We have studied the localization of the small GTPase rab1p in different cell types using polyclonal antibodies prepared against the rab1A isoform of the protein. Immunofluorescence microscopy of normal rat kidney (NRK) and mouse myeloma cells showed the association of the protein with the Golgi complex and peripheral sites where it colocalized with p58, a pre- and cis-Golgi marker protein. Rab1p and p58 also had similar distributions in membrane fractions derived from rat pancreas microsomes. Both were concentrated in two intermediate density subfractions between the rough endoplasmic reticulum and trans-Golgi, whereas rab6p, previously localized to middle and trans-Golgi, was enriched in the light density trans-Golgi fraction. Immunoperoxidase electron microscopy of NRK and myeloma cells revealed the association of rab1p with 1–2 cisternae, vacuolar, and tubulovesicular membranes in the cis-Golgi region. The rab1p-specific staining typically covered the entire lateral surface of the cisternae but, in weakly stained cells, local labeling between closely opposed membranes could also be seen. The rab1p-positive pre-Golgi compartment had a predominantly tubulovesicular appearance in NRK cells whereas in myeloma cells it consisted of vacuoles surrounded by rab1p-positive vesicles and tubules of heterogeneous size. In both cell types the rough ER cisternae and the nuclear envelope contained negligible labeling and no continuities between these and the rab1p-positive membranes were observed. In addition, in myeloma cells the smooth ER subcompartment, containing endogenous retrovirus particles, was devoid of rab1p-labeling. These results indicate that the pre-Golgi (intermediate) compartment consists of different membrane domains and its morphology can vary considerably between different cell types. Further, they suggest that the recruitment of rab1p to membranes occurs predominantly in a post-ER location and that the protein functions in targeting/fusion events within the pre- and cis-Golgi membranes.

REFERENCES

Anthony
C.
,
Cibert
C.
,
Geraud
G.
,
Santa Maria
A.
,
Maro
B.
,
Mayau
V.
,
Goud
B.
(
1992
).
The small GTP-binding protein rab6p is distributed from medial Golgi to the trans -Golgi network as determined by a confocal microscopic approach.
J. Cell Sci
103
,
785
796
Bacon
R.
,
Salminen
A.
,
Ruohola
H.
,
Novick
P.
,
Ferro-Novick
S.
(
1989
).
The GTP-binding protein YPT1 is required for transport in vitro: the Golgi apparatus is defective in YPT1 mutants.
J. Cell Biol
109
,
1015
1022
Bailly
E.
,
McCaffrey
M.
,
Touchot
N.
,
Zahraoui
A.
,
Goud
B.
,
Bornens
M.
(
1991
).
Phosphorylation of two small GTP-binding proteins of the rab family by p34cdc2.
Nature
350
,
715
718
Baker
D.
,
Wuestehube
L.
,
Schekman
R.
,
Botstein
D.
,
Segev
N.
(
1990
).
GTP-binding Ypt1 protein and Ca2+ function independently in a cell-free protein transport reaction.
Proc. Nat. Acad. Sci. USA
87
,
355
359
Balch
W. E.
,
McCaffery
J. M.
,
Plutner
H.
,
Farquhar
M. G.
(
1994
).
Vesicular stomatitis virus glycoprotein is sorted and concentrated during export from the endoplasmic reticulum.
Cell
76
,
841
852
Barlowe
C.
,
Orci
L.
,
Yeung
T.
,
Hosobuchi
M.
,
Hamamoto
S.
,
Salama
N.
,
Rexach
M.
,
Ravazzola
M.
,
Amherdt
M.
,
Schekman
R.
(
1994
).
COPII: a membrane coat formed by sec proteins that drive vesicle budding from the endoplasmic reticulum.
Cell
77
,
895
907
Bennett
M. K.
,
Scheller
R. H.
(
1993
).
The molecular machinery for secretion is conserved from yeast to neurons.
Proc. Nat. Acad. Sci. USA
90
,
2559
2563
Bonatti
S.
,
Torrisi
M.-R.
(
1993
).
The intermediate compartment betweenendoplasmic reticulum and Golgi complex in mammalian cells.
Subcell. Biochem
21
,
121
142
Bourne
H. R.
(
1988
).
Do GTPases direct membrane traffic in secretion?.
Cell
53
,
669
671
Brennwald
P.
,
Novick
P.
(
1993
).
Interactions of three domains distinquishing the ras-related GTP-binding proteins ypt1 and sec4.
Nature
362
,
560
563
Bucci
C.
,
Parton.
R. G.
,
Mather
I. M.
,
Stunnenberg
H.
,
Simons
K.
,
Hoflack
B.
,
Zerial
M.
(
1992
).
The small GTPase rab5 functions as a regulatory factor in the early endocytic pathway.
