(Received for publication, April 6, 1995; and in revised form, August 1, 1995 )
From the
Glycosylphosphatidylinositols (GPIs) are ubiquitous in
eukaryotes and serve to anchor a variety of proteins to the exoplasmic
leaflet of cellular membranes. GPIs are synthesized in the endoplasmic
reticulum (ER), in excess of the amount needed for protein
modification. The fate of the excess GPIs is unknown, but they may be
retained in the ER, transported to other membranes, and/or metabolized.
In relation to this problem, we were interested in determining whether
GPIs were transported to the plasma membrane and whether, like
GPI-anchored proteins, their presence was confined to the apical plasma
membrane domain in polarized epithelial cells. Polarized Madin-Darby
canine kidney epithelial cell monolayers were incubated with
[H]mannose or
[
H]ethanolamine to label GPIs and then infected
with enveloped viruses. We used influenza virus (flu) and vesicular
stomatitis virus (VSV) for these experiments as these viruses are
assembled at the cell surface and acquire their envelope lipids from
the plasma membrane. Furthermore, flu and VSV bud specifically from the
apical and basolateral plasma membrane domains, respectively. Flu and
VSV were isolated from the apical and basolateral media, respectively,
and subjected to lipid analysis. Radiolabeled GPIs were found in both
viruses. Moreover, the membrane concentration of GPIs (i.e. GPI radioactivity normalized to membrane mass) in the two viruses
was essentially the same. These observations suggest that (i)
non-protein-linked GPIs are located at the plasma membrane; (ii) since
GPIs are synthesized in the ER, they must be transported from the ER to
the plasma membrane; and (iii) transport of non-protein-linked GPIs is
not influenced by the sorting processes that target GPI-anchored
proteins exclusively to the apical plasma membrane.
Glycosylphosphatidylinositols (GPIs) ()are a diverse
family of eukaryotic glycolipids containing elements of the sequence
ethanolamine-P-6Man
1-2Man
1-6Man
1-4GlcN
1-6-myo-inositol-1-P-lipid (1, 2) . The core GPI structure is assembled in the
endoplasmic reticulum (ER) (3) via sequential transfer of
components to phosphatidylinositol(1, 2) . Current
models suggest that the biosynthetic pathway is localized to the
cytoplasmic face of the ER (3, 4, 5) and
that an ethanolamine-containing GPI structure flips across the ER
bilayer for transfer to proteins bearing an appropriate
carboxyl-terminal signal sequence(6) .
GPIs are made in
excess of the amount needed for protein modification, and eukaryotic
cells contain significant pools of non-protein-linked GPIs
(10-10
molecules/cell depending on GPI
structure and cell type) in addition to GPI structures (anchors)
covalently linked to protein. Evidence from different experimental
systems suggests that a fraction of the non-protein-linked
GPIs may exit the ER and relocate to other organelles, including the
plasma membrane (PM)(7, 8, 9) . Indeed, some
members of a family of GPI-related glycolipids (glycoinositol
phospholipids) in Leishmania parasites are found at the cell
surface, where they are accessible to antibodies and where they can be
modified by exogenously added
glycosyltransferases(1, 10) .
In renal and intestinal epithelial cell lines such as Madin-Darby canine kidney (MDCK) and Caco-2, GPI-anchored proteins are expressed exclusively at the apical PM domain(11, 12, 13, 14) . It has been proposed that GPI anchors are dominant apical sorting signals for GPI-anchored proteins, acting through biophysical interactions with glycosphingolipid (GSL) clusters in the trans-Golgi network of epithelial cells(11, 15, 16) . In contrast, very little is known about the subcellular distribution and transport of non-protein-linked GPIs. We have studied the composition and distribution of non-protein-linked GPIs in MDCK cells to investigate if the GPI structure per se contains intracellular sorting information, reflected in a polarized distribution of non-protein-linked GPIs on the epithelial cell surface. By analysis of enveloped RNA viruses that bud from the two cell-surface domains of MDCK cells, we show that GPIs are indeed present in the PM, but that they do not display a polarized distribution between the apical and basolateral domains. In the framework of models for epithelial cell polarity(15, 16, 17, 18) , intracellular lipid transport(17, 19) , and GPI biosynthesis(3, 4, 5) , we suggest that non-protein-linked GPIs may be located in the cytoplasmic leaflet of the PM lipid bilayer.
