Owing to the lack of appropriate markers the structural organization of the ER-to-Golgi pathway and the dynamics of its membrane elements have been elusive. To elucidate this organization we have taken a monoclonal antibody (mAb) approach. A mAb against a novel 63 kDa membrane protein (p63) was produced that identifies a large tubular network of smooth membranes in the cytoplasm of primate cells. The distribution of p63 overlaps with the ER-Golgi intermediate compartment, defined by a previously described 53 kDa marker protein (here termed ERGIC-53), as visualized by confocal laser scanning immunofluorescence microscopy and immunoelectron microscopy. The p63 compartment mediates protein transport from the ER to Golgi apparatus, as indicated by partial colocalization of p63 and vesicular stomatitis virus G protein in Vero cells cultured at 15 degrees C. Low temperatures and brefeldin A had little effect on the cellular distribution of p63, suggesting that this novel marker is a stably anchored resident protein of these pre-Golgi membranes. p63 and ERGIC-53 were enriched to a similar degree by the same subcellular fractionation procedure. These findings demonstrate an unanticipated complexity of the ER-Golgi interface and suggest that the ER-Golgi intermediate compartment defined by ERGIC-53 may be part of a greater network of smooth membranes.

REFERENCES

Balch
W.E.
(
1989
).
Biochemistry of interorganelle transport.
J. Biol. Chem
264
,
16965
16968
Balch
W.E.
(
1990
).
Molecular dissection of early stages of the eukaryotic secretory pathway.
Curr. Opin. Cell Biol
2
,
634
641
Balch
W.E.
,
Kahn
R.A.
,
Schwaninger
R.
(
1992
).
ADP-ribosylation factor is required for vesicular trafficking between the endoplasmic reticulum and the cis -Golgi compartment.
J. Biol. Chem
267
,
13053
13061
Beckers
C.J.M.
,
Balch
W.E.
(
1989
).
Calcium and GTP: essential components in vesicular trafficking between the ER and the Golgi apparatus.
J.Cell Biol
108
,
1245
1256
Beckers
C.J.M.
,
Block
M.R.
,
Glick
B.S.
,
Rothman
J.E.
,
Balch
W.E.
) (
1989
).
Vesicular transport between the endoplasmic reticulum and the Golgi stack requires the NEM-sensitive fusion protein.
Nature
339
,
397
398
Beckers
C.J.M.
,
Plutner
H.
,
Davidson
H.W.
,
Balch
W.E.
) (
1990
).
Sequential intermediates in the transport of protein between the ER and the Golgi.
J. Biol. Chem
265
,
18298
18310
Bergmann
J.E.
,
Fusco
P.J.
) (
1990
).
The G protein of vesicular stomatitis virus has free access into and egress from the smooth endoplasmic reticulum of UT-1 cells.
J. Cell Biol
110
,
625
635
Chavrier
P.
,
Parton
R.G.
,
Hauri
H.-P.
,
Simons
K.
,
Zerial
M.
(
1990
).
Localization of low molecular weight GTP-binding proteins to exocytic and endocytic compartments.
Cell
62
,
317
329
Clary
D.O.
,
Griff
I.C.
,
Rothman
J.E.
(
1990
).
SNAPS, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast.
Cell
61
,
709
721
Dean
N.
,
Pelham
H.R.B.
(
1990
).
Recycling of proteins from the Golgi apparatus to the ER in yeast.
J. Cell Biol
111
,
369
377
Duden
R.
,
Griffiths
G.
,
Frank
R.
,
Argos
P.
,
Kreis
T.E.
(
1991
).
Beta-COP, a 110 kd protein associated with non-clathrin-coated vesicles and the Goli complex, shows homology to beta-adaptin.
Cell
64
,
649
665
Fujiki
Y.
,
Hubbard
A.L.
,
Fowler
S.
,
Lazarow
P.B.
(
1982
).
Isolation of intracellular membranes by means of sodium carbonate treatment: Application to endoplasmic reticulum.
J. Cell Biol
93
,
97
102
Gorr
S.V.
,
Stieger
B.
,
Fransen
J.A.M.
,
Kedinger
M.
