3623
Journal of Cell Science 113, 3623-3638 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JCS1697
The p58-positive pre-Golgi intermediates consist of distinct subpopulations
of particles that show differential binding of COPI and COPII coats and
contain vacuolar H+-ATPase
Ming Ying1, Torgeir Flatmark1 and Jaakko Saraste2,*
Departments of Biochemistry and Molecular Biology1 and Anatomy and Cell Biology2, University of Bergen, Norway
*Author for correspondence (e-mail: [email protected])
Accepted 21 August; published on WWW 4 October 2000
SUMMARY
We have studied the structural and functional properties of
the pre-Golgi intermediate compartment (IC) in normal
rat kidney cells using analytical cell fractionation with p58
as the principal marker. The sedimentation profile
(sediterm) of p58, obtained by analytical differential
centrifugation, revealed in steady-state cells the presence
of two main populations of IC elements whose average
sedimentation coefficients, sH=1150±58S (‘heavy’) and
sL=158±8S (‘light’), differed from the s-values obtained for
elements of the rough and smooth endoplasmic reticulum.
High resolution analysis of these subpopulations in
equilibrium density gradients further revealed that the
large difference in their s-values was mainly due to particle
size. The ‘light’ particle population contained the bulk of
COPI and COPII coats, and redistribution of p58 to these
particles was observed in transport-arrested cells, showing
that the two types of elements are also compositionally
distinct and have functional counterparts in intact cells.
Using a specific antibody against the 16 kDa proteolipid
subunit of the vacuolar H+-ATPase, an enrichment of the
Vo domain of the ATPase was observed in the p58-positive
IC elements. Interestingly, these elements could contain
both COPI and COPII coats and their density distribution
was markedly affected by GTPγS. Together with
morphological observations, these results demonstrate
that, in addition to clusters of small tubules and vesicles,
the IC also consists of large-sized structures and
corroborate the proposal that the IC elements contain an
active vacuolar H+-ATPase.
INTRODUCTION
observed at 15°C (Saraste and Kuismanen, 1984) is partly due
to inhibition of this long distance transport step.
In spite of a number of studies employing different cargo
or resident proteins as markers in electron microscopy, the
structural organization and function of the IC still remains
controversial. The individual pre-Golgi intermediates have
been referred to as vesicular tubular clusters (VTCs; Balch et
al., 1994), or transport complexes (TCs; Scales et al., 1997),
terms that derive from their frequent ultrastructural
appearance as assemblies of small, coated or uncoated
vesicles and tubules (Bannykh et al., 1996). However, several
EM studies have demonstrated that the IC also contains a
large-sized component. Such large, frequently vacuolar IC
elements have been described in transport-arrested cells
(Saraste and Kuismanen, 1984; Stinchcombe et al., 1995;
Palokangas et al., 1998), but are also a normal feature of the
IC in untreated cells (Saraste and Svensson, 1991; Lahtinen
et al., 1992; Sesso et al., 1994; Saraste et al., 1995; Ladinsky
et al., 1999). In addition to resident IC proteins, such as p58
and Rab1, the pre-Golgi vacuoles have been shown to contain
cargo proteins (Saraste and Kuismanen, 1984), but their
Protein transport in the early secretory pathway, between the
ER and the Golgi complex, involves pleiomorphic pre-Golgi
structures that constitute the intermediate compartment (IC)
(Saraste and Kuismanen, 1984; Tooze et al., 1988; Bonatti et
al., 1989; Schweitzer et al., 1990; Saraste and Svensson, 1991;
Lahtinen et al., 1992; Lotti et al., 1992; Plutner et al., 1992).
Considerable progress has been made in defining the function
of these elements in bidirectional protein transport at the ERGolgi boundary (see Pelham, 1996; Bannykh and Balch, 1997;
Hong, 1998; Lippincott-Schwartz et al., 1998; Hauri et al.,
2000, for recent reviews). Light microscopic visualization of
the pre-Golgi structures in temperature-shift experiments via
an endogenous marker protein (p58) (Saraste and Svensson,
1991) and, more recently, using a fluorescently tagged cargo
protein in living cells (Presley et al., 1997; Scales et al., 1997),
has shown that they originate at widespread ER sites and
translocate in a microtubule-dependent process to the central
Golgi region. These studies have also demonstrated that the
reversible arrest of protein transport between the ER and Golgi
Key words: Pre-Golgi intermediate compartment, Analytical
differential centrifugation, Sedimentation coefficient, Vacuolar H+ATPase, Coat protein
3624 M. Ying, T. Flatmark and J. Saraste
functional role in ER↔Golgi trafficking has not been studied
in detail.
Two types of small transport vesicles, defined by their
cytoplasmic coat proteins, mediate protein transport between
the ER and the Golgi complex. A sequential function of these
COPII- and COPI-coats/vesicles has been proposed based on
biochemical, morphological and functional studies (Aridor et
al., 1995; Rowe et al., 1996; Scales et al., 1997; MartínezMenárguez et al., 1999). It is generally considered that COPIIcoated vesicles, which are composed of the small GTP-binding
protein Sar1p, as well as Sec23/24 and Sec13/31 protein
complexes in yeast (for reviews, see Kuehn and Schekman,
1997; Barlowe, 1998), function during protein exit from the
ER (Barlowe et al., 1994). After their budding from the ER and
disassembly of the coats, COPII-vesicles are though to
generate the more pleiomorphic IC structures by homotypic
fusion (Aridor et al., 1995; Rowe at al., 1996). Subsequently,
the IC elements bind coatomers (subunits of COPI coats) in a
process that is regulated by the small GTP-binding protein
ARF1 (for a review see Scales et al., 2000). The suggested
functions of COPI coats/vesicles include molecular sorting and
anterograde transport between the ER and Golgi (Pepperkok et
al., 1993; Aridor et al., 1995), bidirectional transport within the
Golgi (Orci et al., 1997), and retrograde transport of selected
components back to the ER (Letourneur et al., 1994; Lewis and
Pelham, 1996; Martínez-Menárguez et al., 1999). Rat p58 and
its human homologue ERGIC53 (Saraste et al., 1987;
Schweitzer et al., 1988), at present probably the best
characterized IC markers, are integral membrane proteins that
utilize the COP machineries for their intracellular transport.
They contain in their cytoplasmic tails motifs that interact with
both COPII and COPI coats (Kappeler et al., 1997; Tisdale et
al., 1997), recycle constitutively between the ER, IC and cisGolgi, and have been suggested to function as cargo receptors
during protein exit from the ER (for review see Hauri et al.,
2000).
To obtain additional information on the structural and
functional organization of the IC, and to facilitate the isolation
and biochemical characterization of these membranes, we have
combined analytical differential and isopycnic gradient
centrifugation to study the polydispersity of the p58-containing
pre-Golgi elements. Analytical differential centrifugation has
been previously established as a powerful tool to estimate the
sedimentation coefficients and size distribution of subcellular
organelles and particles. Accordingly, this method has been
used successfully to estimate the s-values of mitochondria,
lysosomes, peroxisomes/microperoxisomes, and microsomes
in rat liver (for review see Flatmark, 1988), as well as
catecholamine storage granules (Tooze et al., 1991), and
synaptic-like microvesicles (Bauerfeind et al., 1993) in rat
pheochromocytoma (PC12) cells. Our present results obtained
with normal rat kidney (NRK) cells demonstrate for the first
time a high degree of polydispersity of the IC. The p58containing pre-Golgi elements were shown to consist of two
main subpopulations of particles (with average sedimentation
coefficients of sH=1150±58S and sL=158±8S, respectively)
that mainly differ in size. In addition, to gain insight to the
function of the IC in the early secretory pathway, we compared
the distributions of p58, COPII and COPI coats, and the core
proteolipid of vacuolar H+-ATPase using high resolution
density gradient centrifugation. The results show that the Vo
subunit of the ATPase becomes enriched in post-ER
membranes at an early stage of transport, and thus support our
previous studies (Palokangas et al., 1998) indicating that the
p58-positive IC elements contain an active proton pump.
