-HDLp and a chase of 30 minutes in the presence of monensin. The cells
were subsequently incubated with anti-apoLp-I or -II rabbit antibodies
(Schulz et al., 1987
) which
were visualized with a Cy5-labeled goat-anti-rabbit second antibody. Both
apoLp-I (Fig. 6C) and apoLp-II
(Fig. 6D) were predominantly
localized in the ERC and show significant overlap with OG
(Fig. 6E-H). We interpret these
data to indicate that the complete non-exchangeable protein matrix of HDLp,
comprising apoLp-I and -II, is transported to the ERC.
|
Above we showed that iLR is capable of binding and internalizing human RAP
(Fig. 3C). To investigate
whether endocytosed RAP is also transported to the ERC, we repeated the
incubation experiments with monensin using RAP. Subjecting CHO(iLR) cells to a
chase after preincubation with OG-RAP in the presence of monensin resulted in
the convergence of RAP in a single spot near the nucleus
(Fig. 7A). When TMR-Tf was used
in combination with OG-RAP, there was significant colocalization of RAP and Tf
in the ERC (Fig. 7B-D). This
implies that the pathways of ligands that are internalized by iLR are
determined by the intracellular route of the receptor. To visualize the
intracellular localization of iLR, we used anti-iLR antibody and the
Cy5-labeled second antibody to detect iLR in fixed CHO(iLR) cells.
Preincubation of these cells with OG-HDLp followed by a chase in medium
containing monensin shows that the ligand is localized in the ERC
(Fig. 7E), the organelle in
which iLR is also located (Fig.
7F,G). Even in the absence of ligand or monensin, the receptor was
predominantly present in the ERC (Fig.
7H), suggesting constitutive recycling of iLR without antecedent
ligand binding as observed for LDLR
(Anderson et al., 1982;
Brown et al., 1982
) and TfR
(Stein and Sussman, 1986
).
|
To quantify iLR-specific uptake, and subsequent transfer to the ERC of HDLp
and RAP, we incubated wild-type CHO and CHO(iLR) cells with
125I-labeled HDLp and RAP in the presence of monensin. Cells were
preincubated with the 125I-labeled ligands for 45 minutes at
37°C without monensin, followed by a shorter second incubation of 15
minutes at 37°C with the 125I-labeled ligands in the presence
of 25 µM monensin. These experiments revealed an iLR-mediated HDLp uptake
of 112 ng/mg cell protein (means of two duplo experiments, s.e.m.±27),
which corresponds to 350 pmol/mg cell protein. iLR-specific uptake of RAP
was also determined and appeared to be 61.3 ng/mg cell protein (mean of duplo
experiment, s.e.m.±0.44), the equivalent of
1570 pmol/mg cell
protein. This
4.5-fold higher uptake of RAP in comparison to HDLp is in
good agreement with the observation that a 1:1 ratio of RAP to OG-HDLp is
sufficient to completely inhibit HDLp endocytosis
(Fig. 3B). Moreover, it
supports the relatively higher affinity of RAP for iLR in comparison to that
of HDLp, as suggested above.
HDLp is re-secreted from CHO(iLR) cells with a t of
13
minutes
Convergence of HDLp in the ERC implies that the ligand is eventually
re-secreted into the medium (Yamashiro et
al., 1984). Quantitative fluorescence microscopy was used to
determine the exit rate of intracellular HDLp and LDL. CHO(iLR) cells were
analyzed after a preincubation of OG-HDLp and DiI-LDL to label the endocytic
pathway. Shortly after initiating the chase, the clearly visible ERC
predominantly contained HDLp, in which no significant amount of LDL could be
detected (Fig. 8A). In
contrast, the spatially distributed vesicles that were numerously present
contained mainly LDL, some of which harbouring only a minor amount of HDLp.
During the chase, the relative fluorescent intensity of OG-HDLp in the ERC
decreased dramatically compared to that of the individual, LDL-containing
vesicles (Fig. 8B-F). Total
intracellular fluorescence of OG-HDLp and DiI-LDL in cells that were fixed
after a chase at defined time points were determined
(Fig. 8G). The plotted data
show that the relative fluorescence of intracellular OG-HDLp rapidly
decreases, whereas that of DiI-LDL remains constant during a 60 minutes chase.
