p63 is a type II integral membrane protein that has previously been suggested to be a resident protein of a membrane network interposed between the ER and the Golgi apparatus. In the present study, we have produced a polyclonal antibody against the purified human p63 protein to reassess the subcellular distribution of p63 by confocal immunofluorescence, immunoelectron microscopy, and cell fractionation. Double immunofluorescence of COS cells showed significant colocalization of p63 and a KDEL-containing lumenal ER marker protein, except for differences in the staining of the outer nuclear membrane. Immunoelectron microscopy of native HepG2 cells and of COS cells transfected with p63 revealed that both endogenous and overexpressed p63 are predominantly localized in the rough ER. While p63 was colocalized with protein disulfide isomerase, an ER marker protein, very little overlap of p63 was found with ERGIC-53, an established marker for the ER-Golgi intermediate compartment. When rough and smooth membranes were prepared from rat liver, p63 was found to copurify with ribophorin II, a rough ER protein. Both p63 and ribophorin II were predominantly recovered in rough microsomes and were largely separated from the intermediate compartment marker protein p58. From these results it is concluded that p63 is localized in the rough ER.
REFERENCES
Aronson
N.A.
, Touster
O.
(1974
). Isolation of rat liver plasma membrane fragments in isotonic sucrose.
Meth. Enzymol
31
, 90
–102
Banfield
D.K.
, Lewis
M.J.
, Rabouille
C.
, Warren
G.
, Pelham
H.R.B.
(1994
). Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains.
J. Cell Biol
127
, 357
–371
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
63
, 317
–329
Chen
P.S.
, Toribara
T.Y.
, Warner
H.
(1956
). Microdetermination of phosphorous.
Analyt. Chem
28
, 1756
–1758
Chen
Y.-J.
, Hickey
W.F.
, Mezitis
S.G.E.
, Stieber
A.
, Lavi
E.
, Gonatas
J.O.
, Gonatas
N.K.
(1991
). Monoclonal antibody 2H1 detects a 60–65 kD membrane polypeptide of the rough ER of neurons and selectively stain cells of several rat tissues.
J. Histochem. Cytochem
39
, 635
–643
Colbeau
A.
, Nachbauer
J.
, Vignais
P.M.
(1971
). Enzymatic characterization and lipid composition of rat liver subcellular membranes.
Biochim. Biophys. Acta
249
, 462
–492
Crimaudo
C.
, Hortsch
M.
, Gausepohl
H.
, Meyer
D.
(1987
). Human ribophorins I and II: The primary structure and membrane topology of two highly conserved rough ER specific glycoproteins.
EMBO J
6
, 75
–82
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 Golgi complex, shows homology to beta-adaptin.
Cell
64
, 649
–665
Evan
G.
, Lewis
G.K.
, Ramsay
G.
, Bishop
J.M.
(1985
). Isolatin of monoclonal antibodies specific for human c-myc protooncogene product.
Mol. Cell Biol
5
, 3610
–3616
Fullekrug
J.
, Sönnichsen
B.
, Wunsch
U.
, Arseven
K.
, Nguyen Van
P.
, Söling
H.-D.
, Mieskes
G.
(1994
). CaBP1, a calcium binding protein of the thioredoxin family, is a resident KDEL protein of the ER not of the intermediate compartment.
J. Cell Sci
107
, 2719
–2727
Hartmann
E.
, Görlich
D.
, Kostka
S.
, Otto
A.
, Kraft
R.
, Knespel
S.
, Burger
E.
, Rapoport
T.A.
, Prehn
S.
(1993
). A tetrameric complex of membrane proteins in the ER.
Eur. J. Biochem
214
, 375
–381
Hauri
H.-P.
, Schweizer
A.
(1992
). The ER-Golgi intermediate compartment.
Curr. Opin. Cell Biol
4
, 600
–608
Hinman
N.D.
, Phillips
A.H.
(1970
). Similarity and limited multiplicity of membrane proteins from rough and smooth ER.
Science
170
, 1222
–1223
Hortsch
M.
, Meyer
D.I.
(1985
). Immunochemical analysis of rough and smooth microsomes from rat liver: Segregation of docking protein in rough membranes.
Eur. J. Biochem
150
, 559
–569
Hortsch
M.
, Griffith
G.
, Meyer
D.I.
(1985
). Restriction of docking protein to the rough ER: Immunocytochemical localization in rat liver.
Eur. J. Cell Biol
38
, 271
–279
Hortsch
M.
, Avossa
D.
, Meyer
D.I.
(1986
). Characterization of secretory protein translocation: Ribosome-membrane interaction in ER.
J. Cell Biol
103
, 241
–253
Kreibich
G.
, Ulrich
B.L.
, Sabatini
D.D.
(1978
). Proteins of rough microsomal membranes related to ribosome binding. I. Identification of ribophorins I and II, membrane proteins characteristic of rough microsomes.
J. Cell Biol
77
, 464
–487
Krijnse-Locker
J.
, Ericsson
M.
, Rottier
P.J.M.
, Griffiths
G.
(1994
). Characterization of the budding compartment of mouse hepatitis virus: Evidence that transport from the rough ER to the Golgi complex requires only one vesicular transport step.