MATERIALS AND METHODS
Reagents
Cell culture media were obtained from Gibco (Grand, Island, NY).
Thapsigargin was from Calbiochem (Calbiochem, La Jolla, CA).
Affinity purified 125I-Protein A was purchased from Amersham
(Buckinghamshire, UK), the nitrocellulose membrane (0.45 µm) from
Schleicher and Schuell, Inc. (Keene, NJ), and the Slow-Fade
mounting medium for immunofluoresence microscopy from
Molecular Probes (Eugene, Oregon). Except for saponin that was from
Fluka (Buchs, Switzerland), all chemical reagents were purchased
either from Sigma Chemical Co. (St Louis, MO) or Merck
(Darmstadt, Germany).
Antibodies
The following antibodies were used in immunoblotting and immunofluoresence studies: affinity-purified rabbit antibodies directed against
p58 were prepared as described (Saraste and Svensson, 1991). The
polyclonal anti-proteolipid antibodies were raised in rabbits against
the N-terminal peptide (KSEAKNGPEY) of the proteolipid subunit
of bovine chromaffin granule vacuolar H+-ATPase that was coupled
to hemocyanin. The first K was added to the original sequence to
increase the efficiency of coupling. Affinity-purification of antibodies
was carried out using a column containing the peptide coupled to
cyanogen bromide-activated Sepharose 4B. The bound antibodies
were eluted from the column with 0.1 M glycine-HCl (pH 2.65),
whereafter the eluates were neutralized and concentrated. Another
polyclonal anti-proteolipid antibody (anti-T3-L) was kindly provided
by Dr Shoji Ohkuma (Kanazawa University, Japan). The polyclonal
anti-mSEC13 antibodies were a gift from Drs Bor Luen Tang and
Wanjin Hong (National University of Singapore, Singapore). The
polyclonal anti-β-COP antibodies were purchased from Affinity
BioReagents, Inc. (Golden, CO), and the secondary antibodies, FITCor peroxidase-conjugated goat anti-rabbit F(ab)2-fragments, were from
Coulter-Immunotech (Marseille, France).
Cell culture
NRK cells were grown in DMEM supplemented with 10% (v/v) FBS,
100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM L-glutamine.
Mouse myeloma (RPC 5.4) cells were cultivated in DMEM
containing nonessential amino acids, 4500 mg/l glucose, 10% (v/v)
FBS, and the antibiotics in the above concentrations.
Subcellular fractionation
A flow chart for the subcellular fractionation of NRK cells is shown
in Fig. 1. NRK cells (40 dishes of 150 mm diameter) were harvested
at ~90% confluency, washed twice with ice-cold PBS, and then
pelleted by centrifugation for 5 minutes at 300 g in a bench-top
centrifuge. The pellet was resuspended in homogenization buffer A
(HB-A) containing 50 mM sucrose, 0.2 mM EDTA, 5 mM Hepes (pH
7.2) and the following protease inhibitors: 1 mM PMSF, 2 µg/ml
aprotinin, and 10 µg/ml each of chymostatin, leupeptin, antipain and
pepstatin (CLAP). The cells were homogenized by 10 passages
through a ball-bearing cell cracker (Balch and Rothman, 1985) with
a clearance of 0.01 mm. The final concentration of the homogenate
was adjusted to 0.25 M sucrose by adding 2 M sucrose. The nuclei
and cell debris were sedimented by centrifugation for 10 minutes at
660 g in a Sorvall HB-4 rotor, whereafter the postnuclear supernatant
was centrifuged for 15 minutes at 8000 g in the same rotor. The
resulting postmitochondrial supernatant (PMS) was diluted with
Subpopulations of IC elements 3625
homogenization buffer B (HB-B: 0.25 M sucrose, 0.2 mM EDTA, 5
mM Hepes, pH 7.2, supplemented with protease inhibitors at the
concentrations given above) to give a final protein concentration of
0.4-0.5 mg/ml. All operations were performed at 4°C. The further
fractionation of PMS into P1 and P2 membrane fractions, as well as
the corresponding supernatant fractions, S1 and S2 (Fig. 1), is
described below.
Analytical differential centrifugation
PMS was subjected to analytical differential centrifugation using a
standard assay protocol (Slinde and Flatmark, 1973; Strand and
Flatmark, 1998). For each time integral of rpm2 of the centrifugation,
1.0 ml of the PMS was transferred to an ultracentrifuge tube, in a TLS55 rotor, and then centrifuged in Beckman TL-100 tabletop
ultracentrifuge at 4°C. The acceleration was set at 2, and the
deceleration at 0 (brake off). The centrifugal effect [o∫t (rpm)2 dt] was
estimated by the centrifuge integrator. The pellets were carefully
resuspended preventing the generation of air-bubbles. First, each
pellet was resuspended by addition of 50 µl of HB-B, mixed gently
with a micropipette at 0-4°C, whereafter the same buffer was added
to give a final volume of 100 µl, and the pellets were frozen in aliquots
at −80°C. The proteins were separated by SDS-PAGE and detected by
quantitative immunoblotting using the appropriate antibodies and 125IProtein A (see below). The percentage sedimentation of the marker
protein was plotted as a function of the time integrals of rpm2 [o∫t rpm2
dt (minutes−1)] of the individual centrifugations (Slinde and Flatmark,
1973). The s-values were estimated by the equation (Slinde
and Flatmark, 1973): log10 [1−(1–Rmin/Rmax) Y/100]=–s o∫t (rpm)2
dt/3.5×1013, where Rmax (7.52 cm) and Rmin (5.35 cm) are the
distances from the axis of rotation to the bottom and top of the fluid
of the tube, respectively; 100 is the maximum amount of the marker
protein sedimented, and Y represents the percentage sedimentation of
the marker to the bottom of the tube at a certain time integral of rpm2
(convergence value).
Isopycnic sucrose gradient centrifugation
PMS was used as the starting material for centrifugation using a time
integral of rpm2 of 6.8×109 minute−1 (15,000 rpm for 30 minutes),
resulting in a pellet termed P1 (Fig. 1). The resulting supernatant was
again centrifuged at a time integral of rpm2 of 9.5×1010 minute−1
(45,000 rpm for 45 minutes), to give a pellet termed P2 (Fig. 1). These
two centrifugations were carried out using a TLS-55 rotor in a
Beckman TL-100 ultracentrifuge (for selection of time integrals, see
Fig. 3 and the corresponding figure legend). The sedimented particles
in the P1 and P2 pellets were resuspended in the same way as
described for analytical differential centrifugation, except that the
final volume of each pellet was 200 µl, and then layered on top of a
linear sucrose gradient. The gradient was prepared from 1.2 ml layers
of sucrose in 5 mM Hepes buffer (pH 7.2), corresponding to densities
of 1.21, 1.18, 1.16, 1.14, 1.12, 1.10, 1.08, 1.06 and 1.04 g/ml, and left
at 4°C until linear (Flatmark et al., 1985). After centrifugation for 13
hours at 100,000 g in a Beckman SW 41 rotor, with acceleration value
7 and the deceleration at 0 (break off), the bottom of the centrifuge
tube was punctured using a fine hollow needle (21GA). The top of the
centrifuge tube was sealed with a rubber-cap connected to a syringe,
allowing the controlled pumping of air into the top part of the tube.