Cell
70
,
715
728
Chavrier
P.
,
Parton
R. G.
,
Hauri
H.-P.
,
Simons
K.
,
Zerial
M.
(
1990
).
Localization of low molecular weight GTP-binding proteins to exotic and endocytic compartments.
Cell
62
,
317
329
Chavrier
P.
,
Vingron
M.
,
Sander
C.
,
Simons
K.
,
Zerial
M.
(
1990
).
Molecular cloning of YPT1/SEC4-related cDNAs from an epithelial cell line.
Mol. Cell. Biol
10
,
6578
6585
Cooper
M. S.
,
Cornell-Bell
A. H.
,
Chernjavsky
A.
,
Dani
J. W.
,
Smith
S. J.
(
1990
).
Tubulovesicular processes emerge from trans-Golgi cisternae, extend along microtubules, and interlink adjacent trans-Golgi elements into a reticulum.
Cell
61
,
135
145
Cosson
P.
,
Letourneur
F.
(
1994
).
Coatomer interaction with di-lysine endoplasmic reticulum retention motifs.
Science
263
,
1629
1631
Davidson
H. W.
,
Balch
W. E.
(
1993
).
Differential inhibition of multiple vesicular transport steps between the endoplasmic reticulum and the trans-Golgi network.
J. Biol. Chem
268
,
4216
4226
Deretic
D.
,
Papermaster
D. S.
(
1993
).
Rab6 is associated with a compartment that transports rhodopsin from the trans-Golgi to the site of rod outer disk formation in frog retinal photoreceptors.
J. Cell Sci
106
,
803
813
Donaldson
J. G.
,
Lippincott-Schwarz
J.
,
Bloom
G. S.
,
Kreis
T. E.
,
Klausner
R. D.
(
1990
).
Dissociation of a 110-kD peripheral membrane protein from the Golgi apparatus is an early event in brefeldin A action.
J. Cell Biol
111
,
2295
2306
Duden
R.
,
Griffiths
G.
,
Frank
R.
,
Argos
P.
,
Kreis
T. E.
(
1991
).
-COP, a 110 kd protein associated with non-clathrin coated vesicles and the Golgi complex, shows homology to -adaptin.
Cell
64
,
649
665
Dunn
B.
,
Stearns
T.
,
Botstein
D.
(
1993
).
Specificity domains distinquish the ras-related GTPases ypt1 and sec4.
Nature
362
,
563
565
Ferro-Novick
S.
,
Novick
P.
(
1993
).
The role of GTP-binding proteins in transport along the exocytic pathway.
Annu. Rev. Cell Biol
9
,
575
599
Goud
B.
,
Zahraoui
A.
,
Tavitian
A.
,
Saraste
J.
(
1990
).
Small GTP-binding protein associated with Golgi cisternae.
Nature
345
,
553
556
Goud
B.
,
McGaffrey
M.
(
1991
).
Small GTP-binding proteins and their role in transport.
Curr. Opin. Cell Biol
3
,
626
633
Haubruck
H.
,
Prange
R.
,
Vorgias
C.
,
Gallwitz
D.
(
1989
).
The ras-related mouse ypt1 protein can functionally replace the YPT1 gene product in yeast.
EMBO J
8
,
1427
1432
Hauri
H.-P.
,
Schweizer
A.
(
1992
).
The endoplasmic reticulum-Golgi intermediate compartment.
Curr. Opin. Cell Biol
4
,
600
608
Hendricks
L. C.
,
McCaffery
M.
,
Palade
G. E.
,
Farquhar
M. G.
(
1993
).
Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing-COP in pancreatic acinar cells.
Mol. Biol. Cell
4
,
413
424
Hendriks
R. J. M.
,
Fuller
S. D.
(
1994
).
Compartments of the early secretory pathway.
Subcell. Biochem
22
,
101
149
Hobman
T. C.
,
Woodward
L.
,
Farquhar
M. G.
(
1992
).
The rubella virus E1 glyco-protein is arrested in a novel post-ER, pre-Golgi compartment.
J. Cell Biol
118
,
795
811
Huber
L. A.
,
Pimplikar
S.
,
Parton
R.
,
Virta
H.
,
Zerial
M.
,
Simons
K.
(
1993
).
Rab8, a small GTPase involved in vesicular traffic between the TGN and the baso-lateral plasma membrane.
J. Cell Biol
123
,
34
45
Huber
L. A.
,
Ullrich
O.
,
Takai
Y.
,
Lutcke
A.
,
Dupree
P.
,
Olkkonen
V.
,
Virta
H.
,
de Hoop
M. J.
,
Alexandrov
K.
,
Peter
M.
,
Zerial
M.
,
Simons
K.
(
1994
).