From these observations, we conclude that HDLp exits the cells with a
t
of
13 minutes, which is in good agreement with that of Tf
(Mayor et al., 1993
;
Ghosh et al., 1994
). The
clearance of intracellular HDLp strongly suggests that HDLp is re-secreted
after passage through the ERC.
|
Taken together, all the results indicate that HDLp uptake is specifically mediated by iLR. In addition to insect lipoprotein, iLR is capable of binding and internalizing human RAP. In contrast to LDL, which ends up in lysosomes, ligands that are internalized by iLR are not destined for lysosomal degradation. As a result of the intracellular pathway of the receptor, iLR-coupled ligands follow a transferrin-like intracellular recycling route.
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Discussion |
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CHO cells that are transfected with iLR cDNA mediate endocytosis of HDLp,
however, the ligand remains in complex with the receptor in sorting endosomes.
Several LDLR family member mutants have been constructed to identify the
responsible domains and investigate the biochemical mechanisms involved in
ligand uncoupling due to an acidic pH
(Davis et al., 1987;
Mikhailenko et al., 1999
).
Here we present evidence for the first naturally occurring LDLR family member,
the ligands of which remain coupled to iLR in sorting endosomes and are
consequently transported to the ERC to be eventually re-secreted in a
transferrin-like manner.
Re-secretion of HDLp after endocytosis is consistent with the role for HDLp
as a reusable shuttle for selective lipid delivery. The major difference
between insect and mammalian lipoproteins is the selective mechanism by which
insect lipoproteins transfer their hydrophobic cargo. Dependent on the
physiological situation, circulating HDLp particles serve as either DAG
acceptors at the insect fat body during adult stage-restricted flight
activity, or donors during dietary lipid storage in the fat body of larval and
young adult insects (Van der Horst,
1990; Ryan and Van der Horst,
2000
; Van der Horst et al.,
2001
; Van der Horst et al.,
2002
). In the latter case, endocytic uptake of HDLp seems to
conflict with the selective unloading of lipids from HDLp to fat body cells
without concurrent degradation of the ligand
(Arrese et al., 2001
). In
experiments in which fat body tissue from young adult locusts was incubated
with HDLp containing 3H-labeled DAG and apolipoproteins,
3H-DAG appeared to be taken up selectively without substantial
concomitant accumulation of the radiolabeled apolipoproteins
(Dantuma et al., 1997
).
Endocytosis of HDLp for lipid storage in fat body cells had earlier been
postulated for the insect Ashna cyanea (Bauerfeind and Komnick,
1992). However, thus far, evidence for recycling of the ligand had not been
described. Our observations with fluorescently-labeled HDLp strongly support
that, despite receptor-mediated internalization of the ligand, HDLp can be
used as a reusable shuttle in both physiological conditions. Moreover, we
provide preliminary evidence for the existence of a novel selective
lipid-uptake mechanism mediated by an LDLR homologue that takes place
intracellularly.
Despite structural homology between LDL and HDLp at the protein level, we
have shown that iLR specifically internalizes the insect lipoprotein, whereas
LDLR exclusively mediates uptake of LDL. In addition to HDLp, iLR shows a
relatively high affinity for human RAP; a feature that is not shared by LDLR
(Medh et al., 1995). However,
all other members of the LDLR family have been observed to bind RAP with high
affinity and internalize this ligand
(Neels et al., 1998
). The
ability of iLR to bind human RAP is in line with the presence of a RAP
homologous gene identified in the Drosophila genome
(Adams et al., 2000
).
Transition of internalized HDLp to the ERC is mediated by the
membrane-spanning iLR in analogy to Tf recycling
(Yamashiro et al., 1984). In
contrast to the uncoupling of mammalian LDL from LDLR in sorting endosomes,
HDLp remains attached to its receptor despite the decrease in lumenal pH.
Endosome tubulation followed by iterative fractionation of membrane-anchored
recycling receptors results in efficient receptor recycling by default
(Dunn et al., 1989
;
Verges et al., 1999
).
Consequently, ligands that remain coupled to such receptors are recycled as
well. Davis et al. showed that the EGF-precursor homology domain of LDLR is
responsible for acid-dependent ligand dissociation
(Davis et al., 1987
). In
addition, Mikhailenko et al. produced a VLDLR mutant of which the
EGF-precursor homology domain was deleted
(Mikhailenko et al., 1999
).
