Rab6 protein belongs to the Sec4/Ypt/rab subfamily of small GTP-binding proteins involved in intracellular membrane trafficking in yeast and mammalian cells. Its localization both in medial and trans-Golgi network prompted us to study the effects of brefeldin A (BFA) on rab6p redistribution. By two techniques, indirect immunofluorescence and cell fractionation, we investigated the fate of rab6p and compared it to other Golgi or trans-Golgi network markers in BHK-21 and NIH-3T3 cells. BFA, at 5 micrograms/ml, induced redistribution of rab6p according to a biphasic process: during the first 10–15 minutes, tubulo-vesicular structures--colabelled with a bona fide medial Golgi marker called CTR 433--were observed; these structures were then replaced by punctate diffuse staining, which was stable for up to 3 hours. The 110 kDa peripheral membrane protein beta-COP was released much more rapidly from the Golgi membranes, whereas the trans-Golgi network marker TGN 38 relocated to the microtubule organizing center. The kinetics of reversion of BFA action on these antigens was also followed by immunofluorescence. Consistent with these results, rab6 antigen, originally found as 40% in the cytosolic versus 60% in the particulate (P 150,000 g) fraction, became almost entirely cytosolic; moreover, it partitioned in the aqueous phase of Triton X-114 whereas the membrane fraction was detergent-soluble. Rab6p did not become part of the coatomers after its BFA-induced release from Golgi structures. Three requirements seemed to be necessary for such a release: integrity of the microtubules, presence of energy, and a hypothetical trimeric G protein, as revealed by the respective roles of nocodazole, ATP depletion, and sensitivity to aluminium fluoride. Finally, we have shown that BFA does not prevent attachment of newly synthesized rab6p to membranes.

REFERENCES

Allan
V. J.
,
Kreis
T. E.
(
1986
).
A microtubule-binding protein associated with membranes of the Golgi apparatus.
J. Cell Biol
103
,
2229
2239
Antony
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
Bailly
E.
,
McCaffrey
M.
,
Touchot
N.
,
Zahraoui
A.
,
Goud
B.
,
Bornens
M.
(
1991
).
Phosphorylation of two small GTP-binding proteins of the ras family by p34cdc2.
Nature
350
,
715
718
Bigay
J.
,
Deterre
P.
,
Pfister
C.
,
Chabre
M.
(
1987
).
Fluoride complexes of aluminium or beryllium act on G-proteins as reversibly bound analogues of the g phosphate of GTP.
EMBO J
6
,
2907
2913
Bordier
C.
(
1981
).
Phase separation of integral membrane proteins in Triton-X-114.
J. Biol. Chem
256
,
1604
1607
Bourne
H. R.
(
1988
).
Do GTPases direct membrane traffic in secretion?.
Cell
53
,
669
671
Bourne
H. R.
,
Sanders
D. A.
,
McCormick
F.
(
1991
).
The GTPase superfamily: conserved structure and molecular mechanism.
Nature
349
,
117
127
Bradford
M. M.
(
1976
).
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem
72
,
248
254
Bucci
C.
,
Parton
R. G.
,
Mather
I. H.
,
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 exocytic 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 epithelium cell line.
Mol. Cell. Biol
10
,
6578
6585
Doms
R. W.
,
Russ
G.
,
Yewdel
J. W.
(
1989
).
Brefeldin A redistributesresident and itinerant Golgi proteins to the endoplasmic reticulum.
J. Cell Biol
109
,
61
72
Donaldson
J. G.
,
Cassel
D.
,
Kahn
R. A.
,
Klausner
R. D.
(
1992
).
ADP-ribosylation factor, a small GTP-binding protein is required for binding of the coatomer protein-COP to Golgi membranes.
Proc. Nat. Acad. Sci. USA
89
,
6408
6412
Donaldson
J. G.
,
Finazzi
D.
,
Klausner
R. D.
(
1992
).
Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein.
Nature
360
,
350
352
Donaldson
J. G.
,
Kahn
R. A.
,
Lippincott-Schwartz
J.
,
Klausner
R. D.
(
1991
).
Binding of ARF and-COP to Golgi membranes: possible regulation by a trimeric G protein.
Science
254
,
1197
1199
Donaldson
J. G.
,
Lippincott-Schwartz
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
Donaldson
J. G.
,
Lippincott-Schwartz
J.