A total of 32-36 fractions of about 300 µl each were collected and the
density of each fraction determined by refractometry before their
processing for SDS-PAGE and immunoblotting.
SDS-PAGE and quantitative immunoblotting
Aliquots of the fractions obtained either by analytical differential
centrifugation (20 µl) or sucrose gradient centrifugation (120 µl) were
precipitated with acetone (acetone: sample, 4:1), whereafter the
proteins were separated by 10% SDS-PAGE, and electrophoretically
transferred to nitrocellulose membranes. The membranes were
blocked with buffer containing 20 mM Tris-HCl, pH 7.5, 150 mM
NaCl, 5% (w/v) dry milk, and 1% (v/v) Tween-20, and subsequently
reacted with primary antibodies (affinity purified anti-p58 or antiproteolipid antibodies, anti-mSEC13 serum, or polyclonal anti-β-COP
IgG) diluted in the above buffer, followed by incubation with affinity
purified 125I-Protein A (0.2 µCi/ml). The protein-bound radioactivities
were quantitated by scanning in either an Instant Imager (Packard,
USA) or BAS-5000 (Fuji, Japan). The data derived from the analysis
of the density gradients were plotted against the density of each
fraction, and the resulting distribution profile resolved into individual
components, with a gaussian distribution, using the PeakFit software
program (SPSS Inc., Chicago, USA); the ‘AutoFit-Peak I-Residuals’
method was used with the confidence level set at 99%. It should be
noted that both the input densities and the calculated peak densities
of subpopulations (see Figs 4, 6, 8, 9 and 10B) were expressed with
four decimals. However, in the colocalization studies only two
decimals were considered significant. The different profiles were
reproducible; for example in the different experiments the peaks of
p58 immunoreactivity were found in similar density positions (two
decimals significance level) and the variation in integrated peak areas
did not exceed 4%.
Immunocytochemistry
For confocal immunofluorescence microscopy, the NRK cells were
grown for 2 days on glass coverslips to reach ~50% confluency. At
harvest the medium was removed and the cells were fixed for 30
minutes with 3% (w/v) paraformaldehyde in 0.1 M sodium phosphate
buffer, pH 7.4, and then washed 3 times with PBS. The cells were
permeabilized for 5 minutes with 0.2% (w/v) saponin in PBS
containing 0.2% (w/v) BSA (washing buffer) followed by incubation
for 1 hour in blocking buffer (washing buffer supplemented with 10%
(v/v) FBS), and subsequently incubated successively with affinitypurified anti-p58 antibodies and FITC-conjugated goat anti-rabbit
F(ab)2-fragments. The primary and secondary antibodies were diluted
in blocking buffer and each antibody incubation was followed by
extensive washing. The coverslips were mounted in a small drop of
mounting medium on an objective glass and visualized with Bio-Rad
MRC-1000 confocal laser scanning microscope (Bio-Rad
Laboratories, Burlingame, CA).
For immunoperoxidase staining, the cells were grown to ~50%
confluency on 35 mm diameter culture dishes. The conditions used
for the fixation, permeabilization, staining, and processing of cells for
electron microscopy have been described previously (Brown and
Farquhar, 1989; Kuismanen and Saraste, 1989). The samples were
examined in JEOL CX II electron microscope (Tokyo, Japan).
Other analytical methods
Glucose-6-phosphatase (G-6-Pase) activity was measured as
previously described (Harper, 1965).
RESULTS
Two main subpopulations of p58-positive IC
elements based on particle size
Morphological observations
The morphology of the IC has previously been studied in rat
pancreatic exocrine cells and different cultured cell lines using
p58 as the marker protein (see Saraste and Kuismanen, 1992,
for a review). Fig. 2A and B show the localization of p58 in
NRK cells, the cell type used in the present study, as examined
by confocal immunofluorescence microscopy. At steady-state
the protein is detected in punctate pre-Golgi structures of
variable size that are scattered throughout the cytoplasm and
accumulate in the central Golgi area. In addition, antibodies
against p58 give a weak, reticular staining in these cells (Fig.
3626 M. Ying, T. Flatmark and J. Saraste
Fig. 1. Flow chart for the preparation of the different subcellular
fractions from NRK cell homogenates. The PNS and PMS fractions
were obtained by centrifugation in the Sorvall HB-4 rotor (Rmax=14.4
cm and Rmin=6.2 cm). The P1, P2, S1 and S2 fractions were obtained
by centrifugation in the TLS-55 rotor in Beckman TL-100
ultracentrifuge (Rmax=7.52 cm and Rmin=5.35 cm).
2A and B). Immunoelectron microscopy further shows the
presence of p58 in tubular/cisternal elements in the cis-Golgi
region as well as numerous coated or uncoated transport
vesicles (see also Klumperman et al., 1998), ~80 nm in
diameter, that frequently build up larger aggregates called
VTCs (Balch et al., 1994) (Fig. 2C). In addition, both in the
Golgi region and in more peripheral regions of the cell, p58
can be detected in larger vacuolar structures, 0.2 to 0.5 mm in
diameter (Fig. 2C and D). In NRK cells such vacuoles mostly
appear as individual elements, whereas in other cell types
(Fig. 2E-G, showing mouse myeloma cells) they frequently
associate with large membrane clusters that also include
vesicular and tubular elements (Fig. 2F and G). Thus,
according to the morphological data, the pleiomorphic, p58positive IC elements show considerable size heterogeneity.
Moreover, electron microscopy indicates that the punctate
structures resolved by light microscopy (Fig. 2A and B) could
represent either individual p58-containing vacuoles or clusters
of tubulovesicular IC membranes (VTCs).
Analytical differential centrifugation
To further study the polydispersity of the p58-containing IC
elements and to estimate their s-values, the PMS was prepared
from a total NRK cell homogenate and subjected to analytical
differential centrifugation. Aliquots of the resuspended
membrane pellets, obtained by sedimentations at 17 different
time integrals, were precipitated with acetone, followed by
SDS-PAGE and quantitative immunoblotting using affinitypurified antibodies against p58 and 125I-Protein A (see
Materials and Methods). The resulting sedimentation profile
(sediterm) of the marker protein (Fig. 3A) showed a high
degree of polydispersity with two main populations of p58positive IC elements. Since all the p58-containing particles
sedimented at the time integral of o∫t (rpm)2 dt of about
3.8×1010 minute−1, the average amount of p58 sedimented at
this and higher time integrals was used as the 100%
(convergence) value and the amounts sedimented at lower
centrifugal effects were expressed as a percentage of this value
(Slinde and Flatmark, 1973). By regression analysis of the
linearized, logarithmic expression of the data (Fig. 3B), the svalues of the ‘heavy’ and ‘light’ populations of p58-containing
particles were estimated to be sH=1150±58S, and sL=158±8S,
respectively. These two populations contained ~36% (sH) and
~64% (sL) of the total sedimentable, immunoreactive p58
(Table 1). It should be noted that 10.8% of the total subcellular
content of p58-positive elements were removed in the PMS
fraction, corresponding to 9.5% and 1.3% of the heavy and
light populations (sH=1150±58S and sL=158±8S), respectively.