GPIs were radiolabeled by
incubating the cells for 6-12 h with 10 µCi/ml
[H]ethanolamine in Dulbecco's modified
Eagle's medium containing 5% dialyzed fetal bovine serum or with
50 µCi/ml [
H]mannose in glucose-free medium
containing 10% dialyzed fetal bovine serum, 20 mM Hepes (pH
7.4), 0.1 mg/ml glucose, and 10 or 50 µg/ml tunicamycin
(tunicamycin was added from a 10 mg/ml stock in dimethyl sulfoxide to
the cell culture 30 min before adding
[
H]mannose). Glycosphingolipids were labeled by
incubating the cells for 12-14 h with 10 µCi/ml
[
H]galactose in glucose-free medium supplemented
with 5% dialyzed fetal bovine serum, 20 mM Hepes (pH 7.4), and
0.5 mg/ml glucose.
Cells on the Transwell filters were scraped in infection medium, pelleted, and taken for glycolipid extraction. Viruses were isolated from the combined apical or the combined basolateral media. First, detached cells and cellular debris were removed from the media by respective centrifugation for 5 min at 1000 rpm and for 25 min at 5000 rpm at 4 °C in a Sorvall HS-4 rotor. Viruses in the remaining supernatant were pelleted through a 0.25 M sucrose cushion containing 1 mM Tris-HCl (pH 7.4) and 1 mM EDTA (TE buffer) by centrifugation at 4 °C for 3 h at 40,000 rpm in a Beckman SW 41 rotor. The pellet was resuspended in 250 µl of TE buffer, layered on top of a 7-52% linear sucrose gradient, and centrifuged at 4 °C for 150 min at 30,000 rpm in a Beckman SW 41 rotor. Fractions of 0.5 ml were harvested from the top of the gradient using an Auto Densi-Flow IIc pump (Buchler, Lenexa, KA), and radioactivity was measured in 25-µl samples of each fraction by liquid scintillation counting. The sucrose density in each fraction was determined by measuring the refractive index. Fractions containing radiolabeled virus were collected, diluted three to four times with TE buffer, and centrifuged at 4 °C for 2.5 h at 23,000 rpm in a Beckman SW 41 rotor. The resulting virus pellets were resuspended in 0.25 ml of TE buffer or directly taken for lipid extraction.
[H]Ethanolamine- or
[
H]mannose-labeled lipid products were identified
by cochromatography (on thin-layer plates) with previously
characterized mammalian GPIs (22, 23) and by
susceptibility to treatment with GPI-specific phospholipase D and
nitrous acid(24) . Phospholipids on silica TLC plates were
visualized by exposure to iodine vapor, identified by cochromatography
with standards, and quantified by phosphorus analysis of the
appropriate region of silica(25) .
Figure 1:
Analysis of GPIs in MDCK cells. MDCK
cells were radiolabeled with [H]mannose, and
glycolipids were extracted as described under ``Experimental
Procedures.'' The extract was analyzed by TLC directly (A) or after treatment with rabbit serum, a source of
GPI-specific phospholipase D (GPI-PLD) (B). The
chromatogram was developed in chloroform/methanol/water (10:10:3,
v/v/v) and visualized using a radioactivity scanner. The origin (o) and solvent front (f) are indicated, as are the
migration positions of the GPI standards H8 (containing 3
phosphoethanolamine residues as shown; see (22) and (23) ) and H7 (with 2 phosphoethanolamine residues; the middle
mannose is not substituted). The designations GPI-1, GPI-2 (H8 +
another poorly resolved GPI), and GPI-3 (H7) are used throughout this
paper. EtN, ethanolamine.
Figure 3:
Presence of GSLs and GPIs in flu and VSV.
MDCK cells were labeled with [H]galactose (A) or [
H]mannose (B and C) and infected with virus as described in the legend to Fig. 2. Radiolabeled GSLs from
[
H]galactose-labeled cells and viruses were
isolated by sequential extraction in chloroform/methanol (1:2 and 1:1,
v/v, respectively) and were separated by TLC using chloroform,
methanol, 0.2% CaCl
(60:35:8, v/v/v) as the developing
solvent. [
H]GSLs were identified from their R
values and by cochromatography with
unlabeled GSLs that were visualized by spraying the plates with 15%
ethanolic
-naphthol/methanol/sulfuric acid (1.5:1:6, v/v/v) and
heating at 100 °C for 5-15 min. The TLC profiles in A are stacked and truncated to show the differences in GSL content
more clearly; peaks of radioactivity marked A1, A2,
and B1-3 are discussed under ``Results.'' The
large off-scale peak of radioactivity migrates to the position of the
Forssman antigen. GPIs in flu (B) or VSV (C) were
extracted and analyzed as described under ``Experimental
Procedures.'' The origin (o) and solvent front (f) of the chromatograms are
indicated.