Mapping of ras-related GTP-binding proteins by GTP-overlay and 2D-gel analysis.
Proc. Nat. Acad. Sci. USA
91
,
7874
7878
Krijnse-Locker
J.
,
Ericsson
M.
,
Rottier
P.
,
Griffiths
G.
(
1994
).
Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Golgi complex requires only one vesicular transport step.
J. Cell Biol
124
,
55
70
Kuismanen
E.
,
Saraste
J.
(
1989
).
Low temperature-induced transport blocks as tools to manipulate membrane traffic.
Meth. Cell Biol
32
,
257
274
Lahtinen
U.
,
Dahllof
B.
,
Saraste
J.
(
1992
).
Characterization of a 58 kDa cis-Golgi protein in pancreatic exocrine cells.
J. Cell Sci
103
,
321
333
Lledo
P.-M.
,
Vernier
P.
,
Vincent
J.-D.
,
Mason
W.T.
,
Zorec
R.
(
1993
).
Inhibition of rab3B expression attenuates Ca2+ -dependent exocytosis in rat anterior pituitary cells.
Nature
364
,
540
544
Lombardi
D.
,
Soldati
T.
,
Riederer
M. A.
,
Goda
Y.
,
Zerial
M.
,
Pfeffer
S. R.
(
1993
).
Rab9 functions in transport between late endosomes and the trans-Golgi network.
EMBO J
12
,
677
682
Maridonneau-Parini
I.
,
Yang
C.
,
Bornens
M.
,
Goud
B.
(
1991
).
Increase in the expression of a family of small guanosine triphosphate-binding proteins, rab proteins, during induced phagocyte differentiation.
J. Clin. Invest
87
,
901
907
Martinez
O.
,
Schmidt
A.
,
Salamero
J.
,
Hoflack
B.
,
Roa
M.
,
Goud
B.
(
1994
).
The small GTP-binding protein rab6p functions in intra-Golgi transport.
J. Cell Biol
127
,
1575
1588
Nuoffer
C.
,
Davidson
H. W.
,
Matteson
J.
,
Meinkoth
J.
,
Balch
W. E.
(
1994
).
A GDP-bound form of rab1 inhibits protein export from the endoplasmic reticulum and transport between Golgi compartments.
J. Cell Biol
125
,
225
237
Oprins
A.
,
Duden
R.
,
Kreis
T. E.
,
Geuze
H.
,
Slot
J. W.
(
1993
).
-COP localizes mainly to the cis-Golgi side in exocrine pancreas.
J. Cell Biol
121
,
49
59
Ottosen
P. D.
,
Courtoy
P. J.
,
Farquhar
M. G.
(
1980
).
Pathways followed by membrane recovered from the surface of plasma cells and myeloma cells.
J. Exp. Med
152
,
1
19
Pelham
H. R. B.
(
1989
).
Control of protein exit from the endoplasmic reticulum.
Annu. Rev. Cell Biol
5
,
1
23
Pepperkok
R.
,
Scheel
J.
,
Horstmann
H.
,
Hauri
H.-P.
,
Griffiths
G.
,
Kreis
T. E.
(
1993
).
-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo.
Cell
74
,
71
82
Peränen
J.
(
1992
).
Rapid affinity-purification and biotinylation of antibodies.
Biotechniques
13
,
546
–.
Peter
F.
,
Plutner
H.
,
Zhu
H.
,
Kreis
T. E.
,
Balch
W. E.
(
1993
).
-COP is essential for transport of protein from the endoplasmic reticulum to the Golgi in vitro.
J. Cell Biol
122
,
1155
1167
Pfeffer
S.
(
1994
).
Rab GTPases: master regulators of membrane trafficking.
Curr. Opin. Cell Biol
6
,
522
526
Pind
S. N.
,
Nuoffer
C.
,
McCaffery
M. J.
,
Plutner
H.
,
Davidson
H. W.
,
Farquhar
M. G.
,
Balch
W. E.
(
1994
).
Rab1 and Ca2+ are required for the fusion of carrier vesicles mediating endoplasmic reticulum to Golgi transport.
J. Cell Biol
125
,
239
252
Plutner
H.
,
Cox
A. D.
,
Pind
S.
,
Khosravi-Far
R.
,
Bourne
J. R.
,
Schwaninger
R.
,
Der
C. J.
,
Balch
W. E.
(
1991
).
Rab1B regulates vesicular transport between the endoplasmic reticulum and successive Golgi compartments.
J. Cell Biol
115
,
31
43
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
Pryer
N. K.
,
Wuestehube
L. J.
,
Schekman
R.
(
1992
).
Vesicle-mediated protein sorting.
Annu. Rev. Biochem
61
,
471
516
Rexach
M. F.
,
Schekman
R.
(
1991
).