,
Klausner
R. D.
(
1991
).
Guanine nucleotides modulate the effects of Brefeldin A in semipermeable cells: regulation of the association of a 110-kD peripheral membrane protein with the Golgi apparatus.
J. Cell Biol
112
,
579
588
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
Ercolani
L.
,
Stow
J. L.
,
Boyle
J. F.
,
Holtzman
E. J.
,
Lin
H.
,
Grove
J. R.
,
Ausiello
D. A.
(
1990
).
Membrane localization of the pertussis toxin-sensitive G-protein subunitsi-2 and i-3 and expression of a metallothionein-i-2 fusion gene in LLC-PK1 cells.
Proc. Nat. Acad. Sci. USA
87
,
4637
4639
Fischer Von Mollard
G.
,
Sudhof
T.
,
Jahn
R.
(
1991
).
A small GTP-binding protein dissociates from synaptic vesicles during exocytosis.
Nature
349
,
79
81
Fujiwara
T.
,
Oda
K.
,
Yokota
S.
,
Takatsuki
A.
,
Ikehara
Y.
(
1988
).
Brefeldin A causes disassembly of the Golgi complex and accumulation of secretory proteins in the ER.
J. Biol. Chem
263
,
18545
18552
Gorvel
J.-P.
,
Chavrier
P.
,
Zerial
M.
,
Gruenberg
J.
(
1991
).
Rab5 controls early endosome fusion in vitro.
Cell
64
,
915
925
Goud
B.
,
McCaffrey
M.
(
1991
).
Small GTP-binding proteins and their role in transport.
Curr. Opin. Cell Biol
3
,
626
633
Goud
B.
,
Zahraoui
A.
,
Tavitian
A.
,
Saraste
J.
(
1990
).
Small GTP-binding protein associated with Golgi cisternae.
Nature
345
,
553
556
Hancock
J. F.
,
Cadwallader
K.
,
Marshall
C. J.
(
1991
).
Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras (B).
EMBO J
10
,
641
646
Helms
J. B.
,
Rothman
J.
(
1992
).
Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF.
Nature
360
,
352
354
Jasmin
B. J.
,
Cartaud
J.
,
Bornens
M.
,
Changeux
J.-P.
(
1989
).
Golgi apparatus in chick skeletal muscle: changes in its distribution during end plate development and after denervation.
Proc. Nat. Acad. Sci. USA
86
,
7218
7222
Kahn
R. A.
(
1991
).
Fluoride is not an activator of the smaller (20-25 kDa) GTP-binding proteins.
J. Biol. Chem
266
,
15595
15597
Khosravi-Far
R.
,
Lutz
R. J.
,
Cox
A. D.
,
Conroy
L.
,
Bourne
J. R.
,
Sinensky
M.
,
Balch
W. E.
,
Buss
J. E.
,
Der
C. J.
(
1991
).
Isoprenoid modification of rab protein terminating in CC or CXC motifs.
Proc. Nat. Acad. Sci. USA
88
,
6264
6268
Kinsella
B. T.
,
Maltese
W. A.
(
1992
).
Rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.
J. Biol. Chem
267
,
3940
3945
Klausner
R. D.
,
Donaldson
J. G.
,
Lippincott-Schwartz
J.
(
1992
).
Brefeldin A: insights into the control of membrane traffic and organelle structure.
J. Cell Biol
116
,
1071
1080
Leyte
A.
,
Barr
F. A.
,
Kehlenbach
R. H.
,
Huttner
W. B.
(
1992
).
Multiple trimeric G-proteins on the trans -Golgi network exert stimulatory and inhibitory effects on secretory vesicle formation.
EMBO J
11
,
4795
4804
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 an ER recycling pathway.
Cell
60
,
821
836
Lippincott-Schwartz
J.
,
Glickman
J.
,
Donaldson
J. G.
,
Robbins
J.
,
Kreis
T. E.
,
Seamon
K. B.
,
Sheetz
M. P.
,
Klausner
R. D.
(
1991
).
Forskolin inhibits and reverses the effects of Brefeldin A on Golgi802morphology by a cAMP-independent mechanism.
J. Cell Biol
112
,
567
577
Lippincott-Schwartz
J.