They demonstrated that, in contrast to wild-type VLDLR, RAP did not dissociate
from the mutant receptor after internalization and was not degraded. By using
RAP as well as HDLp, we show that iLR is capable of transporting
physiologically unrelated ligands to the ERC, despite having a typical
ligand-dissociating EGF-precursor homology domain. Our results combined with
earlier observations using 3H-labeled HDLp to incubate fat body
cells indicate that iLR-mediated recycling of HDLp plays a physiologically
relevant role in lipid storage (Dantuma et
al., 1997
). A selective lipid extration mechanism would
significantly reduce degradation as well as energy-consuming synthesis of
reusable HDLp.
Cellular uptake of HDLp and human RAP by iLR results in an intracellular
distribution of both ligands that deviates from the classic lysosomal delivery
of mammalian lipoproteins in CHO cells. These observations propose a novel
mechanism for ligand-uptake by an LDLR family member that is present in
insects. It has been suggested that specific mammalian tissues may selectively
take up lipoprotein-bound components with LDLR homologous receptors (e.g.
LRP), however, without endocytosis of the ligand
(Vassiliou et al., 2001;
Swarnakar et al., 2001
).
Additionally, alternative functions for LDLR that deviate from the classic
lysosomal lipoprotein delivery could also depend on the developmental stage or
type of tissue (Dehouck et al.,
1997
). Our model system using iLR and CHO cells provides a
powerful tool to study the molecular basis for the intracellular distribution
and fate of ligands that are internalized by LDL receptors, as well as the
function of individual receptor domains. An important issue to be solved
remains the understanding of the molecular basis for the difference in
targeting behaviour of the mammalian and insect receptors. Although LDLR and
iLR share a 57% sequence similarity, small differences in receptor domains
might determine the fate of bound ligands. Whereas the ligand-binding domain
of LDLR comprises seven cysteine-rich repeats, iLR has eight of these modules.
The larger ligand-binding domain could cause a more stable ligand-receptor
interaction, preventing acid-induced uncoupling in the endosomal compartment
that is mediated by the EGF-precursor homology domain. In addition, the twelve
C-terminal amino acids of the cytoplasmic tail of LDLR are completely
different compared to those of iLR. Moreover, the intracellular portion of iLR
has an additional 10 amino acids. These residues could possibly interact with
cytosolic components involved in processes that direct ligand distribution.
Further analysis of insect lipoproteins and receptors, as well as the
construction of hybrid receptors that are composed of (parts of) insect and
mammalian receptors, will provide new insights into the understanding of
molecular mechanisms that regulate lipoprotein binding and lipid uptake in
mammals.
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Acknowledgments |
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References |
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---|
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A.,
Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A.,
Galle, R. F. et al. (2000). The genome sequence of
Drosophila melanogaster. Science
287,2185
-2195.
Anderson, R. G. W., Brown, M. S., Beisigel, U. and Goldstein, J. L. (1982). Surface distribution and recycling of the low density lipoprotein receptor as visualized with antireceptor antibodies. J. Cell Biol. 93,523 -531.[Medline]
Arrese, E. L., Canavoso, L. E., Jouni, Z. E., Pennington, J. E., Tsuchida, K. and Wells, M. A. (2001). Lipid storage and mobilization in insects: current status and future directions. Insect Biochem. Mol. Biol. 31, 7-17.[CrossRef][Medline]
Babin, P. J., Bogerd, J., Kooiman, F. P., van Marrewijk, W. J. A. and van der Horst, D. J. (1999). Apolipophorin II/I, apolipoprotein B, vitellogenin, and microsomal triglyceride transfer protein genes are derived from a common ancestor. J. Mol. Evol. 49,150 -160.[Medline]
Basu, S. K., Goldstein, J. L., Anderson, R. G. and Brown, M. S. (1981). Monensin interrupts the recycling of low density lipoprotein receptors in human fibroblasts. Cell 24,493 -502.[Medline]
Battey, F., Gafvels, M. E., Fitzgerald, D. J., Argraves, W. S.,
Chappell, D. A., Straus, J. F., III and Strickland, D. K.