For comparison, we also determined the sediterm of G-6Pase, a marker enzyme for rough ER (RER) and smooth ER
(SER) (Harper, 1965), by measuring the enzyme activity from
aliquots of the same fractions. The sediterm revealed the
expected polydispersity (Norseth et al., 1982) related to RERand SER-elements (Fig. 3A) with s-values of 1880±94S and
256±13S, respectively (Fig. 3B; Table 1).
Density gradient centrifugation
The sedimentation behaviour of subcellular particles is
determined by their size, shape and density (Wilson and
Walker, 1994). To further characterize the two main
populations of p58-positive elements, observed by analytical
differential centrifugation, we subjected the subcellular
fractions enriched in the ‘heavy’ and ‘light’ particles,
respectively, to density gradient centrifugation. Accordingly,
PMS prepared from NRK cells was first centrifuged at a time
integral o∫t (rpm)2 dt of 6.8×109 minute−1 to give a P1 pellet
(Fig. 3A), containing the bulk of the membranes belonging
to the heavy population (sH=1150±58S). The resulting
supernatant was then centrifuged at a time integral o∫t (rpm)2
dt of 9.5×1010 minute−1 to obtain a P2 pellet (Fig. 3A),
including the majority of the vesicles of the light population
(sL=158±8S). After resuspension each pellet was layered on
top of a linear sucrose density gradient and, following
centrifugation to equilibrium, the gradients were harvested (see
Materials and Methods) and the refractive index and p58
immunoreactivity of each fraction were determined. As shown
in Fig. 4, the p58-containing IC structures in both the P1 and
P2 fractions were heterogenous in terms of buoyant density.
The distribution profiles of p58 were resolved into individual
gaussian components using non-linear regression analysis
(PeakFit program), with an excellent fit to the experimental
values, i.e. r2=0.995 (P1) and r2=0.998 (P2). The P1 fraction
(Fig. 4A) was resolved into three p58-containing peaks with
Table 1. Estimated average s-values of IC (p58)
populations in homogenates of control and TG-treated
NRK cells as well as s-values of microsomes (G-6-Pase)
Marker
G-6-Pase
p58
p58
Inhibitor
sH (S)
r2
sL (S)
r2
−TG
−TG
+TG
1880±94
1150±58
2957±148
0.92
0.98 (~36%)
0.96 (~12%)
256±13
158±8
161±8
0.99
0.92 (~64%)
0.98 (~88%)
Estimated by linear regression analysis of the logarithmic plots of data (see
Fig. 3B). For calculation, see Materials and Methods. r2: the estimated
coefficient of correlation; sH and sL: s-values of ‘heavy’ and ‘light’
populations, respectively. The relative content of immunoreactive p58 in the
two populations is given in parentheses.
Subpopulations of IC elements 3627
Fig. 2. Morphology and size heterogeneity of the pre-Golgi structures. (A and B) Confocal immunofluorescence microscopy of NRK cells
showing the presence of p58 in punctate pre-Golgi structures of variable size that are scattered throughout the cytoplasm and accumulate in the
Golgi region (asterisks). The arrowheads and arrows in B indicate punctate, p58-positive elements of small and large diameter, respectively.
(C-G) Ultrastructural features of the p58-positive pre-Golgi structures as revealed by immunoperoxidase electron microscopy of NRK (C and
D) and mouse myeloma (E-G) cells. The protein is localized to small tubulovesicular elements (small arrows) as well as to large pleiomorphic
structures (large arrows) both at the cis-side of the Golgi complex (G) (C and D) and at peripheral locations, often close to ER cisternae (E-G).
The small, p58-positive vesicles have an average diameter of ~80 nm, whereas the diameter of the larger, vacuolar structures varies between 0.2
and 0.5 µm. The boxed area in C indicates a cluster of p58-positive tubulovesicular elements (VTC) between the nuclear membrane (NM) and
cis-Golgi and the arrowhead in G a tubular extension of a vacuolar, p58-containing structure. Bars: 10 µm (A and B); 0.2 µm (C-G).
densities of 1.131 g/ml (43% of total p58 in the gradient), 1.146
g/ml (37%) and 1.164 g/ml (20%), whereas the P2 fraction
(Fig. 4B) contained two major, p58-positive components with
densities of 1.119 g/ml (24%) and 1.130 g/ml (49%), as well
as three minor peaks of 1.145 g/ml (11%), 1.162 g/ml (8%)
and 1.179 g/ml (4%). In general, peaks that contained less than
4% of total p58 in the gradient were considered as noise. In
conclusion, although the p58-positive elements in both the P1
and P2 fractions displayed a measurable heterogeneity in terms
of density, in both cases the majority of particles were found
in the same, relatively narrow density range.
Effect of transport inhibition
Previous immunolocalization studies have revealed that
incubation of NRK cells at 15°C results in the accumulation of
p58 both in the peripheral pre-Golgi structures as well as in
tubulovesicular and cisternal membranes in the cis-Golgi
region (Saraste and Svensson, 1991). Based on these
morphological observations, we examined whether the low
temperature-treatment affects the distribution of p58 between
the two main subpopulations of particles, revealed by
analytical differential centrifugation. Since the above results
indicated that rough microsomes do not contain a significant
pool of p58 (Fig. 3), it was possible to further optimize the
sediterm analysis by subjecting the PMS to an additional
centrifugation step (‘RER cut-off’; indicated in Fig. 3A) to
remove most of the RER-elements. Subsequently, the resulting
supernatant was used directly in analytical differential
centrifugation, and the sediterms of p58 were determined as
described above. Interestingly, as shown in Fig. 5, the sediterm
of p58 obtained for the 15°C-treated cells did not significantly
differ from that of control cells incubated at 37°C.
3628 M. Ying, T. Flatmark and J. Saraste
Fig. 3. Analytical differential centrifugation of
NRK cell homogenates revealed two main
populations of p58-positive IC elements.
(A) Sedimentation profile (sediterm) of p58
obtained by centrifugation of PMS prepared
from control cells (filled circles) or cells treated
for 3 hours with TG (open circles). The protein
concentrations of the homogenates were 0.42
and 0.44 mg/ml, respectively. For comparison,
the sediterm of G-6-Pase activity (open
squares), a marker for RER and SER, was
determined using the control cell homogenate.
In the bottom of panel A, the arrowhead, P1 and
P2 indicate the time integrals subsequently
selected to obtain the ‘RER cut-off’ (see Fig. 5)
and the two membrane pellets enriched in
‘heavy’ (P1) and ‘light’ (P2) p58-positive
elements, respectively (see e.g. Fig. 4). (B) The
data in A were used to calculate the average
sedimentation coefficients (s-values) for the G6-Pase-containing particles (open squares), as
well as the p58-positive elements in
homogenates prepared from either control
(filled circles) or TG-treated cells (open circles).
The insets in A show the original western blots
for p58 (control=upper inset; TG=lower inset).
The average amounts of immunoreactive p58
that sedimented at the convergence level derived
from control and TG-treated cells corresponded
to 15956 and 25576 cpm (125I-Protein A),
respectively.
