Download Membrane flow through Golgi compartments

4017
Journal of Cell Science 112, 4017-4029 (1999)
Printed in Great Britain © The Company of Biologists Limited 1999
JCS0606
Membrane flow through the Golgi apparatus: specific disassembly of the cisGolgi network by ATP depletion
Mercedes del Valle, Yolanda Robledo and Ignacio V. Sandoval*
Centro de Biologia Molecular Severo Ochoa, CSIC, Facultad de Ciencias, Universidad Autónoma de Madrid
*Author for correspondence (e-mail: [email protected])
Accepted 2 September; published on WWW 3 November 1999
SUMMARY
Incubation of NRK cells for 30 to 45 minutes with 50 mM
2-deoxy-D-glucose (DOG) in glucose and pyruvate-free
medium results in depletion of the cellular ATP pool and
in specific disassembly of the cis-Golgi network (CGN),
with the stack of Golgi cisternae (SGC) and the trans-Golgi
network (TGN) remaining intact and sensitive to BFA. The
disassembly of the CGN is mediated by long tubular
structures extending outwards from the Golgi complex and
involves microtubules. Upon removal of DOG and addition
of glucose and pyruvate to the culture medium, the
morphology of the CGN is slowly reestablished.
Reconstruction of the CGN involves COPI/COPII-positive
vesicles that resume the transport of proteins and in
particular of CGN membrane proteins out of the ER. Exit
of CGN membrane proteins from the ER is insensitive to
BFA. In cells pretreated with nocodazole, the CGN
membrane proteins are transported to the vicinity of the
SGC fragments dispersed throughout the cytoplasm.
Ultrastructural studies of cells engaged in the
reconstruction of the CGN revealed that the CGN cisterna
emerge as tubular structures extending from 0.2-0.3 µm
uncoated vesicles prior to their organization on the cis-side
of the SGC.
INTRODUCTION
the tubulation of Golgi membranes both in vivo and in vitro
(Cluett et al., 1993; Banta et al., 1995).
The manipulation of the vesicular transport machineries
(Dascher and Balch, 1994; Wilson et al., 1994), the
overexpression or manipulation of proteins resident in
specific compartments (Hsu et al., 1992; Nilsson et al., 1996;
Nelson et al., 1998), and the ability of drugs to disrupt the
membrane traffic (Lippincott-Schwartz et al., 1989; Takizawa
et al., 1993; Deerinck et al., 1993; Sandoval and Carrasco,
1997; Fujiwara et al., 1998) have been used to dissect the
protein transport pathways as well as to define the boundaries,
morphology and links between the Golgi compartments and
adjacent organelles. These approaches nevertheless often
provoked alterations that were extended to part of or to the
whole secretory pathway, and sometimes to the entire
vacuolar system of the cell (Lippincott-Schwartz et al., 1991;
Klausner et al., 1992).
We report here the effect of the controlled depletion of the
ATP cellular pool on the integrity of the Golgi compartments.
We observe that, when the cellular ATP pool is reduced to 1525% of the control, the CGN is specifically disassembled by a
mechanism involving long tubular structures and microtubules.
The energy depletion-insensitive SGC and TGN remain
sensitive to BFA. Upon restoration of the energy levels, the
CGN membrane proteins are transported out of the ER, and the
CGN compartment is slowly reconstituted by a process that is
inhibited by BFA.
The Golgi complex is organized into three functionally distinct
parts: the cis-Golgi network (CGN), the stack of Golgi
cisternae (SGC) and the trans-Golgi network (TGN) (Novikoff
et al., 1971; Palade, 1975; Farquhar and Palade, 1981; Griffiths
and Simons, 1986: Rothman and Orci, 1992; Warren and
Malhotra, 1998). The CGN, as the entry face of the Golgi,
receives the newly synthesized proteins from the ER for their
further transport to the SGC. In addition, the CGN recycles
back to the ER the proteins that escaped the ER and
components of the machinery of vesicular transport that
operates between the ER and the Golgi complex (Dean and
Pelham, 1990; Semenza et al., 1990).
Forty genes have been identified to date in the transport
between ER and the CGN in the yeast (Kaiser et al., 1997),
and the metabolic (Jamieson and Palade, 1968), protein
(Tisdale et al., 1992; Nuoffer et al., 1994; Pind et al., 1994;
Dascher et al., 1994; Dascher and Balch, 1994; Barlowe et al.,
1994; Tisdale and Balch, 1996; Rowe et al., 1998), ion
(Beckers and Balch, 1989; Beckers et al., 1990), nucleotide
(Balch and Rothman, 1985; Balch et al., 1986), and
temperature requirements (Saraste et al., 1986) of this transport
have been determined. ATP-sensitive steps appear to punctuate
the transport of protein between the compartmental boundaries
of the ER and CGN (Jamieson and Palade, 1968; Balch et al.,
1986). Moreover, depletion of the cytosolic pool of ATP causes
Key words: Golgi, ER, ATP, BFA
4018 M. del Valle, Y. Robledo and I. V. Sandoval
Fig. 1. Depletion of the intracellular ATP pool by incubation of
cells with 50 mM 2-deoxy-D-glucose in glucose/pyruvate-free
(DOG) medium. Reversibility by removal of DOG medium and
addition of glucose. NRK cells incubated for 48 hours in
DMEM/10% FCS (normal medium) were washed three times with
glucose- and pyruvate-free DMEM medium before incubation for
45 minutes in DOG medium, and then washed three times in
DMEM and incubated in GLUC medium (A). The open and black
arrows indicate the times at which the cells were shifted from
normal medium to DOG medium and from DOG medium to GLUC
medium, respectively. Cellular ATP levels were measured as
described in Materials and Methods. Molecular mass and cellular
distribution of Helix pomatia binding proteins (HPBP) in NRK
cells. Proteins from a membrane pellet extracted from NRK cells,
washed with 0.1 M Na2CO3 and resolved by SDS-PAGE, blotted
onto nitrocellulose and stained with Helix pomatia lectin
conjugated to horseradish peroxidase. Lanes 1 and 2 were loaded
with 50 and 25 µg protein, respectively. The top and bottom of the
gel are marked with arrows (B). Ultrastructure of NRK cells
immunostained for HPBP and GMPc-1. NRK cells cultured in
normal medium were stained for HPBP using HRPO-Helix pomatia
lectin (C,D) and for GMPc-1 with the 15C8 pAb and HRPO-goat
anti-rabbit IgG (E). Note the confinement of HPBP to the Golgi
ribbons (C) and to the cis-most cisterna of the Golgi complex (D).