,
Marxer
A.
,
Hauri
H.-P.
(
1988
).
A novel marker glycoprotein for the microvillus membrane of surface colonocytes of rat large intestine and its presence in small-intestinal crypt cells.
J. Cell Biol
106
,
1937
1946
Griffiths
G.
,
McDowall
A.
,
Back
R.
,
Dubochet
J.
(
1984
).
On the preparation of cryosections for immunocytochemistry.
J. Ultrastruct. Res
89
,
65
78
Groesch
M.E.
,
Ruohola
H.
,
Bacon
R.
,
Rossi
G.
,
Ferro-Novick
S.
(
1990
).
Isolation of a functional vesicular intermediate that mediates ER to Golgi transport in yeast.
J. Cell Biol
111
,
45
53
Hauri
H.-P.
,
Schweizer
A.
(
1992
).
The endoplasmic reticulum- Golgi intermediate compartment.
Curr. Opin. Cell Biol
4
,
600
608
Hawkes
R.
,
Niday
E.
,
Gordon
J.
(
1982
).
A dot-immunobinding assay for monoclonal and other antibodies.
Anal. Biochem
119
,
142
147
Hendricks
L.C.
,
Gabel
C.A.
,
Suh
K.
,
Farquhar
M.G.
(
1991
).
A 58-kDa resident protein of the cis -Golgi cisterna is not terminally glycosylated.
J. Biol. Chem
266
,
17559
17565
Hicke
L.
,
Schekman
R.
(
1990
).
Molecular machinery required forprotein transport from the endoplasmic reticulum to the Golgi complex.
BioEssays
12
,
253
258
Hsu
V.W.
,
Yuan
L.C.
,
Nuchtern
J.G.
,
Lippincott-Schwartz
J.
,
Hammerling
G.J.
,
Klausner
R.D.
(
1991
).
A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules.
Nature
352
,
441
444
Kaiser
C.A.
,
Schekman
(
1990
).
Distinct sets of sec genes govern transport vesicle formation and fusion early in the secretory pathway.
Cell
61
,
723
733
Karrenbauer
A.
,
Jeckel
D.
,
Just
W.
,
Birk
R.
,
Schmidt
R.R.
,
Rothman
J.E.
,
Wieland
F.T.
(
1990
).
The rate of bulk flow from the Golgi to the plasma membrane.
Cell
63
,
259
267
Kelly
R.B.
(
1985
).
Pathways of protein secretion in eukaryotes.
Science
230
,
25
32
Klausner
R.D.
(
1989
).
Sorting and traffic in the central vacuolar system.
Cell
57
,
703
706
Kreis
T.E.
(
1986
).
Microinjected antibodies against the cytoplasmic domain of vesicular stomatitis virus glycoprotein block its transport to the cell surface.
EMBO J
5
,
931
941
Laemmli
U.K.
(
1970
).
Cleavage of structural proteins during assembly of the head of bacteriophage T4.
Nature
227
,
680
685
Lazzarino
D.A.
,
Gabel
C.A.
(
1988
).
Biosynthesis of the mannose 6-phosphate recognition marker in transport-impaired mouse lymphoma cells. Demonstration of a two-step phosphorylation.
J. Biol. Chem
263
,
10118
10126
Lewis
M.L.
,
Pelham
H.R.B.
(
1990
).
A human homologue of the yeast HDEL receptor.
Nature
348
,
162
163
Lewis
M.J.
,
Pelham
R.B.
) (
1992
).
Ligand-induced redistribution of a human KDEL receptor from the Golgi complex to the endoplasmic reticulum.
Cell
68
,
353
364
Lewis
M.J.
,
Sweet
D.J.
,
Pelham
H.R.B.
(
1990
).
The ERD2 gene determines the specificity of the luminal ER protein retention system.
Cell
61
,
1359
1363
Lindsey
J.D.
,
Ellisman
M.H.
) (
1985
).
The neuronal endomembrane system. I: Direct links between rough ER and the cis-element of the Golgi apparatus.
J. Neurosci
5
,
3111
3123
Lindsey
J.D.
,
Ellisman
M.H.