(1994). The 39 kDa receptor-associated protein regulates ligand
binding by the very low density lipoprotein receptor. J. Biol.
Chem. 269,23268
-23273.
Bauerfind, R. and Komnick, H. (1992). Immunocytochemical localization of lipophorin in the fat body of dragonfly larvae (Aeshna cyanea). J. Insect Physiol. 38,185 -198.
Bogerd, J., Babin, P. J., Kooiman, F. P., Andre, M., Ballagny, C., van Marrewijk, W. J. A. and van der Horst, D. J. (2000). Molecular characterization and gene expression in the eye of the apolipophorin II/I precursor from Locusta migratoria. J. Comp. Neurol. 427,546 -558.[CrossRef][Medline]
Brown, M. S. and Goldstein, J. L. (1986). A receptor-mediated pathway for cholesterol homeostasis. Science 232,34 -47.[Medline]
Brown, M. S., Anderson, R. G., Basu, S. K. and Goldstein, J. L. (1982). Recycling of cell-surface receptors: observations from the LDL receptor system. Cold Spring Harb. Symp. Quant. Biol. 46,713 -721.[Medline]
Brown, M. S., Herz, J. and Goldstein, J. L. (1997). LDL-receptor structure. Calcium cages, acid baths and recycling receptors. Nature 388,629 -630.[CrossRef][Medline]
Bu, G. and Marzolo, M. P. (2000). Role of RAP in the biogenesis of lipoprotein receptors. Trends Cardiovasc. Med. 10,148 -155.[CrossRef][Medline]
Bu, G. and Schwartz, A. L. (1998). RAP, a novel type of ER chaperone. Trends Cell Biol. 8, 272-275.[CrossRef][Medline]
Dantuma, N. P., van Marrewijk, W. J. A., Wynne, H. J. and van der Horst, D. J. (1996). Interaction of an insect lipoprotein with its binding site at the fat body. J. Lipid Res. 37,1345 -1355.[Abstract]
Dantuma, N. P., Pijnenburg, M. A., Diederen, J. H. B. and van der Horst, D. J. (1997). Developmental down-regulation of receptor-mediated endocytosis of an insect lipoprotein. J. Lipid Res. 38,254 -265.[Abstract]
Dantuma, N. P., Potters, M., de Winther, M. P., Tensen, C. P.,
Kooiman, F. P., Bogerd, J. and van der Horst, D. J. (1999).
An insect homolog of the vertebrate very low density lipoprotein receptor
mediates endocytosis of lipophorins. J. Lipid Res.
40,973
-978.
Davis, C. G., Goldstein, J. L., Sudhof, T. C., Anderson, R. G., Russell, D. W. and Brown, M. S. (1987). Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature 326,760 -765.[CrossRef][Medline]
Dehouck, B., Fenart, L., Dehouck, M. P., Pierce, A., Torpier, G.
and Cecchelli, R. (1997). A new function for the LDL
receptor: transcytosis of LDL across the blood-brain barrier. J.
Cell Biol. 138,877
-889.
Dunn, K. W. and Maxfield, F. R. (1992). Delivery of ligands from sorting endosomes to late endosomes occurs by maturation of sorting endosomes. J. Cell Biol. 117,301 -310.[Abstract]
Dunn, K. W., McGraw, T. E. and Maxfield, F. R. (1989). Iterative fractionation of recycling receptors from lysosomally destined ligands in an early sorting endosome. J. Cell Biol. 109,3303 -3314.[Abstract]
Fazio, S., Linton, M. F., Hasty, A. H. and Swift, L. L.
(1999). Recycling of apolipoprotein E in mouse liver.
J. Biol. Chem. 274,8247
-8253.
Ghosh, R. N., Gelman, D. L. and Maxfield, F. R.
(1994). Quantification of low density lipoprotein and transferrin
endocytic sorting HEp2 cells using confocal microscopy. J. Cell
Sci. 107,2177
-2189.
Goldstein, J. L., Brown, M. S., Anderson, R. G. W., Russell, D. W. and Schneider, W. J. (1985). Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu. Rev. Cell Biol. 1,1 -39.[CrossRef]
Griffiths, G., Hoflack, B., Simons, K., Mellman, I. and Kornfeld, S. (1988). The mannose 6-phosphate receptor and the biogenesis of lysosomes. Cell 52,329 -341.[Medline]
Heeren, J., Weber, W. and Beisiegel, U. (1999).