Nucleus (n). Bars, C, 0.5 µM; D, 0.1 µM; E, 0.33 µm.
Fig. 2. Morphology and localization of the CGN, SGC and TGN in
double stained NRK cells. NRK cells incubated in normal medium
were double stained for the CGN-markers HPBP (A) or gp74 (C),
the TGN-marker GMPt-1 (E) and for the SGC-marker, GMPc-1
(B,D,F). Bars, 8.4 µm.
MATERIALS AND METHODS
Cell culture
NRK (normal rat kidney) and Vero (Monkey, African green)
fibroblasts were cultured for a minimum of 72 hours to 80%
confluence on plastic dishes or glass coverslips at 37°C in normal
medium (DMEM, 10% fetal calf serum (FCS), 2 mM glutamine, non
esssential amino acids, 50 U/ml penicillin, 50 µg/ml streptomycin),
in an atmosphere of 93% air, 7% CO2 and 85% humidity.
Membrane flow through Golgi compartments 4019
Fig. 3. Effect of energy depletion on
the integrity and morphology of the
CGN. NRK cells incubated for the
times indicated in DOG medium,
were double stained for HPBP
(A,B,D,E), the ER marker PDI (F) or
GMPc-1 (C,G). Images of the cell
shown in B is an enlargement of the
cell marked with open arrow in A.
Tubular extensions of the CGN are
marked with solid arrows in A and B.
Bars in H indicate the percentage of
cells with recognizable CGN (i.e.
intact or tubulated CGN as shown in
A, B and in Fig. 2A) (ⵧ), SGC (as in
Fig. 2B) (䊏) and TGN (as in Fig.
2E) (䊏) after treatment with 50 mM
DOG for the indicated times. Bars in
I indicate the percentage of cells
stained with FITC-Helix pomatia
lectin that showed staining of intact CGN (as in Fig. 2A) (ⵧ),
tubulated CGN (see A and B) (䊏) or staining of the ER spread
throughout the cytoplasm (see D-E) (䊏). Bars, 10 µm.
Protein probes
The development of CC92, the mouse monoclonal IgM antibody
(mAb) against the CGN marker membrane protein gp74, as well as
the rabbit polyclonal antibodies (pAb) 15C8, against the cis/middle
Golgi membrane protein, GMPc-1, and 18B11, against the trans-Golgi
membrane protein GMPt-1, has been reported (Yuan et al., 1987;
Alcalde et al., 1992, 1994). The mouse mAb P5D4 against the
VSVtsO45 G protein and anti-β-COP maD were purchased from
Sigma. The rat mAb against tubulin and the polyclonal antibody
against sec13 were the gift of Dr J. V. Kilmartin (Kilmartin et al.,
1982) and Dr Hong (Tang et al., 1997), respectively. Helix pomatia
lectin conjugated to fluoresceine isothiocyanate (FITC) or to
horseradish peroxidase (HRPO) was purchased from Sigma. Texas red
and fluorescein-conjugated second antibodies were from
Cooper-Biomedical Inc. (Malvern, PA).
Depletion and replenishment of the ATP intracellular
pools
To lower the ATP, NRK cells were washed three times with warm
glucose-pyruvate-free DMEM containing 1% dialyzed FCS and then
incubated for the required times in the same medium supplemented
with 50 mM 2-deoxy-D-glucose (DOG medium). To restore the ATP
levels, the cells were washed three times with warm DMEM medium,
before subsequent incubation with DMEM/1% FCS supplemented
with 100 mM glucose (GLUC medium). Cellular ATP levels were
meassured by the luciferase-luciferin procedure (Sigma) on
perchloric acid extracts from NRK cells grown on 60 mm plates.
Briefly, the cells were washed with PBS, resuspended in 1 ml cold
perchloric acid, and upon the acid neutralization with KOH the
protein and salt were removed by centrifugation for 10 minutes at
14,000 g in the cold and the ATP levels meassured by the luciferinluciferase procedure in the protein-free supernatant as follows: 1 ml
of 25 mM Hepes/10 mM MgCl2/2 mM EDTA, pH 7.75, were mixed
Fig. 4. Effect of energy depletion on the distribution of β-COP. NRK
cells incubated in normal medium (A,B), or for 45 minutes in DOG
medium (C,D) and then treated for 15 minutes with 10 µg/ml BFA in
the same medium (E,F), were double stained for β-COP and GMPc-1.
Bar, 10 µm.
4020 M. del Valle, Y. Robledo and I. V. Sandoval
with 20 µl (0.8 mg) of luciferin-luciferase (Sigma) and 20 µl of
protein-free supernatant (Castillo et al., 1992), and the photon
emission measured with a Monolight 2010 luminometer (Analytical
Luminescence Laboratory).
Characterization of Helix pomatia binding proteins
(HPBP)
NRK cells grown on 60 mm plates were scraped in 0.5 ml of 0.1 M
Na2CO3 pH 11.3, the nuclei and cellular debris removed by
centrifugation for 10 minutes at 600 g, and the cellular membranes
pelleted by centrifugation for 40 minutes at 4°C at 150,000 g in a
TL100 centrifuge (Beckman). The membranes were resuspended in
Laemmli buffer, run on SDS-PAGE gels, blotted onto nitrocellulose
and the HPBP probed with HRPO-Helix pomatia lectin.