) (
1985
).
The neuronal endomembrane system. II. The multiple forms of the Golgi apparatus cis element.
J. Neurosci
5
,
3124
3134
Lippincott-Schwartz
J.
,
Donaldson
J.G.
,
Schweizer
A.
,
Berger
E.G.
,
Hauri
H.-P.
,
Yuan
L.C.
,
Klausner
R.D.
) (
1990
).
Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests and ER recycling pathway.
Cell
60
,
821
836
Louvard
D.
,
Reggio
H.
,
Warren
G.
(
1982
).
Antibodies to the Golgi complex and the rough endoplasmic reticulum.
J. Cell Biol
92
,
92
106
Matter
K.
,
McDowell
W.
,
Schwartz
R.T.
,
Hauri
H.P.
(
1989
).
Asynchronous transport to the cell surface of intestinal brush border hydrolases is not due to differential trimming of N-linked oligosaccharides.
J. Biol. Chem
264
,
13131
13139
Mellman
I.
,
Simons
K.
(
1992
).
The Golgi complex. In vitro veritas?.
Cell
68
,
829
840
Merisko
E.M.
,
Fletscher
M.
,
Palade
G.E.
) (
1987
).
The reorganization of the Golgi complex in anoxic pancreatic acinar cells.
Pancreas
1
,
95
109
Munro
S.
,
Pelham
H.R.B.
(
1987
).
A C-terminal signal prevents secretion of luminal ER proteins.
Cell
48
,
899
907
Nakano
A.
,
Muramatsu
M.
(
1989
).
A novel GTP-binding protein, Sar1p is involved in transport from the endoplasmic reticulum to the Golgi apparatus.
J. Cell Biol
109
,
2677
2691
Orci
L.
,
Tagaya
M.
,
Amherdt
M.
,
Perrelet
A.
,
Donaldson
J.G.
,
Lippincott-Schwartz
J.
,
Klausner
R.
,
Rothman
J.E.
) (
1991
).
Brefeldin A, a drug that blocks secretion, prevents the assembly of non-clathrin-coated buds on Golgi cisternae.
Cell
64
,
1183
1195
Palade
G.E.
(
1975
).
Intracellular aspects of the process of protein transport.
Science
189
,
347
358
Pelham
H.R.B.
(
1988
).
Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment.
EMBO J
7
,
913
918
Pelham
H.R.B.
(
1989
).
Control of protein exit from the ER.
Annu. Rev. Cell Biol
5
,
1
23
Pelham
H.R.B.
(
1991
).
Recycling of proteins between the endoplasmic reticulum and Golgi complex.
Curr. Opin. Cell Biol
3
,
585
591
Peter
F. P.
,
Nguyen Van Söling
H.-D.
(
1992
).
Differential sorting of Lys-Asp-Glu-Leu proteins in rat liver.
J. Biol. Chem
267
,
10631
10637
Pfeffer
S.R.
,
Rothman
J.E.
) (
1987
).
Biosynthetic protein transport and sorting by the ER and Golgi.
Annu. Rev. Biochem
56
,
829
852
Rambourg
A.
,
Clermont
Y.
(
1990
).
Three-dimensional electron microscopy: structure of the Golgi apparatus.
Eur. J. Cell Biol
51
,
189
200
Rexach
M.F.
,
Schekman
R.W.
) (
1991
).
Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles.
J. Cell Biol
114
,
219
229
Rothman
J.E.
(
1987
).
Protein sorting by selective retention in the endoplasmic reticulum and Golgi stack.
Cell
56
,
521
522
Rothman
J.E.
,
Orci
L.
(
1990
).
Movement of proteins through the Golgi stack: a molecular dissection of vesicular transport.
FASEB J
4
,
1460
1468
Rothman
J.E.
,
Orci
L.
(
1992
).
Molecular dissection of the secretory pathway.
Nature
355
,
409
415
Saraste
J.
,
Kuismanen
E.
(
1984
).
Pre-and post-Golgi vacuoles operate in the transport of Semliki forest virus membrane glycoproteins to the cell surface.
Cell
38
,
535
549
Saraste
J.