,
Yuan
L. C.
,
Bonifacino
J. S.
,
Klausner
R. D.
(
1989
).
Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: evidence for membrane cycling from the Golgi to the ER.
Cell
56
,
801
813
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
Louvard
D.
,
Reggio
H.
,
Warren
G.
(
1982
).
Antibodies to the Golgi complex and the rough endoplasmic reticulum.
J. Cell Biol
92
,
92
107
Lutcke
A.
,
Jansson
S.
,
Parton
R. G.
,
Chavrier
P.
,
Valencia
A.
,
Huber
L. A.
,
Lehtonen
E.
,
Zerial
M.
(
1993
).
Rab17, a novel small GTPase, is specific for epithelial cells and is induced during cell polarization.
J. Cell Biol
121
,
553
564
Luzio
J. P.
,
Brake
B.
,
Banting
G.
,
Howell
K. E.
,
Braghetta
P.
,
Stanley
K. K.
(
1990
).
Identification, sequencing and expression of an integral membrane protein of the trans -Golgi network (TGN 38).
Biochem. J
270
,
97
102
Maridonneau-Parini
I.
,
Yang
C.-Z.
,
Bornens
M.
,
Goud
B.
(
1991
).
Increase in the expression of a family of small GTP-binding proteins, rab proteins, during induced phagocyte differentiation.
J. Clin. Invest
87
,
901
907
Matsui
Y.
,
Kikuchi
A.
,
Araki
S.
,
Hata
Y.
,
Kondo
J.
,
Teranishi
Y.
,
Takai
Y.
(
1990
).
Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.
Mol. Cell. Biol
10
,
4116
4122
Mellman
I.
,
Simons
K.
(
1992
).
The Golgi complex: in vitro veritas?.
Cell
68
,
829
840
Miller
S. G.
,
Carnell
L.
,
Moore
H.-P. H.
(
1992
).
Post-Golgi membrane traffic: Brefeldin A inhibits export from distal Golgi compartments to the cell surface but not recycling.
J. Cell Biol
118
,
267
283
Misumi
Y.
,
Miki
K.
,
Takatsuki
A.
,
Tamura
G.
,
Ikehara
Y.
(
1986
).
Novel blockade by Brefeldin A of intracellular transport of secretory proteins in cultured rat hepatocytes.
J. Biol. Chem
261
,
11398
11403
Narula
N.
,
McMorrow
I.
,
Plopper
G.
,
Doherty
J.
,
Matlin
K. S.
,
Burke
B.
,
Stow
J. L.
(
1992
).
Identification of a 200 kD, Brefeldin-sensitive protein on Golgi membranes.
J. Cell Biol
117
,
27
38
Oberhauser
A. F.
,
Monck
J. R.
,
Balch
W. E.
,
Fernandez
J. M.
(
1992
).
Exocytotic fusion is activated by Rab3A peptides.
Nature
360
,
270
273
Orci
L.
,
Glick
B. S.
,
Rothman
J. E.
(
1986
).
A new type of coated vesicular carrier that appears not to contain clathrin: its possible role in protein transport within the Golgi stack.
Cell
46
,
171
184
Pimplikar
S. W.
,
Simons
K.
(
1993
).
Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein.
Nature
362
,
456
458
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
Reaves
B.
,
Banting
G.
(
1992
).
Perturbation of the morphology of the trans -Golgi network following Brefeldin A treatment: redistribution of a TGN-specific integral membrane protein, TGN 38.
J. Cell Biol
116
,
85
94
Roa
M.
,
Boquet
P.
(
1985
).
Interaction of tetanus toxin with lipid vesicles at low pH. Protection of specific polypeptides against proteolysis.
J. Biol. Chem
260
,
6827
6835
Robinson
M. S.
,
Kreis
T. E.
(
1992
).
Recruitment of coat proteins onto Golgi membranes in intact and permeabilized cells: effects of Brefeldin A and G protein activators.
Cell
69
,
129
138
Rothman
J. E.
,
Orci
L.
(
1992
).
Molecular dissection of the secretory pathway.
Nature
355
,
409
415
Sager
P. R.
,
Brown
P. A.
,
Berlin
R. D.
(
1984
).
Analysis of transferrin recycling in mitotic and interface HeLa cells by quantitative fluorescence microscopy.
Cell
39
,
275
282
Sasaki
T.