Study of the transport of the VSVtsO45 G protein out of
the ER and its acquisition of EndoH resistance
Vero cells grown to 80% confluency on coverglasses or on 60 mm
plates in normal medium were infected for 45 minutes at 31°C with
VSVtsO45 (20-30 pfu/cell) in 200 µl DMEM. Infected cells were
incubated for 4 hours at 39.5°C with normal medium and then for
45 minutes at 39.5°C in either DMEM/1% FCS (DMEMS medium)
or in DOG medium before incubation for different time periods at
31°C in DMEMS, DOG or GLUC medium. The cellular distribution
of the tsO45 G protein was studied by immunofluorescence
microscopy using the mAb P5D4 as described below. To study
the acquisition of EndoH resistance by the tsO45 G protein the
infected cells were labeled for 30 minutes at 39.5°C with
[35S]methionine/cysteine (200 µCi/ml; specific activity >1000
Ci/mmol) (Amersham) before their incubation in DMEMS or DOG
medium at the restrictive temperature. The labeled cells were
scraped in 0.5 ml of 50 mM Tris/0.1 M NaCl/1% NP40/2 mM
EDTA/0.1 mM PMSF, pH 7.4, centrifuged for 15 minutes at 4°C at
11,000 g and the supernatants mixed with 1 volume of the same
buffer without detergent and incubated overnight at 4°C with 10 µl
of mAb P5D4 (Sigma) bound to Protein G (Pharmacia). The
immunoprecipitates were washed, incubated overnight at 37°C with
or without 1 milliunit of EndoH (Boehringer) in 0.1 M sodium
phosphate buffer/2% SDS/2 mM β-mercaptoethanol pH 6.1,
resolved by SDS-PAGE, and the gels soaked for 1 hour in 1 M
sodium salicylate, dried and autoradiographed (Barriocanal et al.,
1986).
Immunofluorescence microscopy
Single and double immunofluorescence microscopy (IFM) studies
were performed on NRK cells grown on coverglasses and fixed and
permeabilized with methanol at −20°C (Barriocanal et al., 1986).
The morphology and localization of the CGN was studied using
the Helix pomatia lectin or the mAb CC92. Similar studies of the
SGC and TGN were performed with the mAb 15C8, and the mAb
18B11, respectively. To quantitate the effects of ATP depletion,
BFA, nocodazole and taxol on the distribution of the CGN
membrane proteins studied by IFM, groups of two hundred cells
grown on separate coverslips and randomly picked under the
microscope were studied. The stained cells were examined in an
Axiovert 135M microscope (Zeiss) and photographed separately
or simultaneously through Texas red or FITC fluorescence
microscopy.
Immunoelectron microscopy
Ultrastructural studies were performed on cells fixed, permeabilized
and stained with HRPO-Helix pomatia lectin, using the preembedding method as previously described (Yuan et al., 1987).
Paraformaldehyde was from Merck (Darmstadt, Germany),
glutaraldehyde (25% in water) from Fluka, (Buchs, Switzerland),
osmium tetroxide from Serva (Heidelberg, Germany) and
diaminobenzidine from Sigma.
RESULTS
Incubation of NRK cells with DOG medium results in
depletion of the ATP pool. Reconstitution of the ATP
levels by glucose addition
Previous studies have shown the blockage of protein traffic
from the ER upon energy depletion by addition of metabolic
inhibitors and inhibitors of oxidative phosphorylation
(Jamieson and Palade, 1968; Weigensberg and Blostein, 1983;
Hendricks et al., 1993) or incubation of cells with culture
medium preflushed with N2 gas (Cluett et al., 1993). To
reversibly deplete the intracellular ATP pool, we incubated
NRK cells with DOG medium for periods of 15, 30 or 60
minutes. In four separate experiments, the intracellular ATP
pool was reduced to 15-25% of normal levels within the first
15 minutes of incubation in DOG medium (Fig. 1A) and
remained stable at these values after 1 hour incubation (data
not shown). Incubation in GLUC medium resulted in recovery
of the ATP pool: the levels of ATP increased by 10-15% within
15 minutes of incubation in GLUC medium and began to level
off after 3 hours incubation reaching 75 to 90% of the initial
levels (Fig. 1A)
Energy depletion provokes the specific disassembly
of the CGN by a mechanism mediated by tubular
structures
Previous studies have demonstrated that all protein export from
the ER is inhibited at 65% of the normal cellular pool of ATP
(Balch et al., 1986). Moreover, depletion of cytosolic ATP
provokes the tubulation of Golgi membranes both in vitro and
in cultured cells (Cluett et al., 1993; Banta et al., 1995).
To study the effects of depleting the intracellular ATP pools
on the integrity of the Golgi complex, we used the Helix
pomatia lectin and the mAb CC92 to study the CGN. Whereas
in human fibroblasts the Helix pomatia lectin reacts with two
major glycoproteins of 40 and 70 kDa confined in the CGN
(Virtanen, 1990), in NRK cells the lectin reacts with two major
membrane glycoproteins of 33 and 37 kDa (HPBP, for Helix
pomatia binding proteins) (Fig. 1B) which are also confined in
the CGN localized to the cis-most Golgi (Fig. 1C,D). Antibody
CC92 reacts with a membrane glycoprotein, gp74, also
contained in the CGN (Alcalde et al., 1994). To study the
effects of depleting the intracellular ATP pools on the SGC and
the TGN the cells were stained with the mAb 15C8 and 18B11,
which react with the membrane glycoproteins GMPc-1 (Fig.
1E) and GMPt-1 (Yuan et al., 1987), respectively.
In untreated NRK cells stained for HPBP (Fig. 2A) and gp74
(Fig. 2C), the CGN is displayed as a compact reticular structure
surrounding the nucleus or capping one of its poles. The double
fluorescence experiment (Fig. 2) shows that the morphology
and localization of the CGN, the SGC (compare Fig. 2A and
C with B and D) and the TGN (compare Fig. 2E with F) were
virtually indistinguishable.
Incubation of NRK cells for 15 minutes with DOG medium
caused a dramatic change in the morphology of the CGN
stained with Helix pomatia lectin (Fig. 3A,B). or with the antigp74 antibody (not shown), as both CGN membrane proteins
appeared in long projections emanating from the perinuclear
reticular structure characteristic of the CGN The projections
extended across the cytoplasm to the cell periphery, or formed
intricate networks over the Golgi area (Fig. 3A,B,I). In
Membrane flow through Golgi compartments 4021
contrast, neither the SGC (Fig. 3C) nor the TGN (not shown),
stained with the anti-GMPc-1 and GMPt-1 antibodies,
respectively, were affected by the 15 minutes incubation in
DOG medium (Fig. 3C). After 30-45 minutes of incubation in
DOG medium, the perinuclear reticular structure stained by
Helix pomatia disappeared (Fig. 3D,H,I) and a haze of
fluorescence was spread throughout most of the area stained
with an antibody against the ER marker protein disulphide
isomerase (PDI) (Fig. 3E and F). The second CGN marker,
gp74, underwent the same redistribution (not shown). Again,
no significant changes in the location and morphology of the
SGC (Fig. 3G) and TGN (not shown) were observed after a
careful quantitative analysis of the effect (Fig. 3H). Longer
incubations of 60 to 90 minutes in DOG medium did not affect
the reticular staining pattern of the SGC, but staining of the
TGN became more diffuse, albeit all the marker protein,
GMPt-1, was localized to the Golgi area (not shown).