Intracellular processing of endocytosed triglyceride-rich lipoproteins
comprises both recycling and degradation. J. Cell Sci.
112,349
-359.
Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K. and
Brown, M. S. (1991). 39-kDa protein modulates binding of
ligands to low density lipoprotein receptor-related
protein/2-macroglobulin receptor. J. Biol.
Chem. 266,21232
-21238.
Hussain, M. M., Strickland, D. K. and Bakillah, A. (1999). The mammalian low-density lipoprotein receptor family. Annu. Rev. Nutr. 19,141 -172.[CrossRef][Medline]
Jin, M. and Snider, M. D. (1993). Role of
microtubules in transferrin receptor transport from the cell surface to
endosomes and the Golgi complex. J. Biol. Chem.
268,18390
-18397.
Kingsley, D. M. and Krieger, M. (1984). Receptor-mediated endocytosis of low density lipoprotein: somatic cell mutants define multiple genes required for expression of surface-receptor activity. Proc. Natl. Acad. Sci. USA 81,5454 -5458.[Abstract]
Kounnas, M. Z., Church, F. C., Argraves, W. S. and Strickland,
D. K. (1992). The 39-kDa receptor-associated protein
interacts with two members of the low density lipoprotein receptor family,
2-macroglobulin receptor and glycorprotein 330. J. Biol.
Chem. 267,21162
-21166.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Mann, C. J., Anderson, T. A., Read, J., Chester, S. A., Harrison, G. B., Kochl, S., Ritchie, P. J., Bradbury, P., Hussain, F. S., Amey, J. et al. (1999). The structure of vitellogenin provides a molecular model for the assembly and secretion of atherogenic lipoproteins. J. Mol. Biol. 285,391 -408.[CrossRef][Medline]
Mayor, S., Presley, J. F. and Maxfield, F. R. (1993). Sorting of membrane components from endosomes and subsequent recycling to the cell surface occurs by a bulk flow process. J. Cell Biol. 121,1257 -1269.[Abstract]
McFarlane, A. S. (1958). Efficient trace-labelling of proteins with iodine. Nature 82, 53.
Medh, J. D., Fry, G. L., Bowen, S. L., Pladet, M. W.,
Strickland, D. K. and Chappell, D. A. (1995). The 39-kDa
receptor-associated protein modulates lipoprotein catabolism by binding to LDL
receptors. J. Biol. Chem.
270,536
-540.
Mellman, I. (1996). Endocytosis and molecular sorting. Annu. Rev. Cell Dev. Biol. 12,575 -625.[CrossRef][Medline]
Mikhailenko, I., Considine, W., Argraves, K. M., Loukinov, D.,
Hyman, B. T. and Strickland, D. K. (1999). Functional domains
of the very low density lipoprotein receptor: molecular analysis of ligand
binding and acid-dependent ligand dissociation mechanisms. J. Cell
Sci. 112,3269
-3281.
Mukherjee, S., Ghosh, R. N. and Maxfield, F. R.
(1997). Endocytosis. Physiol. Rev.
77,759
-803.
Neels, J. G., Horn, I. R., van den Berg, B. M. M., Pannekoek, H. and van Zonneveld, A. J. (1998). Ligand-receptor interactions of the low density lipoprotein receptor-related protein, a multi-ligand endocytic receptor. Fibrinolysis Proteolysis 12,219 -240.
Redgrave, T. G., Roberts, D. C. and West, C. E. (1975). Separation of plasma lipoproteins by density-gradient ultracentrifugation. Anal. Biochem. 65, 42-49.[Medline]
Rensen, P. C. N., Jong, M. C., van Vark, L. C., van der Boom,
H., Hendriks, W. L., van Berkel, T. J. C., Biessen, E. A. L. and Havekes, L.
M. (2000). Apolipoprotein E is resistant to intracellular
degradation in vitro and in vivo. Evidence for retroendocytosis. J.
Biol. Chem. 275,8564
-8571.
Rodenburg, K. W., Kjøller, L., Petersen, H. H. and
Andreasen, P. A. (1998). Binding of urokinase-type
plasminogen activator/plasminogen activator inhibitor-1 complex to endocytosis
receptors 2-macroglobulin receptor/low density lipoprotein
receptor-related protein and very low density lipoprotein receptor involves
basic residues in the inhibitor. Biochem. J.
