It has been shown previously that whilst the rat type I integral membrane protein TGN38 (ratTGN38) is predominantly localised to the trans-Golgi network this protein does reach the cell surface from where it is internalised and delivered back to the trans-Golgi network. This protein thus provides a suitable tool for the investigation of trafficking pathways between the trans-Golgi network and the cell surface and back again. The human orthologue of ratTGN38, humTGN46, behaves in a similar fashion. These proteins are internalised from the cell surface via clathrin mediated endocytosis, a process which is dependent upon the GTPase activity of dynamin. We thus reasoned that humTGN46 would accumulate at the surface of cells rendered defective in clathrin mediated endocytosis by virtue of the fact that they express a GTPase defective mutant of dynamin I. It did not. In fact, expression of a dominant negative GTPase defective mutant of dynamin I had no detectable effect on the steady state distribution of humTGN46. One explanation for this observation is that humTGN46 does not travel directly to the cell surface from the trans-Golgi network. Further studies on cells expressing the dominant negative GTPase defective mutant of dynamin I indicate that the major recycling pathway for humTGN46 is in fact between the trans-Golgi network and the early endosome.

REFERENCES

Bleil
J. D.
,
Bretscher
M. S.
(
1982
).
Transferrin receptor and its recycling in HeLa-cells.
EMBO J
1
,
351
355
Bos
K.
,
Wraight
C.
,
Stanley
K. K.
(
1993
).
TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain.
EMBO J
12
,
2219
2228
Clague
M. J.
,
Urbe
S.
,
Aniento
F.
,
Gruenberg
J.
(
1994
).
Vacuolar ATPase activity is required for endosomal carrier vesicle formation.
J. Biol. Chem
269
,
21
24
Damke
H.
,
Baba
T.
,
Warnock
D. E.
,
Schmid
S. L.
(
1994
).
Induction of mutant dynamin specifically blocks endocytic coated vesicle formation.
J. Cell Biol
127
,
915
934
Damke
H.
,
Gossen
M.
,
Freundlieb
S.
,
Bujard
H.
,
Schmid
S. L.
(
1995
).
Tightly regulated and inducible expression of dominant interfering dynamin mutant in stably transformed Hela-cells.
Meth. Enzymol
257
,
209
220
Futter
C. E.
,
Connolly
C. N.
,
Cutler
D. F.
,
Hopkins
C. R.
(
1995
).
Newly synthesized transferrin receptors can be detected in the endosome before they appear on the cell-surface.
J. Biol. Chem
270
,
10999
11003
Girotti
M.
,
Banting
G.
(
1996
).
TGN38-green fluorescent protein hybrid proteins expressed in stably transfected eukaryotic cells provide a tool for the real-time, in vivo study of membrane traffic pathways and suggest a possible role for ratTGN38.
J. Cell Sci
109
,
2915
2926
Gossen
M.
,
Bujard
H.
(
1992
).
Tight control of gene expression in mammalian cells by tetracycline responsive promoters.
Proc. Nat. Acad. Sci. USA
89
,
5547
5551
Herskowits
J. S.
,
Burgess
C. C.
,
Obar
R. A.
,
Vallee
R. B.
(
1993
).
Effects of mutant rat dynamin on endocytosis.
J. Cell Biol
122
,
565
578
Hinshaw
J. E.
,
Schmid
S. L.
(
1995
).
Dynamin self-assembles into rings suggesting a mechanism for coated vesicle budding.
Nature
374
,
190
192
Hopkins
C. R.
(
1983
).
Intracellular routing of transferrin and transferrin receptors in epidermoid carcinoma A431-cells.
Cell
35
,
321
330
Hopkins
C. R.
,
Trowbridge
I. S.
(
1983
).
Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431-cells.
J. Cell Biol
97
,
508
521
Humphrey
J. S.
,
Peters
P. J.
,
Yuan
L. C.
,
Bonifacino
J. S.
(
1993
).
Localization of TGN38 to the trans-Golgi network- involvement of a cytoplasmic tyrosine-containing sequence.
J. Cell Biol
120
,
1123
1135
Jones
S. M.
,
Crosby
J. R.
,
Salamero
J.
,
Howell
K. E.
(
1993
).