As another means to affect the distribution of p58 among the
two subpopulations of particles we tested thapsigargin (TG), a
drug that selectively inhibits the ER Ca2+-ATPase (Thastrup et
al., 1990) and results in the depletion of the ER Ca2+-stores.
The inclusion of TG in the present experiments was based on
our recent observations showing that this drug causes an
accumulation of p58-containing vesicular structures at the ERGolgi boundary, suggesting that it inhibits the normal recycling
pathway of the protein (Ying, M. et al., unpublished). NRK
cells were incubated for 3 hours with TG (1 µM) before
harvest, whereafter the PMS was prepared and subjected to
analytical differential centrifugation. Although the sediterm of
p58, obtained for TG-treated cells, still displayed a biphasic
profile similar to that seen in control cells (Fig. 3A), the relative
amounts of the two populations of p58-positive elements were
clearly different. Thus, the recovery of p58 in the ‘heavy’
population was reduced to ~12% of the total, as compared to
~36% in control cells (Table 1) and, in parallel, the absolute
amount of p58 that sedimented together with the ‘light’
particles showed an about 1.6-fold increase (data not shown).
In addition, treatment of cells with TG specifically increased
the estimated s-value of the ‘heavy’ particles (sH=2957±148S;
see Fig. 3B and Table 1), whereas that of the ‘light’ particles
remained unaffected. In contrast to p58, the sedimentation
behaviour of an ER marker was not significantly affected by
TG (data not shown).
The p58-positive IC structures contain vacuolar H+ATPase
Our recent studies suggested that the p58-positive pre-Golgi
Subpopulations of IC elements 3629
Fig. 4. Comparison of the density profiles of the P1
and P2 membrane fractions observed by
equilibrium density gradient centrifugation. The P1
and P2 membrane fractions were prepared from
NRK cells as described in the text and indicated in
Fig. 3A and, following resuspension, subjected to
equilibrium centrifugation in linear sucrose
gradients. 32-33 fractions were collected and
analyzed by SDS-PAGE and western blotting with
antibodies directed against p58. The total
distributions of p58 in the gradients (upper graphs
in A and B) were determined by scanning of the
exposed films (see the inset in B). The distribution
profiles were resolved into individual components
with a gaussian distribution by non-linear regression
analysis using the PeakFit program (lower graphs in
A and B) for the P1 and P2 fractions, respectively.
The arrows indicate the top (left) and bottom (right)
of the gradients, respectively. The inset in B shows
the original western blots with antibodies against
p58 for the P2 fractions.
structures contain an active vacuolar V-type H+ATPase that regulates retrograde transport at
the ER-Golgi boundary (Palokangas et al.,
1998). To obtain further evidence for the
presence of the H+-ATPase in the IC, we
examined the subcellular localization of the 16
kDa integral membrane proteolipid, a subunit
of the proton-translocating Vo-domain of the
pump. To increase resolution, the P2 fraction
was selected as the starting material, since it
lacks most of the RER and heterogenous
‘heavy’ lysosomal particles of high s-value
(Flatmark at al., 1985), but (due to the presence
of cross-contaminating P1 elements) provides a
representative sample containing all the p58positive components of varying density (Fig.
4). Interestingly, the anti-proteolipid antibodies
recognized in NRK cell homogenates a major
band of molecular mass ~48 kDa, which,
together with a minor band of ~32 kDa and
a very faint ~16 kDa band, most likely
correspond to the trimeric, dimeric and monomeric forms of
the proteolipid, respectively (Fig. 6, inset). This finding is not
unexpected since the proteolipid is an extremely hydrophobic
protein (Mandel et al., 1988) and a similar oligomerisation has
been observed for the proteolipid of mitochondrial H+-ATPase
(Kopecký et al., 1986). It can be explained by thermal- and/or
reductant-dependent aggregation of the protein (Hyman et al.,
1993), its high binding capacity for SDS (Miyake et al., 1978),
and/or the observed dependency of its electrophoretic mobility
on the conditions of SDS-PAGE (Kopecký et al., 1986; Ruppert
et al., 1999). Moreover, the same bands were also recognized
in NRK cells by another peptide antibody (Nezu et al., 1992),
directed against the Vo proteolipid, indicating that the major
~48 kDa band represents an oligomeric form of the subunit.
As seen from Fig. 6, the proteolipid displayed a broad
distribution in the density gradient and, by non-linear
regression analysis, the signal could be resolved into 9 main
peaks (r2=0.995) between densities 1.072 and 1.194 g/ml.
The light density components most likely represent H+-
ATPase-containing endosomal/Golgi membranes (Tycko and
Maxfield, 1982; Glickman et al., 1983), whereas some of the
minor peaks of higher density probably represent residual
lysosomes or ER elements (Ohkuma et al., 1982; Rees-Jones
and Al-Awqati, 1984) in the P2 fraction. Of particlar interest
was the detection of the proteolipid in the two main p58containing components at densities 1.119 and 1.133 g/ml
(Fig. 4; see also Fig. 8), corroborating previous results on the
presence of a functional proton pump in the IC elements
(Palokangas et al., 1998). Importantly, these two peaks
represented a substantial fraction (~27%) of the total
immunoreactive proteolipid in the gradient.
Colocalization of p58, proteolipid and coat proteins
in IC elements
The above results suggested that the P2 fraction is specifically
enriched in small transport vesicles. Therefore, we determined
the distributions of COPII and COPI coat proteins in the P1 and
P2 fractions (and the corresponding supernatant fractions S1
3630 M. Ying, T. Flatmark and J. Saraste
Fig. 5. Incubation of cells at 15°C does not
affect the sedimentation profile of p58. NRK
cells were either maintained at 37°C or
incubated for 3 hours at 15°C before
homogenization. PMS fractions were
prepared and centrifuged at a time integral of
rpm2 of 1.366×109 minute−1 to remove most
of RER (‘RER cut-off’; see the text and Fig.
3A), whereafter the supernatants were
subjected to analytical differential
centrifugation as described in the legend to
Fig. 3A. Aliquots of the individual membrane
pellets were analyzed by SDS-PAGE and their
p58 content quantitated by western blotting
(insets). The upper inset/open circles, and
lower inset/closed circles, correspond to
37°C- and 15°C-treated cells, respectively.
and S2; see Fig. 1) using antibodies against the mammalian
homologue of yeast Sec13p (mSEC13) (Swaroop et al., 1994;
Shaywitz et al., 1995; Tang et al., 1997) and the β-COP subunit
of coatomer, respectively. As shown in Fig. 10A (control), the
bulk of membrane-bound mSEC13 (93%) and β-COP (87%)
sedimented together with the P2 membranes, with a minor pool
found in the P1 fraction. The relative amounts of mSEC13 and
β-COP in the P2 and P1 fractions, as normalized to their
p58 content, were estimated to be 7:1 and 3.6:1, respectively.
Moreover, the sediterm of mSEC13, as determined by analytical
differential centrifugation, resembled the sedimentation
behavior of the ‘light’ population of p58-positive membranes
(Fig. 7, compare to Fig. 3A).
Density gradient centrifugation of P2 membranes was used
to examine the association of the COPII and COPI coat
structures with IC elements at high resolution. As shown
in Fig. 8B, the mSEC13-containing membranes displayed
considerable heterogeneity and could be resolved into 7 peaks
(r2=0.962) at the density range between 1.050 and 1.178 g/ml.