,
Svensson
K.
(
1991
).
Distribution of the intermediate elements operating in ER to Golg transport.
J. Cell Sci
100
,
415
430
Schmitt
H.D.
,
Puzichia
M.
,
Gallwitz
D.
(
1988
).
Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium.
Cell
53
,
635
647
Schweizer
A.
,
Fransen
J.A.M.
,
Bächi
T.
,
Ginsel
L.
,
Hauri
H.-P.
(
1988
).
Identification, by a monoclonal antibody, of a 53 kD protein associated with a tubular-vesicular compartment at the cis-side of the Golgi appartus.
J. Cell Biol
107
,
1643
1653
Schweizer
A.
,
Fransen
J.A.M.
,
Matter
K.
,
Kreis
T.E.
,
Ginsel
L.
,
Hauri
H.-P.
(
1990
).
Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus.
Eur. J. Cell Biol
53
,
185
196
Schweizer
A.
,
Matter
K.
,
Ketcham
C.M.
,
Hauri
H.-P.
(
1991
).
The isolated ER-Golgi intermediate compartment exhibits properties that are different from ER and cis -Golgi.
J. Cell Biol
113
,
45
54
Segev
N.
,
Mulholland
J.
,
Botstein
D.
(
1988
).
The yeast GTP-bindingYPT1 protein and a mammalian counterpart are associated with the secretion machinery.
Cell
52
,
915
924
Semenza
J.C.
,
Hardwick
K.G.
,
Dean
N.
,
Pelham
H.R.B.
(
1990
).
ERD2, a yeast gene required for the receptor-mediated retrieval of luminal ER proteins from the secretory pathway.
Cell
61
,
1349
1357
Serafini
T.
,
Orci
L.
,
Amherdt
M.
,
Brunner
M.
,
Kahn
R.A.
,
Rothman
J.E.
) (
1991
).
ADP-ribosylation factor is a subunit of the coat of Golgi-derived COP-coated vesicles: A novel role for a GTP-binding protein.
Cell
67
,
239
253
Slot
J.W.
,
Geuze
H.J.
,
Gigengack
S.
,
Lienhard
G.E.
,
James
D.E.
(
1991
).
Immunolocalization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.
J. Cell Biol
113
,
123
135
Tanaka
K.
,
Fukudome
H.
(
1991
).
Three-dimensional organization of the Golgi complex observed by scanning electron microscopy.
J. Electron Microsc. Tech
17
,
15
23
Tartakoff
A.M.
(
1986
).
Temperature and energy dependence of secretory protein transport in the exocrine pancreas.
EMBO J
5
,
1477
1482
Tooze
S.A.
,
Tooze
J.
,
Warren
G.
(
1988
).
Site of addition of N-acetyl-galactosamine to the E1 glycoprotein of a mouse hepatitits virus-A59.
J. Cell Biol
106
,
1475
1487
Wandinger-Ness
A.
,
Bennett
M.K.
,
Antony
C.
,
Simons
K.
(
1990
).
Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells.
J. Cell Biol
111
,
987
1000
Warren
G.
(
1987
).
Signals and salvage sequences.
Nature
327
,
17
18
Wieland
F.T.
,
Gleason
M.L.
,
Serafini
T.A.
,
Rothman
J.E.
(
1987
).
The rate of bulk flow from ER to the cell surface.
Cell
50
,
289
300
Wilson
D.W.
,
Whiteheart
S.W.
,
Orci
L.
,
Rothman
J.E.
(
1991
).
Intracellular membrane fusion.
Trends Biochem Sci
16
,
334
337
Wilson
D.W.
,
Wilcox
C.A.
,
Flynn
G.C.
,
Chen
E.
,
Kuang
W.-J.
,
Henzel
W. J.
,
Block
M.R.
,
Ullrich
A.
,
Rothman
J.E.
(
1989
).
A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast.
Nature
339
,
355
359
Xiao
L.
,
Storrie
B.
(
1991
).
Behavior of a transitional tubulovesicular compartment at the cis-side of the Golgi apparatus in in vivo fusion studies of mammalian cells.
Exp. Cell Res
193
,
213
218
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