Incubation of cells in DOG medium thus resulted in the rapid
relocation of CGN membrane proteins, a phenomenon that
probably reflected the disassembly of this Golgi compartment.
The intact SGC retains the β-COP attached to its
membranes following energy depletion
The pattern of CGN disassembly was similar to that of Golgi
disassembly described in BFA-treated cells (LippincottSchwartz et al., 1989). It has been shown that BFA causes the
reversible redistribution of β-COP from Golgi membranes into
the cytosol (Donaldson et al., 1990; Orci et al., 1991). To
further define the effects of ATP depletion, we studied whether
incubation of cells in DOG medium caused redistribution of βCOP from the SGC membranes into the cytosol. When cells
were incubated for 45 minutes in DOG medium and double
stained for β-COP and GMPc-1, a significant amount of β-COP
remains attached to the Golgi membranes as shown by
immunofluorescence microscopy (Fig. 4C,D). This contrasted,
therefore, with the effect of BFA which induced the release of
all the Golgi-associated β-COP before the redistribution of the
Golgi proteins to the ER (Donaldson et al., 1990; Orci et al.,
1991). Moreover, treatment of cells preincubated for 45
minutes in DOG with 10 µg/ml BFA for 15 minutes resulted
in release of the β-COP associated with the Golgi membranes
to the cytoplasm, before complete disassembly of the SGC, a
process which in cells incubated with DOG proceeds more
slowly than in cells incubated in normal medium (Fig. 4E,F).
Role of microtubules in the disassembly of the CGN
The perinuclear location and integrity of the Golgi complex
requires the web of microtubules organized by the centrosome
and extended throughout the cytoplasm (Wehland and
Sandoval, 1983; Sandoval et al., 1984; Turner and Tartakoff,
1989), as does the vesicle-mediated anterograde and retrograde
transport of materials through the secretory pathway
(Lippincott-Schwartz et al., 1995; Lippincott-Schwartz and
Cole, 1995; Cole et al., 1996; Presley et al., 1997). To test
whether low ATP affected the integrity and organization of
cytoplasmic microtubules, we incubated NRK cells for 60
minutes in DOG medium. As shown in the fluorescence
experiment (Fig. 5A) the cells stained with the anti-tubulin
antibody displayed the characteristic web of long cytoplasmic
microtubules organized by the centrosome as in normal cells.
Therefore, under the conditions that caused CGN disassembly
the integrity and organization of the cytoplasmic microtubules
were not significantly affected, with the exception of some
bundling, thus excluding the possibility that disassembly of the
CGN was provoked by extensive microtubule alterations.
Moreover, cells treated for 60 minutes with the microtubule
polymerization inhibitor nocodazole (Fig. 5B), and then for 45
minutes more with the drug in DOG medium, were able to
reconstitute substantially the network of cytoplasmic
microtubules after being washed free of nocodazole (data not
shown). As described before, addition of nocodazole to cells
incubated in DMEM was followed by the rapid relocation of
gp74 from the perinuclear reticulum characteristic of the CGN,
into punctuate structures scattered throughout the cytoplasm
(Fig. 5C) (Alcalde et al., 1994).
We next studied the effect of DOG on the HPBP
accumulated in the punctuate structures. For this purpose cells
incubated for 60 minutes in nocodazole were further incubated
for 0, 20, 45 or 60 minutes with the drug in DOG medium (Fig.
5D,E,I). We observed that whereas the punctuate structures
still retained a great part of HPBP a diffuse cytoplasmic
fluorescence became visible in a significant number of cells,
suggesting some translocation of HPBP from the punctuate
structures to the ER (Fig. 5D). In these cells the distribution of
HPBP and the SGC marker GMPc-1 was strikingly different,
the latter being localized to large fragments near the
perinuclear area (Fig. 5E) (Alcalde et al., 1994).
It has been reported that reorganization of microtubules into
bundles disconnected from the centrosome by incubation of
cells with taxol (Fig. 5F) caused a profound change in the Golgi
structure: the drug provoked the disruption of the Golgi into
large fragments that became associated with the microtubule
bundles (Wehland et al., 1983). To further investigate whether
disruption of the microtubule organization had any effect on
the redistribution of HPBP induced by ATP depletion, cells
were incubated for 3 hours with 10 µM taxol and the incubation
with the drug continued in DOG medium for 0, 20 or 45
minutes before cells were fixed, permeabilized and stained for
HPBP and GMPc-1. Disruption of microtubule organization
severely retarded, but did not block, the HPBP redistribution
(Fig. 5G,I). Following 20 minutes incubation of taxol-treated
cells in DOG medium, most cells retained the HPBP in large
perinuclear structures that were also stained with the SGC
marker GMPc-1 (compare Fig. 5G and H). Even after 45
minutes incubation with taxol in DOG medium, roughly half
of the cells still retained HPBP in the reticular perinuclear
structures characteristic of the CGN. Interestingly, many of
these structures displayed still the tubule-like expansions (Fig.
5G; arrows) observed in cells incubated for 15 minutes in DOG
medium without drug (Fig. 3A,B). It is likely, therefore, that
the taxol-induced microtubule rearrangement and the resulting
Golgi and ER reorganization may affect the biogenesis and
function of the tubules involved in the redistribution of the
CGN membranes.