329, 55-63.[Medline]
Russell, D. W., Schneider, W. J., Yamamoto, T., Luskey, K. L., Brown, M. S. and Goldstein, J. L. (1984). Domain map of the LDL receptor: sequence homology with the epidermal growth factor precursor. Cell 37,577 -585.[Medline]
Ryan, R. O. and van der Horst, D. J. (2000). Lipid transport biochemistry and its role in energy production. Annu. Rev. Entomol. 45,233 -568.[CrossRef][Medline]
Schulz, T. K. F., van der Horst, D. J., Amesz, H., Voorma, H. O. and Beenakkers, A. M. T. (1987). Monoclonal antibodies specific for apoproteins of lipophorins from the migratory locust. Arch. Insect Pysiol. Biochem. 6, 97-107.
Segrest, J. P., Jones, M. K., de Loof, H. and Dashti, N.
(2001). Structure of apolipoprotein B-100 in low density
lipoproteins. J. Lipid Res.
42,1346
-1367.
Shelness, G. S. and Sellers, J. A. (2001). Very-low-density lipoprotein assembly and secretion. Curr. Opin. Lipidol. 12,151 -157.[CrossRef][Medline]
Soulages, J. L. and Wells, M. A. (1994). Lipophorin: the structure of an insect lipoprotein and its role in lipid transport in insects. Adv. Protein Chem. 45,371 -415.[Medline]
Stein, B. S. and Sussman, H. H. (1986).
Demonstration of two distinct transferrin receptor recycling pathways and
transferrin-independent receptor internalization in K562 cells. J.
Biol. Chem. 261,10319
-10331.
Stein, B. S., Bensch, K. G. and Sussman, H. H.
(1984). Complete inhibition of transferrin recycling by monensin
in K562 cells. J. Biol. Chem.
259,14762
-14772.
Sullivan, P. C., Ferris, A. L. and Storrie, B. (1987). Effects of temperature, pH elevators, and energy production inhibitors on horseradish peroxidase transport through endocytic vesicles. J. Cell Physiol. 131, 58-63.[Medline]
Swarnakar, S., Beers, J., Strickland, D. K., Azhar, S. and
Williams, D. L. (2001). The apolipoprotein E-dependent low
density lipoprotein cholesteryl ester selective uptake pathway in murine
adrenocortical cells involves chondroitin sulfate proteoglycans and an alpha
2-macroglobulin receptor. J. Biol. Chem.
276,21121
-21128.
Van der Horst, D. J. (1990). Lipid transport function of lipoproteins in flying insects. Biochim. Biophys. Acta 1047,195 -211.[Medline]
Van der Horst, D. J., van Marrewijk, W. J. A. and Diederen, J. H. B. (2001). Adipokinetic hormones of insect: Release, signal transduction, and responses. Int. Rev. Cytol. 211,179 -240.[Medline]
Van der Horst, D. J., van Hoof, D., van Marrewijk, W. J. A. and Rodenburg, K. W. (2002). Alternative lipid mobilization: the insect shuttle system. Mol. Cell. Biol. (in press).
Vassiliou, G., Benoist, F., Lau, P., Kavaslar, G. N. and
McPherson, R. (2001). The low density lipoprotein
receptor-related protein contributes to selective uptake of high density
lipoprotein cholesteryl esters by SW872 liposarcoma cells and primary human
adipocytes. J. Biol. Chem.
276,48823
-48830.
Verges, M., Havel, R. J. and Mostov, K. A.
(1999). A tubular endosomal fraction from rat liver: biochemical
evidence of receptor sorting by default. Proc. Natl. Acad. Sci.
USA 96,10146
-10151.
Weers, P. M. M., van Marrewijk, W. J. A., Beenakkers, A. M. T.
and van der Horst, D. J. (1993). Biosynthesis of locust
lipophorin. Apolipophorins I and II originate from a common precursor.
J. Biol. Chem. 268,4300
-4303.
Yamashiro, D. J., Tycko, B., Fluss, S. R. and Maxfield, F. R. (1984). Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell 37,789 -800.[Medline]