Figure 2:
Polarized budding of flu and VSV from the
apical and basolateral surface domains of MDCK cells. MDCK cells were
seeded on 75-mm Transwell filters, incubated with
[H]galactose (to label GSLs), and infected with
flu (A), VSV (B), or no virus (C). Released
viruses in the apical (
) or basolateral (
) media were
purified and analyzed by centrifugation in 7-52% linear sucrose
gradients (A-C). Gradient fractions (0.5 ml) were
collected from the top of the gradients and taken for liquid
scintillation counting (A-C, radioactivity). The sucrose
density in each fraction was determined by measuring the refractive
index (C, sucrose density). As a control, MDCK cells were
infected with flu or VSV for 4 h as described under ``Experimental
Procedures,'' followed by metabolic labeling for 4 h at 37 °C
with 150 µCi/ml [
S]methionine. Released
viruses were analyzed as described above, and 1.0-ml fractions were
monitored for the marker proteins influenza HA2 and VSV-G by
immunoprecipitation, followed by SDS-PAGE and densitometry (C,
relative intensity percent).
The remaining
[H]mannose-labeled products (running in the upper
half of the chromatogram in Fig. 1A) were not sensitive
to various tunicamycin pretreatments and were GPI-specific
phospholipase D-, nitrous acid-, and mild acid-resistant, but mild
base-sensitive. Their identity is currently unknown, but based on their
response to the various treatments, it is clear that they are not
dolichol-P-monosaccharides, dolichol-PP-oligosaccharides, or GPIs. Work
is in progress to identify the nature of these lipids.
Following
published work on domain-specific biotinylation of cell-surface
proteins in MDCK and other polarized cells(11) , we
investigated whether ethanolamine-containing GPIs could be derivatized
at the cell surface by membrane-impermeant amine-reactive probes.
Consistent with previous observations on GPI modification under
approximately physiological conditions(3, 4) , these
attempts were unsuccessful (data not shown). We therefore decided to
use enveloped RNA viruses to investigate the presence of GPIs in the
plasma membrane. These viruses obtain their envelope lipids by budding
from select membranes in their host cells; flu and VSV bud from the PM,
and in polarized MDCK cells, the two viruses bud specifically from the
apical (flu) and basolateral (VSV) PM domains(20) . Thus,
analyses of flu and VSV budded from [H]mannose-
or [
H]ethanolamine-labeled MDCK cells provide an
opportunity to investigate whether non-protein-linked GPI
structures are present at the PM and whether they display a polarized
surface distribution.
Analysis of
[H]galactose-labeled GSLs in the two viruses
provided another test of the polarity of the MDCK cell monolayers used
for our experiments. Viruses budded from
[
H]galactose-labeled MDCK cell monolayers were
collected from sucrose gradients as in Fig. 2(A and B) (only basolaterally budded VSV was used for analysis), and
GSLs were extracted and analyzed by TLC. As shown in Fig. 3A, there are clear differences in the
radiolabeled GSL profiles of flu and VSV. For example, the structures
denoted B1, B2, and B3 (comigrating on TLC
with G
, lactosylceramide, and glucosylceramide,
respectively) are found solely or predominantly in VSV, while those
marked A1 and A2 (comigrating with G
and
globosides, respectively) are enriched in flu. Although we did not
characterize individual GSL species in any detail, the characteristic
differences in GSL expression at the apical (flu) and basolateral (VSV)
PM domains are consistent with previous reports (26, 27) and confirm that the MDCK cell monolayers
exhibit glycolipid polarity under our experimental conditions.
The [H]mannose-labeled
GPI/PE ratio (cpm/nmol) in purified viruses, representing the plasma
membrane, was 0-30% lower than in cells (Table 1,
``Enrichment''), indicating that the free GPIs are probably
not preferentially enriched in the plasma membrane. Moreover,
comparison of the GPI/PE ratio in flu versus VSV showed that
the GPIs were similarly concentrated in both viruses (polarity values
of
1) and hence did not display a polarized distribution in the
MDCK cell plasma membrane (Table 1, ``Polarity'').
We also investigated whether GPI-anchored proteins could be
recovered in enveloped viruses in a manner reflecting their polarized
cell-surface distribution. Although cellular glycoproteins are
generally not assembled into enveloped viruses(28) ,
GPI-anchored proteins may be incorporated via the same mechanisms used
to recruit plasma membrane lipids into viral envelopes. Indeed, Calafat et al.(28) reported the presence of a GPI-anchored
protein, Thy-1 (29) , in VSV and murine leukemia virus virions
recovered from infected thymoma cells. Since Thy-1 is not found in
epithelial cells (30) and as we wished to maximize our chances
of detecting GPI-anchored proteins in viruses, we chose to analyze a
stably transfected MDCK cell clone expressing high levels of a chimeric
GPI-anchored protein, gD1-DAF (14) . This cell line has been
previously characterized and is known to express gD1-DAF exclusively at
the apical cell-surface domain(14) . SDS-PAGE and Western
blotting of solubilized MDCK(gD1-DAF) cells clearly showed a band
corresponding to the 40-kDa gD1-DAF precursor and a smear (50-55
kDa) corresponding to mature forms of the chimera (data not shown).