J. Cell Biol
124
, 55
–70
Laemmli
U. K.
(1970
). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227
, 680
–685
Lahtinen
U.
, Dahllöf
B.
, Saraste
J.
(1992
). Characterization of a 58 kDa cis-Golgi protein in pancreatic exocrine cells.
J. Cell Sci
103
, 321
–333
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 reveals an ER cycling pathway.
Cell
60
, 821
–836
Lotti
L.V.
, Torrissi
M.-R.
, Pascale
M.C.
, Bonatti
S.
(1992
). Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein to the Golgi complex.
J. Cell Biol
118
, 43
–50
Oprins
A.
, Duden
R.
, Kreis
T.E.
, Geuze
H.J.
, Slot
J.W.
(1993
). -COP localizes mainly to the cis-Golgi side in exocrine pancreas.
J. Cell Biol
121
, 49
–59
Palade
G.E.
(1975
). Intracellular aspects of the process of protein transport.
Science
189
, 347
–358
Pelham
H.R.B.
(1988
). Evidence that lumenal ER proteins are sorted from secreted proteins in a post-ER compartment.
EMBO J
7
, 913
–918
Plutner
H.
, Davidson
H.W.
, Saraste
J.
, Balch
W.E.
(1992
). Morphological analysis of protein transport from the ER to Golgi membranes in digitonin-permeabilized cells: Role of the p58 containing compartment.
J. Cell Biol
119
, 1097
–1116
Saraste
J.
, Kuismanen
J.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 Golgi transport.
J. Cell Sci
100
, 415
–430
Saraste
J.
, Kuismanen
J.E.
(1992
). Pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the Golgi complex.
Sem. Cell Biol
3
, 343
–355
Schindler
R.
, Itin
C.
, Zerial
M.
, Lottspeich
F.
, Hauri
H.-P.
(1993
). ERGIC-53, a membrane protein of the ER-Golgi intermediate compartment, carries an ER retention motif.
Eur. J. Cell Biol
61
, 1
–9
Schweizer
A.
, Fransen
J.A.M.
, Bächi
T.
, Ginsel
L.
, Hauri
H.-P.
(1988
). Identification, by a monoclonal antibody, of a 53 kDa protein associated with a tubular-vesicular compartment at the cis-side of the Golgi apparatus.
J. Cell Biol
108
, 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 the ER to the Golgi apparatus.
Eur. J. Cell Biol
53
, 185
–196
Schweizer
A.
, Ericsson
M.
, Bächi
T.
, Griffiths
G.
, Hauri
H.-P.
(1993
). Characterization of a novel 63 kDa membrane protein: Implications for the organization of the ER-to-Golgi pathway.
J. Cell Sci
104
, 671
–683
Schweizer
A.
, Rohrer
J.
, Jenö
P.
, Demaio
A.
, Buchman
T.G.
, Hauri
H.-P.
(1993
). A reversibly palmitoylated resident protein (p63) of an ER-Golgi intermediate compartment is related to a circulatory shock resuscitation protein.
J. Cell Sci
104
, 685
–694
Schweizer
A.
, Rohrer
J.
, Hauri
H.-P.
, Kornfeld
S.
(1994
). Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers.
J. Cell Biol
126
, 25
–39
Sharma
R.N.
, Behar-Bannelier
M.
, Rolleston
F.S.
, Murray
R.K.
(1978
). Electrophoretic studies on liver ER membrane polypeptides and their phosphorylation in vivo and in vitro.
J. Biol. Chem
253
, 2033
–2043
Slot
J.W.
, Geuze
H.J.
(1985
). A new method of preparing gold probes for multiple-labeling cytochemistry.
Eur. J. Cell Biol
38
, 87
–93
Sodeik
B.
, Doms
R.W.
, Ericsson
M.
, Hiller
G.
, Machamer
C.E.
, van't Hof
W.
, van Meer
G.
, Moss
B.
, Griffiths
G.
(1993
). Assembly of vaccinia virus: Role of the intermedoate compartment between the endoplasmic reticulum and the Golgi stacks.
J. Cell Biol
121
, 521
–541
Stieger
B.
, Matter
K.
, Bauer
B.
, Bucher
K.
, Hoechli
M.
, Hauri
H.-P.
(1988
). Disection of the asynchronous transport of intestinal microvillar hydrolases to the cell surface.
J. Cell Biol
106
, 1853
–1861
Tooze
S.A.
, Tooze
J.
, Warren
G.
(1988
). Site of addition of N-acetyl-galactosamine to the E1 glycoprotein of mouse hepatitis virus-A59.
J. Cell Biol
106
, 1475
–1487
Towbin
H.
, Staehelin
T.
, Gordon
J.
(1979
). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocelluose sheets: Procedure and applications.
Proc. Nat. Acad. Sci. USA
70
, 4350
–4354
Venable
J.H.
, Cogeshall
R.
(1965
). A simple lead citrate stain for use in electron microscopy.
J. Cell Biol
25
, 407
–408
Vogel
F.
, Hartmann
E.
, Görlich
D.
, Rapoport
T.A.
(1990
). Segregation of the signal sequence receptor protein in the rough ER membrane.
Eur. J. Cell Biol
53
, 197
–202
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