Although mSEC13 and p58 colocalized in several peaks (Fig.
8B), interestingly, no coenrichment of the two proteins was
observed. Accordingly, the bulk of mSEC13 associated with
Fig. 6. Distribution of the Vo
proteolipid of the vacuolar H+ATPase in equilibrium density
gradients. PMS fraction was
prepared from NRK cells and
centrifuged successively to sediment
the P1 and P2 fractions as described
in Fig. 3A, whereafter the P2
fraction was subjected to
equilibrium density gradient
centrifugation. 36 fractions were
collected and analyzed by SDSPAGE and western blotting with
affinity purified antibodies directed
against the proteolipid subunit of Vtype H+-ATPase (see inset on the
right; the numbers refer to the
fractions from the top (left, arrow)
to the bottom (right, arrow) of the
gradient). The radioactivities of the
bands were quantitated by scanning,
and from the distribution profile of
the proteolipid in the gradient
(upper graph), the individual
components were resolved by nonlinear regression analysis using the PeakFit program. The inset on the left shows that the affinity-purified peptide antibody reacts predominantly
with a 48 kDa band (lane A) that is also recognized by the alternative T3-L antiserum prepared against the proteolipid (lane B). The proteins in
PMS of NRK cells were separated by 12.5% SDS-PAGE and transferred to nitrocellulose membranes. After Ponceau S staining a single, broad
lane was cut vertically and the two halves were reacted with the two anti-proteolipid antibodies. After immunostaining, the two halves were
again mounted together for autoradiography. The molecular mass markers (from top to bottom) were 97 kDa, 66 kDa, 45 kDa, 31 kDa, 21 kDa
and 14 kDa.
Subpopulations of IC elements 3631
Fig. 7. Sedimentation profile of
mSEC13 obtained by analytical
differential centrifugation. PMS
prepared from NRK cells was
subjected to centrifugation as
described in the legend to Fig. 3A and
the resulting 17 membrane pellets
were analyzed by SDS-PAGE and
western blotting with antibodies
directed against the 36 kDa mSEC13
subunit (inset) of COPII coats.
membranes of relatively high density (1.143-1.178 g/ml) that
contained little p58 and, conversely, only one of the two major
p58-peaks (at 1.119 g/ml) contained mSEC13, however, only
~8% of the total mSEC13-signal in the gradient.
The distribution profile of β-COP revealed that only
relatively small amounts of β-COP colocalized with the major
peaks of p58 (1.119 and 1.135 g/ml; Fig. 8B). In summary, the
comparison of the distributions of the different markers in these
gradients (Fig. 8B) showed that both major p58-containing
components also contain the proteolipid. Interestingly, one of
them (Fig. 8B, asterisks) also contained low levels of the both
coat proteins, mSEC13 and β-COP, whereas the other
contained only a small amount of β-COP (Fig. 8B, stars) and,
therefore, represents predominantly non-coated, p58-positive
vesicles. In addition, all four markers colocalized in a minor
p58-positive component (density 1.162 g/ml; Fig. 8B, clovers),
representing a large IC structure that is predominantly found
in the P1 fraction (see Fig. 4A).
Unexpectedly, major mSEC13- and β-COP-containing
peaks, representing ~28% (mSEC13) and ~56% (β-COP) of
the total immunoreactive protein, were found at the top of the
gradients (Fig. 8). To identify these peaks, a cytosolic fraction
(S2; see Fig. 1) containing soluble mSEC13 and coatomer
complexes was prepared and subjected to density gradient
centrifugation. As shown in Fig. 9, cytosolic mSEC13 was
resolved into two major peaks, corresponding to the previously
described low and high molecular mass complexes (Tang et al.,
1997), whereas the bulk of cytosolic β-COP sedimented as a
single peak representing the 14S coatomer (Duden et al., 1991;
Waters et al., 1991). The positions of these peaks (Fig. 9)
verified that the low density peaks seen in Fig. 8 indeed
represent free coat complexes, indicating that considerable
release of the coat proteins takes place during the fractionation
of the P2 membranes.
To increase the stability of the coats, we used GTPγS, a
GTP-analog that inhibits the dissociation of coatomer from
membranes (Melançon et al., 1987). The presence of GTPγS
(100 µM) during the homogenization and fractionation of the
cells increased the recovery of both coat proteins in the P2
fraction by about 50% (Fig. 10A, GTPγS), and also stabilized
the coats against the mechanical effects related to resuspension
and high speed pelleting (fractions P3 and S3 in Fig. 10A). For
reasons that are not presently understood, but may relate to the
propensity of the protein to aggregate under the conditions
used, it also increased the recovery of mSEC13 in the S2
fraction (Fig. 10A).
Analysis of the GTPγS-treated P2 membranes in the density
gradients showed that, as a result of the stabilization of the
COPI coats, the β-COP-positive membranes shifted towards
higher densities, constituting a heterogenous population of
vesicles (8 peaks) at the density range between 1.134 and 1.201
g/ml (Fig. 10B). The specific association of β-COP with the
major p58-containing components increased about 2.4-fold
and, in parallel, the latter increased in density with the major
peak of p58 (1.161 g/ml) colocalizing with the major peak of
β-COP (Fig. 10B, stars). As compared to untreated membranes
(Fig. 8B), the mSEC13-containing elements displayed a more
homogenous pattern in response to GTPγS. The major peak of
mSEC13 was now found at a lighter density (1.142 g/ml),
where it colocalized with both p58 and β-COP (Fig. 10B;
asterisks). The specific association of mSEC13 with p58 at this
peak position was increased ~4-fold.
DISCUSSION
Methodological aspects
Previous light and electron microscopic studies have
demonstrated the pleiomorphic structure of the p58-containing
pre-Golgi intermediates and their rapid and often reversible
rearrangements and redistribution in transport-arrested cells
(Saraste and Svensson, 1991). In addition, recent studies on
living cells (Presley et al., 1997; Scales et al., 1997) have
revealed that the IC consists of highly dynamic structures. In
the present study the structural organization of the IC has been
further studied using two analytical methods of cell
fractionation. In contrast to electron microscopy, which usually
is carried out on a rather limited number of cells, the present
results describe the steady-state distribution of the IC marker
p58 in a large population of cells (in most experiments
about 7×108 cells). The first method, analytical differential
centrifugation, was used to define two main populations of
p58-containing particles and determine their respective svalues. After its introduction (Slinde and Flatmark, 1973),
this method has been improved both theoretically and
experimentally (Strand and Flatmark, 1998). Secondly, a high
3632 M. Ying, T. Flatmark and J. Saraste
Fig. 8. Comparison of the distributions of p58, mSEC13, β-COP and the proteolipid in equilibrium density gradients. The P2 fraction was
prepared from NRK cells and subjected to centrifugation in linear sucrose gradients, as in Figs 4 and 6, whereafter the distributions of p58,
mSEC13, β-COP and the proteolipid in the gradient fractions were determined by quantitative immunoblotting. Panels in A show the total
distributions of the proteins, whereas for the panels in B the distribution profiles were analyzed by the PeakFit program to demonstrate better
the constituent peaks including hidden peaks. The peaks corresponding to free mSEC13-complex or coatomer are labeled with (f) and the major
p58-positive peaks containing all three (mSEC13, β-COP and proteolipid) or two (β-COP and proteolipid) of the other markers are indicated
with asterisks and stars, respectively. In addition, a minor p58-containing peak, most likely representing a large-sized IC element (Fig. 4A), that
colocalized with all three other markers, is indicated with a clover.
resolution separation of the p58-containing particles in
equilibrium density gradients was achieved by (i) collecting a
large number (an average 34) of fractions from the gradients,
(ii) plotting the fractional content of the selected marker
proteins as a function of density, and (iii) resolving the overall
distribution profile into individual components with a gaussian
distribution by non-linear regression analysis. The combination
of these two approaches resulted in novel and complementary
information on the functional organization of the p58containing pre-Golgi intermediates.