These bands were not detected in wild-type cells. The
gD1-DAF-expressing MDCK cells were then infected with virus (flu or
VSV) under conditions chosen to maximize virus yield. Western blotting
analysis of purified virus samples recovered from the infected cultures
detected none of the gD1-DAF forms, indicating that gD1-DAF is not
incorporated into the viral envelopes. Thus, the viral assay is not
useful in probing the plasma membrane distribution of GPI-anchored
proteins in general. These results contrast with those of Calafat et al.(28) described above. One explanation for these
apparently conflicting observations is that relatively small
GPI-anchored proteins such as Thy-1 (24 kDa) may behave
essentially as large glycolipids and escape the potential steric
constraints or ectodomain interactions that result in the elimination
of gD1-DAF (
50-55 kDa) from virus budding
sites(28) .
We used enveloped RNA viruses to obtain purified samples of
the compositionally distinct PM domains of polarized MDCK cell
monolayers in order to assay for the presence of non-protein-linked GPIs at the cell surface. The observation
that particular classes of enveloped viruses bud from specific cellular
membranes has been exploited to obtain very pure membrane samples of
subcellular compartments(21, 31, 32) . In
this way, flu and VSV, which bud from the apical and basolateral PM
domains, respectively, have been used to study the phospholipid and GSL
composition of the PM domains in
[P]phosphate-labeled (21) or
[
H]galactose-labeled (26) MDCK cells.
Elegant control experiments with non-epithelial cells or MDCK cells
under nonpolarized conditions showed that these viruses do not select
for the lipids in their envelope, but that their lipid content reflects
the composition of the membrane from which they bud(21) . The
viral assay offers unique advantages for lipid analyses: pure membrane
preparations may be obtained without contamination from intracellular
membranes, and unlike the amine modification protocols that we
attempted without success in preliminary work, information is obtained
on lipid constituents present in both leaflets of the parent
membrane. The results presented here are based on this assay and show
clearly that non-protein-linked GPIs may be found in the PM of
polarized MDCK cells and that the GPIs are similarly concentrated in
the apical and basolateral PM domains.
The presence of GPIs in flu and VSV and, by implication, in the PM indicates that GPIs are not retained at their site of synthesis in the ER. Consistent with this observation, previous work has shown that GPIs may be found in lipid extracts of rat liver PM preparations (9) and that members of a family of GPI-related glycolipids (glycoinositol phospholipids) are expressed on the surface of the protozoan parasite Leishmania major(10) . Since GPIs are synthesized in the cytoplasmic leaflet of the ER(3, 4, 5) , they may be transported to the PM via monomeric diffusion or protein-mediated exchange through the cytosol or via a vesicular transfer mechanism. All three transport mechanisms would result in expression of GPIs at the cytoplasmic face of the PM (see below), unless transbilayer movement (flip-flop) occurs. Since differences in lipid composition between the apical and basolateral PM domains are confined to the exoplasmic leaflet of the membrane bilayer(17, 18) , the nonpolarized distribution of GPIs at the PM (Table 1) suggests that GPIs may indeed be located in the cytoplasmic leaflet of the PM. We were unable to confirm this directly by examining the orientation of the various GPI structures in the MDCK cell PM or in purified viruses. The probes (phosphatidylinositol-specific phospholipase C and concanavalin A) that have been used successfully in the analysis of transmembrane orientation of protozoan and mammalian GPIs lacking inositol acyl and side chain phosphoethanolamine groups (3, 4) do not hydrolyze or recognize the mammalian GPIs that we describe. Therefore, it cannot be ruled out that some or all of the non-protein-linked GPIs in the PM are expressed at the cell exterior, albeit without polarity. This would involve flipping of GPIs into the exoplasmic membrane leaflet of an intracellular organelle such as the ER, followed by vesicular transport without sorting at the trans-Golgi network. The results and hypotheses presented in this paper demonstrate the contrast between the distribution and transport of free GPIs and GPI-anchored proteins. The latter are confined to the exoplasmic leaflet of cellular membranes and are presumably sorted in the trans-Golgi network for selective delivery to the apical PM domain via a vesicular mechanism (11, 16) . Although we were unable to use our viral assay to confirm the cell-surface polarity of GPI-anchored proteins, the exclusive localization of these proteins to the apical plasma membrane domain in MDCK cells has been extensively documented using immunofluorescence (14) , domain-selective biotinylation(12, 14) , domain-selective radioiodination(30) , and cell-surface immunoprecipitation(13) .
This work is dedicated to the memory of Jan T. van't Hof(1924-1994) and Elsa V. Boulan-Rodriguez(1920-1995).