,
Kikuchi
A.
,
Araki
S.
,
Hata
Y.
,
Isomura
M.
,
Kuroda
S.
,
Takai
Y.
(
1990
).
Purification and characterization from bovine braincytosol of a protein that inhibits the dissociation of GDP form and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein.
J. Biol. Chem
265
,
2333
2337
Seabra
M. C.
,
Brown
M. S.
,
Slaughter
C. A.
,
Sudhof
T. C.
,
Goldstein
J. L.
(
1992
).
Purification of component A of rab geranyl-geranyl transferase: possible identity with the choroideremia gene product.
Cell
70
,
1049
1057
Seabra
M. C.
,
Goldstein
J. L.
,
Sudhof
T. C.
,
Brown
M. S.
(
1992
).
Rab geranyl-geranyl transferase: a multisubunit enzyme that prenylates GTP-binding proteins terminating in Cys-X-Cys or Cys-Cys.
J. Biol. Chem
267
,
14497
14503
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
Serafini
T.
,
Stenbeck
G.
,
Brecht
A.
,
Lottspeich
F.
,
Orci
L.
,
Rothman
J. E.
,
Wieland
F. T.
(
1991
).
A coat subunit of Golgi derived non-clathrin-coated vesicle with homology to the clathrin-coated vesicle coat protein-adaptin.
Nature
349
,
215
220
Sternweis
P. C.
,
Gilman
A. G.
(
1982
).
Aluminium: a requirement for activation of the regulatory component of adenylate cyclase by fluoride.
Proc. Nat. Acad. Sci. USA
79
,
4888
4891
Stow
J. L.
,
Bruno de Almeida
J.
,
Narula
N.
,
Holtzman
E. J.
,
Ercolani
L.
,
Ausiello
D. A.
(
1991
).
A heterotrimeric G protein, Gi-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epiphelial cells.
J. Cell Biol
114
,
1113
1124
Takai
Y.
,
Kaibuchi
K.
,
Kaibuchi
A.
,
Kawata
M.
(
1992
).
Small GTP-binding proteins.
Int. Rev. Cytol
133
,
187
230
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
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
Ueda
T.
,
Takeyama
Y.
,
Ohmori
T.
,
Ohyanagi
H.
,
Saitoh
Y.
,
Takai
Y.
(
1991
).
Purification and characterization from rat liver cytosol of a GDP dissociation inhibitor (GDI) for liver 24 K G, a ras p21-like GTP-binding protein, with properties similar to those of smg p25A GDI.
Biochemistry
30
,
909
917
Van der Sluijs
P.
,
Hull
M.
,
Huber
L. A.
,
Goud
B.
,
Mellman
I.
(
1992
).
Reversible phosphorylation-dephosphorylation determines the localization of rab4 during the cell cycle.
EMBO J
11
,
4379
4389
Van der Sluijs
P.
,
Hull
M.
,
Zahraoui
A.
,
Tavitian
A.
,
Goud
B.
,
Mellman
I.
(
1991
).
The small GTP-binding protein rab4 is associated with early endosomes.
Proc. Nat. Acad. Sci. USA
88
,
6313
6317
Walworth
N. C.
,
Goud
B.
,
Kabcenell
A. K.
,
Novick
P. J.
(
1989
).
Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic.
EMBO J
8
,
1685
1693
Warren
G.
,
Davoust
J.
,
Cockroft
A.
(
1984
).
Recycling of transferrin receptors in A431 cells is inhibited during mitosis.
EMBO J
3
,
2217
2225
Waters
M. G.
,
Serafini
T.
,
Rothman
J. E.
(
1991
).
‘Coatomer’: a cytosolic protein complex containing subunits of non-clathrin-coated Golgi transport vesicles.
Nature
349
,
248
251
Wood
S. A.
,
Park
J. E.
,
Brown
W. J.
(
1991
).
Brefeldin A causes a microtubule-mediated fusion of the trans -Golgi network and early endosomes.
Cell
67
,
591
600
Yang
C.-Z.
,
Mayau
V.
,
Godeau
F.
,
Goud
B.
(
1992
).
Characterization of the unprocessed and processed forms of rab6 expressed in baculovirus/insect cell systems.
Biochem. Biophys. Res. Commun
182
,
1499
1505
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
This content is only available via PDF.