A cytosolic complex of p62 and rab6 associates with TGN38/41 and is involved in budding of exocytic vesicles from the trans-Golgi network.
J. Cell Biol
122
,
775
788
Jones
S. M.
,
Howell
K. E.
,
Henley
J. R.
,
Cao
H.
,
McNiven
M. A.
(
1998
).
Role of dynamin in the formation of transport vesicles from the trans Golgi network.
Science
279
,
573
577
Karin
M.
,
Mintz
B.
(
1981
).
Receptor-mediated endocytosis of transferrin in developmentally totipotent mouse teratocarcinoma stem-cells.
J. Biol. Chem
256
,
3245
3252
Leitinger
B.
,
Hille Rehfeld
A.
,
Spiess
M.
(
1995
).
Biosynthetic transport of the asialoglycoprotein receptor H1 to the cell surface occurs via endosomes.
Proc. Nat. Acad. Sci. USA
92
,
10109
10113
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 (TGN38).
Biochem. J
270
,
97
102
Octave
J. N.
,
Schneider
Y. J.
,
Crichton
R. R.
,
Trouet
A.
(
1981
).
Transferrin uptake by cultured rat embryo fibroblasts- the influence of temperature and incubation-time, subcellular-distribution and short-term kinetic studies.
Eur. J. Biochem
115
,
611
618
Ohno
H.
,
Stewart
J.
,
Fournier
M. C.
,
Bosshart
H.
,
Rhee
I.
,
Miyatake
S.
, et al. 
(
1995
).
Interaction of tyrosine-based sorting signals with clathrin-associated proteins.
Science
269
,
1872
1875
Ponnambalam
S.
,
Girotti
M.
,
Yaspo
M.-L.
,
Owen
C. W.
,
Perry
A. C. F.
,
Suganuma
T.
, et al. 
(
1996
).
Primate homologues of rat TGN38: primary structure expression and functional implications.
J. Cell Sci
109
,
675
685
Reaves
B.
,
Horn
M.
,
Banting
G.
(
1993
).
TGN38/41 recycles between the cell-surface and the TGN- Brefeldin-A affects its rate of return to the TGN.
Mol. Biol. Cell
4
,
93
105
Reaves
B.
,
Banting
G.
(
1994
).
Vacuolar ATPase inactivation blocks recycling to the trans-Golgi network from the plasma-membrane.
FEBS Lett
345
,
61
66
Reaves
B. J.
,
Bright
N. A.
,
Mullock
B. M.
,
Luzio
J. P.
(
1996
).
The effect of wortmannin on the localisation of lysosomal type I integralmembrane glycoproteins suggest a role for phosphoinositide 3-kinse activity in regulating membrane traffic late in the endocytic pathway.
J. Cell Sci
109
,
749
762
Roquemore
E. P.
,
Banting
G.
(
1998
).
Efficient trafficking of TGN38 between the endosome and the TGNrequires a free serine hydroxyl group at position 331 in the cytosolic domain.
Mol. Biol. Cell
9
,
2125
2144
Stephens
D. J.
,
Crump
C. M.
,
Clarke
A. R.
,
Banting
G.
(
1997
).
Serine-331 and tyrosine-333 are both involved in the interaction between the cytosolic domain of TGN38 and the2 subunit of the AP2 clathrin adaptor complex.
J. Biol. Chem
272
,
14104
14109
Takei
K.
,
McPherson
P. S.
,
Schmid
S. L.
,
Decamilli
P.
(
1995
).
Tubular membrane invaginations coated by dynamin rings are induced by GTP-gamma-S in nerve-terminals.
Nature
374
,
186
190
van der Bliek
A. M.
,
Redelmeier
T. E.
,
Damke
H.
,
Tisdale
E. J.
,
Meyerowitz
E. M.
,
Schmid
S. L.
(
1993
).
Mutations in human dynamin block an intermediate stage in coated vesicle formation.
J. Cell Biol
122
,
553
560
Wong
S. H.
,
Hong
W. J.
(
1993
).
The SXYQRL sequence in the cytoplasmic domain of TGN38 plays a major role in trans-Golgi network localization.
J. Biol. Chem
268
,
22853
22862
This content is only available via PDF.