Reassembly of the CGN upon energy repletion
The process of CGN reconstitution is illustrated in Fig. 6, in
which cells incubated for 45 minutes in DOG medium were
washed free of DOG and incubated in GLUC medium for
periods between 15 minutes and 2 hours. The double
fluorescence experiment shows that after 15 minutes of
incubation in GLUC medium, the HPBP were redistributed
4022 M. del Valle, Y. Robledo and I. V. Sandoval
Fig. 5. Effect of microtubule depolymerization on the redistribution of HPBP elicited by ATP depletion. NRK cells incubated for 60 minutes in
DOG medium (A), or treated for 60 minutes with 20 µM nocodazole in normal medium (B,C), prior to incubation for 60 minutes in DOG
medium containing nocodazole (D,E) were fixed, permeabilized and stained for tubulin (A,B), stained for HPBP alone (C) or double-stained for
HPBP and GMPc-1 (D,E). Bars, 8 µm. Effect of the disruption of microtubule organization on the redistribution of HPBP elicited by ATP
depletion. NRK cells treated for 3 hours with 10 µM taxol in normal medium and stained for tubulin (F), or treated 3 hours with 10 µM taxol
and then incubated for 45 minutes with the drug in DOG medium before being permeabilized and double-stained for HPBP (G) and GMPc-1
(H). Tubular extensions are marked with arrows (G) Bars, 9 µm. Quantitation of the effects of nocodazole and taxol on the redistribution of
HBPB in ATP depleted cells. Cells incubated in normal DMEM medium without drugs (a), or for 60 minutes with 20 µM nocodazole (b), or for
3 hours with 10 µM taxol (c), were further incubated for 20 (ⵧ) or 45 ( ) minutes in DOG medium with or without the corresponding drugs,
processed and studied by fluorescent microscopy. Bars in I indicate the percentage of cells that showed HPBP staining in vesicle-like structures
as in C, or in perinuclear reticular structures as in G.
from a diffuse staining pattern to large vesicle-like structures
(i.e. pre-CGN elements), which were often arranged in a
semicircle around the intact SGC (Fig. 6A,B,C).
Reconstruction of the CGN was a relatively slow process; for
completion it required incubations of more than 90 minutes in
GLUC medium for most cells (D-I).
To further study the interaction between the structures
precursor of the CGN and the SGC, we compared the cellular
distributions of pre-CGN elements and of the elements derived
from the SGC disrupted with nocodazole. Cells were
preincubated for 45 minutes in DOG medium and subsequently
incubated for 60 minutes in the same medium with 20 µM
nocodazole before being washed free of DOG and subsequent
incubation for 90 minutes with nocodazole in GLUC medium.
In contrast to the cells incubated without nocodazole, the cells
incubated with the anti-microtubule drug were unable to
completely reconstruct the continuous reticulum of the CGN
in the vicinity of the nucleus, and the HPBP were localized to
large pleiomorphic structures scattered throughout the
cytoplasm (Fig. 6J). Interestingly, most of these structures were
found to codistribute and to be shaped in the same fashion as
the SGC fragments produced by nocodazole treatment (Fig.
6F,G). This observation again, suggests that the CGN and the
SGC elements interact in an unknown manner during the
process of CGN reconstruction.
gp74 codistributes with COPI and COPII shortly after
replacing the DOG medium by GLUC medium
Recent studies have documented the involvement of COPII
coated vesicles in the anterograde movement of membranes out
of the ER (Barlowe et al., 1994; Campbell and Schekman 1997;
Gaynor and Emr, 1997) and of COPI in retrograde transport
(Scheel et al., 1997; Orci et al., 1997). In addition, COP I
appears to replace COPII within the tubulovesicular clusters
Membrane flow through Golgi compartments 4023
Fig. 6. Reconstitution of the
CGN upon incubation of ATPdepleted cells with glucose.
NRK cells incubated for 45
minutes in DOG medium and
then in GLUC medium for the
indicated times (A-I), or
incubated for 45 minutes in
DOG medium and then for 60
minutes with 20 µM
nocodazole in DOG medium
before incubation for 60
minutes with the drug in
GLUC medium (J,K). The
cells were fixed, permeabilized
and stained for HPBP and
GMPc-1 as indicated in the
panels. The image in C was
produced by merging the
images shown in A and B.
Images in F and G are
enlargements of the cell
marked with white arrow in D
and E. The image in H was
photographed through
fluorescence microscopy by
simultaneously recording
fluorescein and Texas red
fluorescence. Arrows in J and
K mark Golgi-derived
fragments stained by FITCHelix pomatia and the mAb
15C8. Bars in I show the
percentage of cells that
incubated for 45 minutes in
DOG medium and then for 15
minutes, 1 hour or 2 hours in
GLUC medium when stained
for HPBP showed diffuse
cytoplasmic staining as in Fig.
4D (ⵧ), or staining of
punctuate structures as in A
( ), or perinuclear reticular
structures as in D (䊏). Bars
6 µm.
that form the intermediate compartment, to prime the budding
of vesicles that are targeted to the CGN (Aridor et al., 1995;
Bannykh et al., 1996; Rowe et al., 1996; Scheel et al., 1997;
Orci et al., 1997) (for reviews see Kuehn and Schekman, 1997;
Schekman and Mellman, 1997). To further characterize the
structures involved in CGN reconstitution, we compared the
distribution of gp74, sec 13 (i.e. COPII) and β-COP (COPI) in
cells cultured in normal medium and in cells preincubated for
45 minutes in DOG medium prior to their incubation in GLUC
medium for 15 minutes. A large amount of COPI and COPII
was localized to the Golgi and perinuclear area of cells
incubated in normal medium, but whereas sec 13 was also
4024 M. del Valle, Y. Robledo and I. V. Sandoval
found in scattered punctuate structures outside the Golgi area,
β-COP was more uniformly distributed throughout the
cytoplasm (compare Figs 4A and 7B). Incubation of cells for
45 minutes with DOG did not alter significantly the patterns of
COPI (compare Fig. 4A and C) and COP II (compare Fig. 7B
and D) distribution found in cells incubated in normal medium.
Double fluorescence experiments revealed that, in cells
preincubated for 45 minutes in DOG medium and then
incubated for 15 minutes in GLUC medium, the major part of
the structures involved in the redistribution of gp74 and in the
reconstruction of the CGN contained sec 13 (Fig. 7E,F) and βCOP (Fig. 7G,H). Somehow, the overlapping between gp74
and β-COP was more complete than between gp74 and sec 13,
since a significant number of the sec 13-positve punctuate
structures was not stained by gp74. The same observations
were made when the experiment was repeated and the cells
stained for HPBP instead of gp74 (not shown).
Transport of the VSVtsO45 G protein through the
secretory pathway is restored by energy repletion
To study the effect of energy repletion on the transport of
proteins out of the ER and through the Golgi, we infected Vero
cells with the temperature-sensitive mutant vesicular stomatitis
virus (VSV) and studied the cellular distribution of the tsO45
G protein and HPBP in cells incubated for 45 minutes at 39.5°C
(i.e. restrictive temperature) in DOG medium and then
transferred to 31°C (permissive temperature) and incubated for
different time periods in DOG or GLUC medium. The double
fluorescence study of the tsO45 G protein and the HPBP in
cells incubated for 45 minutes at 31°C in DOG medium (Fig.