Distinct biochemical requirements for the budding, targeting, and fusion of ER-derived transport vesicles.
J. Cell Biol
114
,
219
229
Roth
J.
,
Taatjes
D. J.
,
Lucocq
J. M.
,
Weinstein
J.
,
Paulson
J. C.
(
1985
).
Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation.
Cell
43
,
287
295
Rothman
J. E.
,
Orci
L.
(
1992
).
Molecular dissection of the secretory pathway.
Nature
355
,
409
415
Salama
N.
,
Yeung
T.
,
Schekman
R. W.
(
1993
).
The sec13p complex and reconstitution of vesicle budding from the ER with purified cytosolic proteins.
EMBO J
12
,
4073
4082
Salminen
A.
,
Novick
P.
(
1987
).
A ras-like protein is required for a post-Golgi event in yeast secretion.
Cell
49
,
527
538
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.
,
Palade
G. E.
,
Farquhar
M. G.
(
1986
).
Temperature-sensitive steps in the transport of secretory proteins through the Golgi complex in pancreatic exocrine cells.
Proc. Nat. Acad. Sci. USA
83
,
6425
6429
Saraste
J.
,
Palade
G. E.
,
Farquhar
M. G.
(
1987
).
Antibodies to rat pancreas Golgi subfractions: Identification of a 58-kD cis-Golgi protein.
J. Cell Biol
105
,
2021
2030
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
E.
(
1992
).
Pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the Golgi complex.
Semin. Cell Biol
3
,
343
355
Schwaninger
R.
,
Plutner
H.
,
Bokoch
G. M.
,
Balch
W. E.
(
1992
).
Multiple GTP-binding proteins regulate vesicular transport from the ER to Golgi membranes.
J. Cell Biol
119
,
1077
1096
Segev
N.
,
Mulholland
J.
,
Botstein
D.
(
1988
).
The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery.
Cell
52
,
915
924
Segev
N.
(
1991
).
Mediation of the attachment or fusion step in vesicular transport by the GTP-binding ypt1 protein.
Science
252
,
1553
1556
Söllner
T.
,
Rothman
J. E.
(
1994
).
Neurotransmission: harnessing fusion machinery at the synapse.
Trends Neurosci
17
,
344
348
Søgaard
M.
,
Tani
K.
,
Ye
R. R.
,
Geromanos
S.
,
Tempst
P.
,
Kirchhausen
T.
,
Rothman
J. E.
,
Söllner
T.
(
1994
).
A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles.
Cell
78
,
937
948
Takatsuki
A.
,
Tamura
G.
(
1985
).
Brefeldin A, a specific inhibitor of intracellular translocation of vesicular stomatitis virus G-protein: Intracellular accumulation of high-mannose type G-protein and inhibition of its surface expression.
Agric. Biol. Chem
49
,
899
902
Tisdale
E. J.
,
Bourne
J. R.
,
Khosravi-Far
R.
,
Der
C. J.
,
Balch
W. E.
(
1992
).
GTP-binding mutants of rab1 and rab2 are potent inhibitors of vesicular transport from the endoplasmic reticulum to the Golgi complex.
J. Cell Biol
119
,
749
761
Tixier-Vidal
A.
,
Barret
A.
,
Picart
R.
,
Mayau
V.
,
Vogt
D.
,
Wiedenmann
B.
,
Goud
B.
(
1993
).
The small GTP-binding protein, Rab6p, is associated with both Golgi and post-Golgi synaptophysin-containing membranes during synaptogenesis of hypothalamic neurons in culture.
J. Cell Sci
105
,
935
947
Touchot
N.
,
Chardin
P.
,
Tavitian
A.
(
1987
).
Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library.
Proc. Nat. Acad. Sci. USA
84
,
8210
8214
van der Sluijs
P.
,
Hull
M.
,
Webster
P.
,
Male
P.
,
Goud
B.
,
Mellman
I.
(
1992
).
The small GTP-binding ptotein rab4 controls an early sorting event in the endocytic pathway.
Cell
70
,
729
740
Vielh
E.
,
Touchot
N.
,
Zahraoui
A.
,
Tavitian
A.
(
1989
).
Nucleotide sequence of a rat cDNA: Rab1B, encoding a Rab1-YPT related protein.
Nucl. Acids Res
17
,
1770
–.
Warren
G.
(
1987
).
Signals and salvage sequences.
Nature
327
,
17
18
Zahraoui
A.
,
Touchot
N.
,
Chardin
P.
,
Tavitian
A.
(
1989
).
The human rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion.
J. Biol. Chem
264
,
12394
12401
Zerial
M.
,
Stenmark
H.
(
1993
).
Rab GTPases in vesicular transport.
Curr. Opin. Cell Biol
5
,
613
620
This content is only available via PDF.