Distinct subpopulations of p58-positive IC elements
The sedimentation profile (sediterm) of p58, obtained by
analytical differential centrifugation of homogenates prepared
from steady-state NRK cells, revealed a high degree of
polydispersity and two main populations, i.e. ‘heavy’ (fastsedimenting) and ‘light’ (slowly-sedimenting) particles, were
identified. The sedimentation coefficients (s-values) of these
populations (sH=1150±58S and sL=158±8S, respectively)
differed from those obtained for RER and SER using G-6-Pase
as the marker (Table 1), indicating that at steady-state p58 is
Subpopulations of IC elements 3633
Fig. 9. Sedimentation of soluble coat complexes in sucrose density gradients. A cytosolic fraction was prepared from NRK cells as described in
Materials and Methods, loaded on top of linear sucrose gradients, and subjected to centrifugation under the same conditions that were used for
equilibrium density gradient centrifugation of the P2 fraction. 34 fractions were collected and the positions of cytosolic, free mSEC13containing complexes and coatomer in the gradients were determined by quantitative immunoblotting using antibodies against mSEC13 and βCOP, respectively. Panels in A show the total distributions of mSEC13 and β-COP and the panels in B the individual components as resolved
using the PeakFit program. The peaks corresponding to free mSEC13-complex or coatomer are labeled with (f).
not enriched in ER-derived vesicles. This finding is in contrast
to a recent report on the fractionation of HepG2 cells, showing
the presence of a major pool of human ERGIC-53 in the ER
fraction (Klumperman et al., 1998). To determine the density
properties of the two s-populations, differential centrifugation
was used to prepare a P1 (containing most of the ‘heavy’
particles) and a P2 fraction (enriched in the ‘light’ particles).
On isopycnic density gradient centrifugation the two
populations revealed a different distribution pattern, although
the main peaks were recovered at similar buoyant densities.
Therefore, it can be concluded that the large difference in svalues between the two populations is predominantly due to a
difference in particle size, with only a small contribution of a
difference in the average buoyant density. Moreover, on the
basis of the determined s-values of the two populations a
diameter ratio (sH/sL) of ~2.7 was calculated from the equation
s=2 r2 (ρp – ρm)/9η (De Duve and Berthet, 1953). Thus, a good
correlation exists between the analytical centrifugation data
and morphological results showing the presence of p58 in
relatively large, pleiomorphic structures (average diameter
~230 nm) and small, tubulovesicular elements (average
diameter ~80 nm).
The bulk of mSEC13 and β-COP cosedimented with the
‘light’ population of p58-positive membranes, supporting the
notion that the P2 fraction is enriched in small transport
vesicles/tubular fragments. Furthermore, the sediterm of
p58 obtained for TG-treated cells revealed considerable
redistribution of the marker protein to the P2 fraction (Table
1), in accordance with electron microscopic observations
showing the accumulation of small, p58-positive vesicles in the
drug-treated cells (Ying, M. et al., unpublished). Thus, the two
subpopulations of p58-positive elements resolved by analytical
differential centrifugation represent authentic membrane
structures with morphological/functional correlates in the
intact cells. Previously, similar criteria have been used to
distinguish between immature and mature secretory granules
in rat pheochromocytoma (PC12) cells (Tooze et al., 1991).
Interestingly, although the long-distance transport of pre-Golgi
membranes to the Golgi region is inhibited at 15°C (Saraste
and Svensson, 1991; Presley et al., 1997), incubation of NRK
cells at this low temperature did not affect the sediterm of p58,
i.e. its distribution between the two main subpopulations of IC
elements. This finding indicates that the pre-Golgi elements
maintain their characteristic properties despite their altered
intracellular localization caused by the 15°C block.
Possible functions of the large pre-Golgi structures
The present results, together with our previous morphological
findings in different cell types (Saraste and Kuismanen, 1992)
support the conclusion that the fast-sedimenting, large-sized
membranous elements are an ubiquitous component of the IC,
and thus stand in contrast to the description of the IC as clusters
of small vesicles and tubules (VTCs; Bannykh and Balch,
1997). The VTC model is partly based on the experimental
systems and electron microscopic methods that have been used.
Namely, the use of permeabilized cell systems (Balch et al.,
1994), and even the fixation of intact cells using chemical
fixatives (Hess et al., 2000), could induce the vesiculation of
3634 M. Ying, T. Flatmark and J. Saraste
membranes, thus emphasizing the vesicular morphology of the
pre-Golgi elements. Moreover, in immunogold labeling the
antibody probes predominantly bind to the surface of the thin
sections, whereas during pre-embedding immunoperoxidase
labeling the reaction product of the enzyme fills the lumen of
the structure of interest. Thus, the latter method has facilitated
the visualization of the large IC structures which, according
to the present results, are also less abundant than the
tubulovesicular component.
Together with the VTCs, the individual large pre-Golgi
structures, which can obtain vacuolar dimensions (up to ~0.5
µm in diameter), could give rise to the punctate IC elements
Subpopulations of IC elements 3635
that can be resolved by light microscopy. It is possible that the
vacuolar structures, with their tubular extensions (Saraste and
Kuismanen, 1984; Presley et al., 1997; Palokangas et al., 1998;
present results), correspond to mobile pre-Golgi structures that
have been visualized in living cells (Presley et al., 1997; Scales
et al., 1997; Shima et al., 1999), whereas the membranes with
predominantly vesiculotubular appearance (VTCs) represent
more proximal, and possibly more stationary elements of the
IC, that are localized close to the ER exit sites (Bannykh et al.,
1996; Chao et al., 1999).
Of particular interest is the finding showing that the largesized pre-Golgi elements, containing about one third of total
p58, were largely devoid of coat proteins. Moreover, it is likely
that the minor pools of mSEC13 and β-COP that were found
to be associated with the P1 fraction are at least partly due to
contamination by P2 vesicles (see above). The relative scarcity
of coat structures in the large p58-positive elemens supports
the conclusion that they represent a functionally distinct part
of the IC. These elements may represent COPI and COPII coatfree, fusogenic domains involved in homotypic fusion (Rowe
et al., 1998). It is also possible that they are transient structures
that, after formation at the ER exit sites and transport to the
Golgi region, are consumed at the cis-Golgi (Bannykh
and Balch, 1997). The morphological and compositional
characteristics of these large IC structures, however, raise the
possibility that they have a specialized function in transport.