8A,B), showed that both infected and uninfected cells
displayed the homogeneous cytoplasmic distribution of HPBP
characteristic of cells that suffered the disassembly of the CGN.
In contrast, in cells subsequently incubated for 20 minutes at
31°C in GLUC medium, both the tsO45 G protein and the
HPBP were found to co-localize in punctuate structures often
arranged in a semicircle at one pole of the nucleus (Fig. 8C,D).
Extension of the incubation in GLUC medium to 3 hours
resulted in their transport to a compact perinuclear structure,
probably the Golgi (Fig. 8E and F) since the HPBP were
retained in that whereas some of the the tsO45 G protein
molecules were transported to the cell periphery, as shown by
the staining of the plasma membrane (Fig. 8E, arrows). The
effect of the incubation at 31°C in GLUC medium on the
redistribution of the tsO45 G protein, was consistent with the
resume of the protein transport between the ER and Golgi and
strongly suggested that the viral and the HPBP were
transported out of the ER through the same pathway.
Monitoring of the acquisition of EndoH resistance by the
tsO45 G protein, an event that marks the transport of
glycoproteins through the SGC (Fig. 8G) showed that most of
the newly synthesized viral protein became resistant to the
glycosidase between 1 and 2 hours after transfer of the cells
incubated in DMEMS from 39.5°C to 31°C. Incubation of cells
for 45 minutes at 39.5°C in DOG medium and then at 31°C in
DOG medium completely inhibited the acquisition of EndoH
resistance by the protein. Interesting, much of the protein
recovered from cells incubated for 45 minutes at 39.5°C in
DOG medium and then incubated for 10 hours at 31°C in
GLUC medium, remained sensitive to EndoH. The slow
acquisition of EndoH resistance and the rapid transport of the
tsO45 G protein to the cell surface after transfer of cells to the
permissive temperature and GLUC medium, may reflect
different sensitivities of the protein transport and the
glycosylation systems to changes in the ATP levels.
Alternatively, though less likely, the tsO45 G protein might be
transported to the cell surface by a path that excludes the stack
of Golgi cisternae.
Ultrastructure of NRK cells incubated in DOG
medium before and after their transfer to GLUC
medium
To better define the CGN membrane protein distribution in
NRK cells with low ATP levels, we examined cells incubated
in DOG medium by electron microscopy. Cells incubated for
the times indicated in DOG medium were fixed, permeabilized,
and labeled with HRPO-Helix pomatia lectin using the
preembedding immunoperoxidase procedure. After incubation
of the cells for 15 minutes in DOG medium, staining was often
localized within the Golgi to a few convoluted tubular
structures area and, less often, to long smooth tubules (Fig. 9A
and inserts). Moreover, after incubation in DOG medium for
30 to 45 minutes the peroxidase-staining virtually disappeared,
a phenomenon similar to that observed in NRK cells treated
with BFA and stained for the medial-Golgi marker
mannosidase II (Lippincott-Schwartz et al., 1989), and
numerous stain-free SGC were localized to the vicinity of the
nucleus (Fig. 9B).
To further characterize the structures involved in CGN
reconstruction, cells incubated for 45 minutes in DOG medium
were incubated for time periods between 15 minutes and 3
hours in GLUC medium (Fig. 9C-F). Among the HRPO-Helix
pomatia stained structures observed in cells incubated for 30
minutes in GLUC medium, we found a significant number of
ER cisternae with the characteristic ribosomes (inserts in Fig.
9C,D) attached to their surface, an indication that the HPBP
and/or the CGN enzymes involved in O-glycosylation were
translocated to the ER of the ATP depleted cells. The
peroxidase staining was also localized to cisterna-like
structures that emerged as finger-like extensions from large
uncoated vesicles localized to the Golgi area (Fig. 9D, and
lower insert). Finally, cells incubated in GLUC medium for 90
minutes or longer accumulated the HRPO staining product in
tubulovesicular elements often lining one of the sides of the
SGC that remained stain-free (Fig. 9E,F). Serial sections of
these cells (Fig. 9E,F) showed that the reconstruction of the
CGN along the Golgi ribbon was not uniform.
Differential effects of ATP depletion and BFA on the
trafficking of CGN and SGC membrane proteins
Previous studies performed with cells stained for gp74 showed
that, after treatment with BFA, the reticular CGN was replaced
by a punctuate staining that appear to reflect an engagement of
the CGN membrane glycoprotein in an idle cycle of continuous
entry and exit from the ER mediated by vesicles (Fig. 10A)
(Alcalde et al., 1994). The localization of gp74 to punctuate
structures was in contrast with the homogenous distribution of
the SGC marker, GMPc-1, throughout the cytoplasm (Fig. 10B;
a quantitative analysis of the experiment is shown in P). To
study whether the SGC of cells incubated in DOG medium
remained BFA sensitive, cells preincubated for 45 minutes in
DOG medium were further incubated with 10 µg/ml BFA for
Membrane flow through Golgi compartments 4025
30 or 60 minutes in the same medium before being fixed and
stained with antibodies against gp74 (Fig. 10C) and GMPc-1
(Fig. 10D). Counting of the cells displaying distinct GMPc-1positive perinuclear structures, such as those shown in Fig.
10D, demonstrated that though the SGC was disassembled
significantly more slowly in cells with low ATP levels it
remained sensitive to BFA (Fig. 10P).
To further characterize the pathway of transport of CGN
membranes out of the ER, the distribution of gp74 and the SGC
marker GMPc-1 was investigated in cells preincubated for 45
minutes in DOG medium and then treated for 30 minutes with
10 µg/ml BFA in GLUC medium. It can be seen that the gp74
reappeared in punctuate structures scattered throughout the
cytoplasm (Fig. 10E,P), a pattern of distribution similar to that
observed when BFA was added to cells incubated in normal
medium (compare A and E), whereas in most of the cells the
SGC marker GMPc-1 showed the homogeneous distribution
expected for its redistribution from the SGC to the ER (Fig.
10F,P). When cells were incubated for 45 minutes with DOG
and then for 60 minutes with BFA in the same medium,
replacement of GLUC for DOG also resulted in reappearance
of gp74 in punctuate structures (Fig. 10G,H), however, the
number of these was significantly less than when BFA was
added together with GLUC (Fig. 10E,F), the difference being
probably due to the prolonged incubation of the cells in DOG
medium. As expected, in cells preincubated for 60 minutes
with BFA in normal medium and then incubated for 60 minutes
with the drug in DOG, both the gp74 and the GMPc-1 were
homogeneously distributed throughout the cytoplasm,
indicating that they were retained in the ER (Fig. 10I,J,P).
Moreover, in cells washed-free of the drug and transferred to
GLUC medium, both gp74 and GMPc-1 were localized to the
same cytoplasmic structures (Fig. 10K to M), suggesting that
they were transported out of the ER by the same pathway. The
size and frequent arrangement of these structures in a semicircle
around the Golgi area was in fact reminiscent of the structures
that housed the CGN membrane proteins after the substitution
of DOG for glucose in the culture medium and their release
from the ER (Fig. 6A). Finally, in cells incubated for 60 minutes
with BFA in normal medium before incubation for 60 minutes
with BFA in DOG medium and then incubated for 60 minutes
in BFA-free DOG medium, the gp74 was uniformly distributed
throughout the cytoplasm whereas the GMPc-1 was localized to
punctuate and elongated structures clustered near the nucleus,
indicating that GMPc-1 was transported out of the ER that
retained the gp74 (Fig. 10N,O,P).
DISCUSSION
The studies reported here extend previous observations that
ATP-sensitive steps punctuate protein transport between the ER
and CGN (Jamieson and Palade, 1968; Balch and Rothman,
1985; Balch and Keller, 1986), and that depletion of the ATP
cellular pool causes the tubulation of Golgi membranes both in
vivo and in vitro (Cluett et al., 1993; Banta et al., 1995).
Immunofluorescence microscopy studies show that incubation
of cells with DOG in glucose- and pyruvate-free medium results
in rapid and complete redistribution of membrane proteins
normally restricted to the CGN. Redistribution is specific in that
only the CGN membrane proteins redistribute to the ER (see
below), whereas the SGC and TGN membrane proteins are
retained in apparently intact Golgi elements. This is in contrast
with the indiscriminate redistribution of membrane proteins
from Golgi to the ER produced by BFA, redistribution that
resulted in complete disassembly of the Golgi complex.
Nevertheless, since we have studied only the redistribution of
HPBP and gp74, the conclusion that all the CGN membrane
proteins undergo the same redistribution in ATP-depleted cells
must await definitive confirmation from independent studies
using other CGN membrane markers
Immunoultrastructural studies using HRPO-conjugated Helix
pomatia show that after incubation of cells for 15 minutes in
DOG medium, the typical CGN reticular structure in the vicinity
of the nucleus is replaced by convoluted tubular structures as
well as by long smooth tubules that are more rarely seen. Longer
incubations with DOG result in localization of the CGN
membrane markers HPBP and gp74 to a delicate reticulum that
extends throughout the cytoplasm and is stained by the ER
marker PDI as shown by immunofluorescence microscopy.
Immunoelectron microscopy studies of these cells, however, did
not show clear ER staining with HRPO-Helix pomatia lectin,
probably for the same technical reasons that impeded the
visualization of the Golgi proteins translocated to the ER of
NRK cells treated with BFA (Lippincott-Schwartz et al., 1989).
The staining of ER cisternae with HRPO-Helix pomatia lectin
and the localization of HPBP and gp74 to the structures that
transport the VSVtsO45 G protein out of the ER upon washing
the cells free of DOG and their incubation for 15 minutes in
GLUC medium, further support the possibility that the CGN
membrane markers are translocated to the ER upon ATP
depletion.
The specific redistribution of CGN membranes that follows
the ATP depletion strongly suggests that within the Golgi
complex the CGN is a separate compartment with distinct
boundaries and which bears a distinct membrane flow. The
massive exchange of membranes between the ER and the CGN
(Wieland et al., 1987) and their retrieval by recycling, probably
determine much of the size and structure of the CGN, and may
explain its sensitivity and distinct response to ATP depletion.
The disassembly of the CGN and the lack of response of the
SGC membranes to ATP depletion may be explained by the
retention of COPI by the SGC membranes of the cells treated
with DOG, a fact which is in contrast with the rapid release of
COPI from the Golgi membranes in BFA-treated cells
(Donaldson et al., 1990).
As with BFA, the redistribution of CGN proteins in ATPdepleted cells is mediated by long tubular structures. It is
noteworthy that whereas the tubules promoted by lowering
ATP levels have been shown to be substrates for the rebinding
of vesicle-associated coat proteins (Cluett et al., 1993), the
tubules extended in response to BFA appeared to form after the
release of COPI components from the Golgi membranes
(Donaldson et al., 1990). These observations suggest that the
tubules formed after DOG or BFA treatment may be different
structures and suggest that they may have different functions.
The transport of the SGC marker, GMPc-1, out of the ER
after washing free of BFA the cells cultured in DOG medium,
indicate that levels of ATP as low as 15-25% below the normal
level do not inhibit completly the transport out of the ER
mediated by vesicles, and shows that vesiculation is not always
suppressed under conditions that favor tubulation (Klausner et
al., 1992; Cluett et al., 1993).
4026 M. del Valle, Y. Robledo and I. V. Sandoval
Fig. 7. Transport of gp74 out
of the endoplasmic reticulum is
mediated by COPI/COPII
positive vesicles. NRK cells
incubated in normal medium
(A,B) or for 45 minutes in
DOG medium (C,D) prior to
incubation for 15 minutes in
GLUC medium (E-H) were
fixed-permeabilized and
double stained for gp74 and for
sec 13 (i.e. COPII) or β-COP
(COPI) as indicated in the
panels. The cells in G were
directly stained with Texas redconjugated gp74. Fluoresceineand Texas red-conjugated
second antibodies were used to
stain the cells shown in the
remaining panels. Enclosed in
squares in E to H are the areas
enlarged in the inserts. When
photographed the cells shown
in H were underexposed to
eliminate the fluorescence due
to cytoplasmic β-COP pool
(see Fig. 4A) and facilitate the
study of its co-distribution with
the membrane protein gp74.
Arrows in inserts mark doublestained vesicles. Bar, 11.2 µm.