Accordingly, they could represent the initial containers for
concentrated anterograde cargo (Martínez-Menárguez et al.,
1999) and, assuming that transport through the Golgi involves
cisternal maturation, could continue to serve this function also
during later stages of transport. Thus, as compared to more
transient tubulovesicular IC elements, they could have a
relatively long half-life. Because of their large diameter (up to
~0.5 µm) they would also fulfill the geometric needs for the
transport of large cargo molecules, for example lipoprotein
particles in liver cells (Dahan et al., 1994), collagen fibrils in
fibroblasts (Leblond, 1989; Bonfanti et al., 1998), and virus
Fig. 10. GTPγS enhances the association of mSEC13 and β-COP
with membranes and alters the density distribution of coated, p58containing vesicles. (A) NRK cells were homogenized in the
presence or absence of GTPγS (100 µM), whereafter the PMS, P1,
S1 (supernatant of P1), P2, and S2 (supernatant of P2; cytosol)
fractions were prepared. The P2 pellets were further resuspended in
the homogenization medium with or without GTPγS and
recentrifuged to yield the fractions designated P3 (pellet) and S3
(supernatant). To determine the distributions of mSEC13 and β-COP
in the different fractions, samples were run in SDS-PAGE and
subjected to immunoblotting with specific antibodies. The grey and
black columns indicate fractions prepared in the absence or presence
of GTPγS, respectively. (B) The P2 fraction, prepared in the presence
of GTPγS, was resuspended and analyzed by equilibrium density
gradient centrifugation (see Fig. 4) in the presence of the analogue,
whereafter the distributions of p58, mSEC13 and β-COP in the
gradient fractions were determined by immunoblotting. The panels
on the left, where the top (left) and bottom (right) of the gradients are
indicated by arrows, show the total distributions of the three markers,
whereas the panels on the right demonstrate the individual
components as resolved by the PeakFit program. The peaks
corresponding to free mSEC13-complex or coatomer are labeled
with (f) and the major peaks containing all three markers (p58,
mSEC13 and β-COP), or two of the markers (p58 and β-COP) are
indicated with asterisks and stars, respectively.
particles that bud in pre-Golgi membranes (Tooze et al., 1984;
Jäntti et al., 1997). In conclusion, the present results are in
accordance with previous views of the pre-Golgi intermediates
as maturing, functionally complex structures, which have an
important role in the biogenesis of the Golgi complex (Saraste
and Kuismanen, 1992; Lippincott-Schwartz, 1993; LippincottSchwartz et al., 1998).
The same p58-positive IC elements contain both
COPI and COPII coats
Previous studies have concluded that the IC is the major site
for the binding of COPI coats (Oprins et al., 1993; Griffiths et
al., 1995; Martínez-Menárguez et al., 1999), whereas the
function of COPII coats is generally associated with protein
exit from the ER (Barlowe, 1998). Recent immunolocalization
of mSEC13 and β-COP in pancreatic exocrine cells showed
that, although the majority (70 to 80%) of both coats are
associated with the intermediate compartment (VTCs), they
were largely concentrated in distinct membrane subdomains
(Martínez-Menárguez et al., 1999). The authors concluded that
the mSEC13 immunoreactivity in the VTCs was due to long,
tubular ER buds that intermingle with COPI-positive IC
elements. In the present study the major pool of mSEC13 was
found in relatively dense membranes that contained negligible
amounts of p58 and β-COP and most likely correspond to
membranes that operate in protein exit from the ER.
However, our results also support the proposal that, in
addition to their role in ER exit, COPII coats associate with the
IC (Tang et al., 1997; Scales et al., 1997; Füllekrug et al.,
1999). This is in accordance with previous results showing that
the cytoplasmic tail of p58 binds both coatomer and the COPII
subunit sec23 in vitro (Kappeler et al., 1997; Tisdale et al.,
1997), as well as the present data that demonstrate the specific
enrichment of p58 in the IC. Interestingly, we observed that
distinct subpopulations of p58-containing structures contained
both mSEC13 and β-COP, indicating that dissociation of
COPII coats does not precede the binding of coatomer to
membranes. This is in contrast to current ideas of the sequential
operation of these coats in the early stages of protein secretion
(see Barlowe, 1998; Scales et al., 2000, for reviews). Moreover,
the finding of p58-positive vesicles that contained either both
mSEC13 and β-COP, or β-COP only, is in accordance with the
dual function of COPI coats both during ER exit (Dascher and
Balch, 1994; Lavoie et al., 1999), and at later stages of ERGolgi trafficking (for reviews see Lippincott-Schwartz et al.,
1998; Scales et al., 2000).
When the P2 fraction was analyzed by density gradient
centrifugation, considerable dissociation of the membranebound coat complexes, particularly the coatomer, was
observed. To stabilize the coats cells were homogenized and
fractionated in the presence of GTPγS, a poorly hydrolyzable
GTP-analogue, which results in a persistent activation of
ARF1, and inhibits the release of coatomer from membranes
(Melançon et al., 1987). When added to in vitro systems that
reconstitute the formation of COPI vesicles, GTPγS has been
reported to result in missorting of cargo molecules (Nickel et
al., 1998; Lanoix et al., 1999; Pepperkok et al., 2000), whereas
the analogue was used here to stabilize pre-existing coat
structures, making artefactual localization of p58 unlikely. As
expected, GTPγS increased the membrane-binding of β-COP
and, in parallel, the densities of the p58- and β-COP-positive
3636 M. Ying, T. Flatmark and J. Saraste
IC elements. It also enhanced the detection of mSEC13 in
vesicles containing both p58 and β-COP, but, simultaneously,
lead to the depletion of mSEC13 from the putative ER exit
membranes. This latter effect could be due to the presence in
cells of multiple types of mSEC13-containing coat structures
(Tang et al., 1997; see Figs 9 and 10) that have different
stabilities in the presence and absence of GTPγS.
Enrichment of the Vo domain of vacuolar H+-ATPase
in IC membranes
The vacuolar H+-ATPase is widely distributed within animal
cells and is found in endosomes, lysosomes, Golgi membranes,
clathrin-coated vesicles and several types of secretory granules,
where the enzyme is responsible for maintaining an acidic
internal environment (for review, see Stevens and Forgac,
1997). The lumenal pH of the different intracellular
compartments varies considerably with lysosomes being most
acidic, and ER nearly neutral (Mellman et al., 1986). Previous
studies have indicated that the assembly of the vacuolar H+ATPase complex can take place already in the ER (Myers and
Forgac, 1993; Graham et al., 1998) and subcellular fractions
prepared from rat liver, enriched in rough and smoth ER
vesicles, have been reported to contain proton ATPase activity
(Rees-Jones and Al-Awqati, 1984). The present results,
showing the enrichment of the Vo proteolipid in p58containing vesicles strongly support our previous conclusion,
based on the colocalization of p58 and the weak base DAMP,
that the IC contains an active H+-ATPase (Palokangas et al.,
1998). Moreover, the association of both mSEC13 (COPII) and
β-COP (COPI) with the proteolipid-containing IC elements
indicates that the proton pump is incorporated into IC elements
already at an early stage of transport, most likely at ER exit
sites. The function of the ATPase in the early secretory
pathway could relate to the determination of the directionality
of transport, protein segregation in pre-Golgi intermediates,
and concentration of cargo molecules, i.e. processes that are
likely to involve the regulated binding of COPII and COPI
coats to IC membranes.
We thank Drs Bor Luen Tang and Wanjin Hong (National
University of Singapore, Singapore), and Dr Shoji Ohkuma
(Kanazawa University, Japan) for generously providing antibodies.
This work was financially supported by the Norwegian Cancer Society
(T.F. and J.S.), the Novo Nordisk Foundation (T.F. and J.S.) and the
Research Council of Norway (T.F.).
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