Fig. 8. HPBP and VSVts045G protein are co-transported out of the ER by the same vesicles. Vero cells incubated for 4 hours at 39.5°C upon
infection with the tsO45 VSV were further incubated for 45 minutes at 39.5°C in DOG medium and then for the indicated time periods at 31°C in
DOG or in GLUC medium. Cells were fixed permeabilized with methanol and double stained for the tsO45 G protein and HPBP as indicated in the
panels. In E the VSVts045 G protein localized to the plasma membrane is marked with arrows. Bars, 8.4 µm. Kinetics of acquisition of EndoH
resistance by the tsO45 G protein. tsO45 VSV infected Vero cells were labeled with [35S]methionine/cysteine and incubated as described in G.
Incubations in DMEMS medium (green arrows, a, c-e), in DOG medium (red arrows, b,f,g) and in GLUC medium (blue arrows, b,h-j) were
stopped at the indicated times and the EndoH resistance of tsO45 G protein (samples a-j) analyzed as described in Materials and Methods.
Membrane flow through Golgi compartments 4027
Fig. 9. Distribution of HPBP in ATP-depleted and
in ATP-replenished cells studied by EM. NRK cells
incubated in DOG medium for 15 minutes (A), or
for 45 minutes (B) prior to subsequent incubation
for 45 minutes (C,D) or for 90 minutes (E,F; serial
sections) in GLUC medium. The cells were
processed and stained with HRPO-Helix pomatia
lectin using the pre-embedding procedure. Arrows
in A mark Helix-pomatia-stained convoluted tubular
structures and cisternae remnants in the vicinity of
the centrosome (Ct) and nucleus (N). Two of these
HRPO-stained convoluted structures and a long
smooth tubule are shown in the inserts. Arrows in B
mark stain-free SGC. Arrows in C mark four
cisternae with the characteristic spiny surface of the
RER. Three of these RER cisternae are shown at
large magnification in the insert of C and in the
upper insert of D. Arrows in D mark stained
vesicles with finger-like expansions, one of these
structures is shown at large magnification in the
lower insert. (E and F) Stretches of a Golgi ribbon
(arrows) displaying different extents of CGN
reconstitution on the cis-side (E,F). Bars: A, 0.43
µm; A, inserts, 0,17 µm; B, 0.34 µm; C, 0.6 µm;
D, 0.33 µm; D, insert, 0.15 µm; E and F, 0.67 µm.
Fig. 10. ATP depletion
and BFA treatment affect
differently the trafficking
of CGN and SGC
membranes. BFA
interferes with the
pathway of CGN
reconstitution. NRK
cells incubated 30
minutes with 10 µg/ml
BFA in normal medium
(A,B), or for 45 minutes
in DOG medium prior to
their treatment for 30
minutes with 10 µg/ml
BFA in DOG (C,D) or
GLUC medium (E,F).
After a 45 minute
incubation in DOG
medium, cells were also
treated for 60 minutes
with BFA in DOG
medium prior to their
treatment for 30 minutes
with BFA in GLUC
medium (G,H). Cells were also incubated for 60 minutes with 10 µg/ml BFA in normal medium before their incubation with the drug in DOG
medium for 60 minutes (I,J), and subsequent incubation for 30 minutes in drug-free GLUC medium (K-M) or for 60 minutes in DOG medium
(N,O). The cells were fixed, permeabilized, and stained for gp74 and GMPc-1 as indicated in the panels. Bars: A-D and I, J, 16 µm; E-H and KO, 7.5 µm;. Bars in P show in the described experiments the percentage of cells in which gp 74 (䊏) and GMPc-1 (䊏
䊏) were not relocated to the
ER and were displayed as punctuate or perinuclear reticular structures.
4028 M. del Valle, Y. Robledo and I. V. Sandoval
We find that low ATP and BFA cause the specific retention
of CGN and SGC membrane proteins, respectively, in the ER.
Their different effects probably reflect differences in the
mechanisms of anterograde and/or retrograde transport that
determine the distribution of those proteins between the ER
and the Golgi.
The labeling of the vesicles that transport gp74/HPBP out of
the ER with antibodies against COPI and COPII, and the
evidence that COPI appears to replace COPII coat on the
surface of the membranes of the tubulovesicular clusters that
constitute the intermediate compartment (Rowe et al., 1996),
strongly suggests that after their release from the ER the CGN
membrane proteins traffic through the same pathway described
for secretory proteins (Schweitzer et al., 1990; Hauri and
Schweitzer, 1992), The localization of CGN and SGC
membrane proteins to the same large structures that encircle
the perinuclear area that host the Golgi upon removal of BFA
and the replacement of GLUC for DOG medium, shows that
at some point, their pathways from the ER to the area in which
the Golgi is reconstructed converge. With regard to this further
research is necessary to establish the origin of the large
uncoated vesicles that appear to mother the CGN cisternae.
With respect to the question of whether the CGN and
SGC can be independently assembled, at least under the
experimental conditions here used, the answer is no. Neither
the CGN nor the SGC were reconstituted when the membrane
components of the other compartment were retained in the ER.
It was interesting, however, that reconstitution of the CGN
upon restoration of ATP levels was conditioned by the
structural integrity and distribution of the SGC elements. Thus,
in untreated cells with intact perinuclear SGC, the CGN was
completely reconstructed, to the extent that the newly
assembled elements exactly matched the intricate laced
morphology of the SGC. In contrast, in cells treated with
nocodazole and as result with the SGC broken into fragments
dispersed throughout the cytoplasm, the HPBP as well as gp74
were always localized to structures that exactly matched
the shape and distribution of the SGC fragments, with
independence of their size or distribution. These results and the
ability of BFA to impede the assembly of the CGN by retaining
the components of the SGC in the ER, suggest that some
interaction between the CGN and SGC elements must exist
during the process of Golgi reconstruction.
Finally, the protocol used here to specifically disassemble
the CGN may be helpful to study the ultrastructure and
function of this part of the Golgi apparatus.
This work was supported by grants from the EC (FMRX-CT960058) and the Comision Interministerial de Ciencia y Tecnología (940035) to I.V.S. We thank Maria Teresa Rejas Marcos and Milagros
Guerra Rodriguez for their skillful help in the processing of cells for
the EM studies and to Drs J. V. Kilmartin and W. Hong for the gift
of antibodies against tubulin and sec 13p. The help of Mariano
Esteban with the art work is acknowleged.
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